G2Cdb::Gene report

Gene id
G00001397
Gene symbol
YWHAE (HGNC)
Species
Homo sapiens
Description
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide
Orthologue
G00000148 (Mus musculus)

Databases (7)

Gene
ENSG00000181399 (Ensembl human gene)
7531 (Entrez Gene)
83 (G2Cdb plasticity & disease)
YWHAE (GeneCards)
Literature
605066 (OMIM)
Marker Symbol
HGNC:12851 (HGNC)
Protein Sequence
P62258 (UniProt)

Synonyms (1)

  • FLJ45465

Literature (112)

Pubmed - other

  • Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment.

    Nagamani SC, Zhang F, Shchelochkov OA, Bi W, Ou Z, Scaglia F, Probst FJ, Shinawi M, Eng C, Hunter JV, Sparagana S, Lagoe E, Fong CT, Pearson M, Doco-Fenzy M, Landais E, Mozelle M, Chinault AC, Patel A, Bacino CA, Sahoo T, Kang SH, Cheung SW, Lupski JR and Stankiewicz P

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

    Background: Deletions in the 17p13.3 region are associated with abnormal neuronal migration. Point mutations or deletion copy number variants of the PAFAH1B1 gene in this genomic region cause lissencephaly, whereas extended deletions involving both PAFAH1B1 and YWHAE result in Miller-Dieker syndrome characterised by facial dysmorphisms and a more severe grade of lissencephaly. The phenotypic consequences of YWHAE deletion without deletion of PAFAH1B1 have not been studied systematically.

    Methods: We performed a detailed clinical and molecular characterization of five patients with deletions involving YWHAE but not PAFAH1B1, two with deletion including PAFAH1B1 but not YWHAE, and one with deletion of YWHAE and mosaic for deletion of PAFAH1B1.

    Results: Three deletions were terminal whereas five were interstitial. Patients with deletions including YWHAE but not PAFAH1B1 presented with significant growth restriction, cognitive impairment, shared craniofacial features, and variable structural abnormalities of the brain. Growth restriction was not observed in one patient with deletion of YWHAE and TUSC5, implying that other genes in the region may have a role in regulation of growth with CRK being the most likely candidate. Using array based comparative genomic hybridisation and long range polymerase chain reaction, we have delineated the breakpoints of these nonrecurrent deletions and show that the interstitial genomic rearrangements are likely generated by diverse mechanisms, including the recently described Fork Stalling and Template Switching (FoSTeS)/Microhomology Mediated Break Induced Replication (MMBIR).

    Conclusions: Microdeletions of chromosome 17p13.3 involving YWHAE present with growth restriction, craniofacial dysmorphisms, structural abnormalities of brain and cognitive impairment. The interstitial deletions are mediated by diverse molecular mechanisms.

    Journal of medical genetics 2009;46;12;825-33

  • Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

    Mangin PH, Receveur N, Wurtz V, David T, Gachet C and Lanza F

    INSERM UMR_S 949, EFS-Alsace, Université de Strasbourg, Strasbourg, France. pierre.mangin@efs-alsace.fr

    Background: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway.

    Objective: The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX.

    Results: In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain.

    Conclusions: Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

    Journal of thrombosis and haemostasis : JTH 2009;7;9;1550-5

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Isoform-specific cleavage of 14-3-3 proteins in apoptotic JURL-MK1 cells.

    Kuzelová K, Grebenová D, Pluskalová M, Kavan D, Halada P and Hrkal Z

    Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, Prague 2, Czech Republic. kuzel@uhkt.cz

    The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways.

    Journal of cellular biochemistry 2009;106;4;673-81

  • Identification of YWHAE, a gene encoding 14-3-3epsilon, as a possible susceptibility gene for schizophrenia.

    Ikeda M, Hikita T, Taya S, Uraguchi-Asaki J, Toyo-oka K, Wynshaw-Boris A, Ujike H, Inada T, Takao K, Miyakawa T, Ozaki N, Kaibuchi K and Iwata N

    Department of Psychiatry, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.

    Schizophrenia is a complex mental disorder with a fairly high degree of heritability. Although the causes of schizophrenia remain unclear, it is now widely accepted that it is a neurodevelopmental and neurodegenerative disorder involving disconnectivity and disorder of the synapses. Disrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene involved in neurodevelopment, including maturation of the cerebral cortex. To identify other susceptibility genes for schizophrenia, we screened for DISC1-interacting molecules [NudE-like (NUDEL), Lissencephaly-1 (LIS1), 14-3-3epsilon (YWHAE), growth factor receptor bound protein 2 (GRB2) and Kinesin family 5A of Kinesen1 (KIF5A)], assessing a total of 25 tagging single-nucleotide polymorphisms (SNPs) in a Japanese population. We identified a YWHAE SNP (rs28365859) that showed a highly significant difference between case and control samples, with higher minor allele frequencies in controls (P(allele) = 1.01 x 10(-5) and P(genotype) = 4.08 x 10(-5) in 1429 cases and 1728 controls). Both messenger RNA transcription and protein expression of 14-3-3epsilon were also increased in the lymphocytes of healthy control subjects harboring heterozygous and homozygous minor alleles compared with homozygous major allele subjects. To further investigate a potential role for YWHAE in schizophrenia, we studied Ywhae(+/-) mice in which the level of 14-3-3epsilon protein is reduced to 50% of that in wild-type littermates. These mice displayed weak defects in working memory in the eight-arm radial maze and moderately enhanced anxiety-like behavior in the elevated plus-maze. Our results suggest that YWHAE is a possible susceptibility gene that functions protectively in schizophrenia.

    Human molecular genetics 2008;17;20;3212-22

  • Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein.

    Brummer T, Larance M, Herrera Abreu MT, Lyons RJ, Timpson P, Emmerich CH, Fleuren ED, Lehrbach GM, Schramek D, Guilhaus M, James DE and Daly RJ

    Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

    Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2(S210A/T391A) exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14-3-3-binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site-selective binding of 14-3-3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.

    The EMBO journal 2008;27;17;2305-16

  • Chibby cooperates with 14-3-3 to regulate beta-catenin subcellular distribution and signaling activity.

    Li FQ, Mofunanya A, Harris K and Takemaru K

    Department of Pharmacological Sciences and 2Graduate Program in Genetics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA. li@pharm.stonybrook.edu

    beta-Catenin functions in both cell-cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of beta-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although beta-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits beta-catenin-mediated transcriptional activation. Here, we identified 14-3-3epsilon and 14-3-3zeta as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3-binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with beta-catenin, causing beta-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of beta-catenin, thereby antagonizing beta-catenin signaling.

    Funded by: NIDDK NIH HHS: R01 DK073191

    The Journal of cell biology 2008;181;7;1141-54

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • 14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization.

    Tak H, Jang E, Kim SB, Park J, Suk J, Yoon YS, Ahn JK, Lee JH and Joe CO

    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.

    The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.

    Cellular signalling 2007;19;11;2379-87

  • 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase.

    Mizuno E, Kitamura N and Komada M

    Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-16 Nagatsuta, Yokohama, Japan.

    The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 epsilon, gamma, and zeta as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS(680)SP, by phosphatase treatment, and by competition with the Ser(680)-phosphorylated RSYS(680)SP peptide. Metabolic labeling with [(32)P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser(680) is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser(680) in a phosphorylation-dependent manner. The mutation at Ser(680) led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3epsilon inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser(680) and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.

    Experimental cell research 2007;313;16;3624-34

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast.

    Yahyaoui W, Callejo M, Price GB and Zannis-Hadjopoulos M

    McGill Cancer Centre, Montreal, Quebec, Canada. wafaa.yahyaoui@mcgill.ca <wafaa.yahyaoui@mcgill.ca&gt;

    Background: Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner.

    Results: By expressing the human 14-3-3epsilon, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3epsilon complements the S. cerevisiae Bmh1/Bmh2 double knockout, conserves its cruciform binding activity, and associates in vivo with the yeast replication origins ARS307. Deletion of the alpha5-helix, the potential cruciform binding domain of 14-3-3, decreased the cruciform binding activity of the protein as well as its association with the yeast replication origins ARS307 and ARS1. Furthermore, the mutant cells had a reduced ability to stably maintain plasmids bearing one or multiple origins.

    Conclusion: 14-3-3, a cruciform DNA binding protein, associates with yeast origins of replication and functions as an initiator of DNA replication, presumably through binding to cruciform DNA forming at yeast replicators.

    BMC molecular biology 2007;8;27

  • The epsilon isoform of 14-3-3 protein is a component of the prion protein amyloid deposits of Gerstmann-Sträussler-Scheinker disease.

    Di Fede G, Giaccone G, Limido L, Mangieri M, Suardi S, Puoti G, Morbin M, Mazzoleni G, Ghetti B and Tagliavini F

    Department of Neuropathology and Neurology, Istituto Nazionale Neurologico Carlo Besta, Milan, Italy.

    The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic events, such as transduction pathway modulation, cell cycle control, and apoptosis. Seven isoforms have been identified that are abundant in the brain, preferentially localized in neurons. Remarkable increases in 14-3-3 are seen in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease (CJD), and it has been found in pathologic inclusions of several neurodegenerative diseases. Moreover, the zeta isoform has been detected in prion protein (PrP) amyloid deposits of CJD patients. To further investigate the cerebral distribution of 14-3-3 in prion-related encephalopathies, we carried out an immunohistochemical and biochemical analysis of brain tissue from patients with Gerstmann-Sträussler-Scheinker disease (GSS) and sporadic, familial and acquired forms of CJD, using specific antibodies against the seven 14-3-3 isoforms. The study showed a strong immunoreactivity of PrP amyloid plaques of GSS patients for the 14-3-3 epsilon isoform, but not for the other isoforms. The epsilon isoform of 14-3-3 was not found in PrP deposits of CJD. These results indicate that the epsilon isoform of 14-3-3 is a component of PrP amyloid deposits of GSS and suggest that this is the sole 14-3-3 isoform specifically involved in the neuropathologic changes associated with this disorder.

    Funded by: NIA NIH HHS: AG010133

    Journal of neuropathology and experimental neurology 2007;66;2;124-30

  • Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.

    Chi A, Valencia JC, Hu ZZ, Watabe H, Yamaguchi H, Mangini NJ, Huang H, Canfield VA, Cheng KC, Yang F, Abe R, Yamagishi S, Shabanowitz J, Hearing VJ, Wu C, Appella E and Hunt DF

    Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.

    Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.

    Funded by: NCRR NIH HHS: RR01744; NHGRI NIH HHS: U01-HG02712; NICHD NIH HHS: HD40179; NIGMS NIH HHS: GM 37537

    Journal of proteome research 2006;5;11;3135-44

  • C-terminal HERG (LQT2) mutations disrupt IKr channel regulation through 14-3-3epsilon.

    Choe CU, Schulze-Bahr E, Neu A, Xu J, Zhu ZI, Sauter K, Bähring R, Priori S, Guicheney P, Mönnig G, Neapolitano C, Heidemann J, Clancy CE, Pongs O and Isbrandt D

    Institute for Neural Signal Transduction, ZMNH, Department of Pediatrics, University Hospital Hamburg-Eppendorf, Hamburg, Germany.

    Beta-adrenergic receptor-mediated cAMP or protein kinase A (PKA)-dependent modulation of cardiac potassium currents controls ventricular action potential duration (APD) at faster heart rates. HERG (KCNH2) gene mutations are associated with congenital long-QT syndrome (LQT2) and affect IKr activity, a key determinant in ventricular repolarization. Physical activity or emotional stress often triggers lethal arrhythmias in LQT2 patients. Beta-adrenergic stimulation of HERG channel activity is amplified and prolonged in vitro by the adaptor protein 14-3-3epsilon. In LQT2 families, we identified three novel heterozygous HERG mutations (G965X, R1014PfsX39, V1038AfsX21) in the C-terminus that led to protein truncation and loss of a PKA phosphorylation site required for binding of 14-3-3epsilon. When expressed in CHO cells, the mutants produced functional HERG channels with normal kinetic properties. We now provide evidence that HERG channel regulation by 14-3-3epsilon is of physiological significance in humans. Upon co-expression with 14-3-3epsilon, mutant channels still bound 14-3-3epsilon but did not respond with a hyperpolarizing shift in voltage dependence as seen in wild-type channels. Co-expression experiments of wild-type and mutant channels revealed dominant-negative behavior of all three HERG mutations. Simulations of the effects of sympathetic stimulation of HERG channel activity on the whole-cell action potential suggested a role in rate-dependent control of APD and an impaired ability of mutant cardiac myocytes to respond to a triggered event or an ectopic beat. In summary, the attenuated functional effects of 14-3-3epsilon on C-terminally truncated HERG channels demonstrate the physiological importance of coupling beta-adrenergic stimulation and HERG channel activity.

    Human molecular genetics 2006;15;19;2888-902

  • HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation.

    Matsuda N, Tanaka H, Yamazaki S, Suzuki J, Tanaka K, Yamada T and Masuda M

    Department of Microbiology, Dokkyo Medical University School of Medicine, Kita-kobayashi 880, Mibu, Tochigi 321-0293, Japan.

    Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.

    Microbes and infection 2006;8;12-13;2736-44

  • Efficient ADAM22 surface expression is mediated by phosphorylation-dependent interaction with 14-3-3 protein family members.

    Gödde NJ, D'Abaco GM, Paradiso L and Novak U

    Department of Surgery, University of Melbourne, Parkville 3050, Australia.

    ADAM22 is one of three catalytically inactive ADAM family members highly expressed in the brain. ADAM22 has numerous splice variants, all with considerable cytoplasmic tails of up to 148 amino acids. ADAM22 can act to inhibit cell proliferation, however, it has been suggested that it also acts as an adhesion protein. We identified three 14-3-3 protein members by a yeast two-hybrid screen and show by co-immunoprecipitation that the cytoplasmic domain of ADAM22 can interact with all six 14-3-3 proteins expressed in the brain. In addition, we show that 14-3-3 proteins interact preferentially with the serine phosphorylated precursor form of ADAM22. ADAM22 has two 14-3-3 protein binding consensus motifs; the first binding site, spanning residues 831-834, was shown to be the most crucial for 14-3-3 binding to occur. The interaction between ADAM22 and 14-3-3 proteins is dependent on phosphorylation of ADAM22, but not of 14-3-3 proteins. ADAM22 point mutants lacking functional 14-3-3 protein binding motifs could no longer accumulate efficiently at the cell surface. Deletion of both 14-3-3 binding sites and newly identified ER retention motifs restored localization of ADAM22 at the cell surface. These results reveal a role for 14-3-3 proteins in targeting ADAM22 to the membrane by masking ER retention signals.

    Journal of cell science 2006;119;Pt 16;3296-305

  • Inhibitory interaction of the plasma membrane Na+/Ca2+ exchangers with the 14-3-3 proteins.

    Pulina MV, Rizzuto R, Brini M and Carafoli E

    Venetian Institute of Molecular Medicine, and Department of Biochemistry, University of Padova, 35100 Padova, Italy.

    The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.

    Funded by: Telethon: GP0193Y01

    The Journal of biological chemistry 2006;281;28;19645-54

  • A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins.

    Gao Y, Jiang M, Yang T, Ni J and Chen J

    State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China.

    hPFTAIRE1 (PFTK1), a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals (NLSs) in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms (beta, epsilon, eta, tau) were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif (RHSSPSS) in the hPFTAIRE1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser119 with Ala in the conserved binding motif abolished the specific interaction between the hPFTAIRE1 and the 14-3-3 proteins. The mutant S120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser119 is crucial for the interaction between hPFTAIRE1 and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells (SH-SY5Y) when fused to the C-terminus of a green fluorescent protein (GFP), indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.

    Cell research 2006;16;6;539-47

  • The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation.

    Petretti C, Savoian M, Montembault E, Glover DM, Prigent C and Giet R

    CNRS UMR 6061 Université de Rennes I, Equipe Labellisée Ligue Nationale Contre le Cancer, IFR140 GFAS, Faculté de Médecine, France.

    The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. gamma-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11(p58) isoform, but not the CDK11(p110) isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11(p58) in centrosome maturation and bipolar spindle morphogenesis.

    EMBO reports 2006;7;4;418-24

  • Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.

    Gong W, Russell M, Suzuki K and Riabowol K

    Southern Alberta Cancer Research Institute, Dept. of Biochemistry, University of Calgary, #370 Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.

    ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

    Molecular and cellular biology 2006;26;8;2947-54

  • 14-3-3gamma binds to MDMX that is phosphorylated by UV-activated Chk1, resulting in p53 activation.

    Jin Y, Dai MS, Lu SZ, Xu Y, Luo Z, Zhao Y and Lu H

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA.

    It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14-3-3gamma and Chk1. 14-3-3gamma was identified as an MDMX-associated protein via an immuno-affinity purification-coupled mass spectrometry. Consistently, 14-3-3gamma directly interacted with MDMX in vitro, and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild-type, but not the kinase-dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14-3-3gamma-MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN-01 repressed all of these effects. Moreover, overexpression of 14-3-3gamma, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX-enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14-3-3gamma by siRNA reduced UV-induced p53 level and G1 arrest. Thus, these results demonstrate 14-3-3gamma and Chk1 as two novel regulators of MDMX in response to UV irradiation.

    Funded by: NCI NIH HHS: CA079721, CA095441, CA93614, R01 CA079721, R01 CA093614, R01 CA095441

    The EMBO journal 2006;25;6;1207-18

  • Regulation of MDMX nuclear import and degradation by Chk2 and 14-3-3.

    LeBron C, Chen L, Gilkes DM and Chen J

    Molecular Oncology Program, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.

    The MDM2 homolog MDMX is an important regulator of p53 during mouse embryonic development. DNA damage promotes MDMX phosphorylation, nuclear translocation, and degradation by MDM2. Here we show that MDMX copurifies with 14-3-3, and DNA damage stimulates MDMX binding to 14-3-3. Chk2-mediated phosphorylation of MDMX on S367 is important for stimulating 14-3-3 binding, MDMX nuclear import by a cryptic nuclear import signal, and degradation by MDM2. Mutation of MDMX S367 inhibits ubiquitination and degradation by MDM2, and prevents MDMX nuclear import. Expression of 14-3-3 stimulates the degradation of phosphorylated MDMX. Chk2 and 14-3-3 cooperatively stimulate MDMX ubiquitination and overcome the inhibition of p53 by MDMX. These results suggest that MDMX-14-3-3 interaction plays a role in p53 response to DNA damage by regulating MDMX localization and stability.

    The EMBO journal 2006;25;6;1196-206

  • Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation.

    Fritz A, Brayer KJ, McCormick N, Adams DG, Wadzinski BE and Vaillancourt RR

    Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Tucson, Arizona 85721, USA.

    MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.

    Funded by: NIA NIH HHS: AG19710; NIEHS NIH HHS: ES12007, P30 ES06694, P42 ES04940; NIGMS NIH HHS: GM51366, GM62265

    The Journal of biological chemistry 2006;281;10;6236-45

  • 14-3-3 proteins integrate E2F activity with the DNA damage response.

    Milton AH, Khaire N, Ingram L, O'Donnell AJ and La Thangue NB

    Laboratory of Cancer Biology, Division of Medical Sciences, University of Oxford, UK.

    The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.

    Funded by: Cancer Research UK: 13058; Medical Research Council: G0500905, G9400953

    The EMBO journal 2006;25;5;1046-57

  • Protein kinase A phosphorylates and regulates dimerization of 14-3-3 epsilon.

    Gu YM, Jin YH, Choi JK, Baek KH, Yeo CY and Lee KY

    College of Pharmacy, Chonnam National University, Yongbong-dong 300, Buk-Gu, Gwangju 500757, Republic of Korea.

    Recognition of phosphorylated serine/threonine-containing motifs by 14-3-3 depends on the dimerization of 14-3-3. However, the molecular cues that control 14-3-3 dimerization are not well understood. In order to identify proteins that control 14-3-3 dimerization, we analyzed proteins that have effects on 14-3-3 dimerization and report that protein kinase A (PKA) phosphorylates 14-3-3zeta at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14-3-3zeta dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14-3-3zeta by PKA. A phospho-mimic mutant of 14-3-3zeta, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14-3-3epsilon and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14-3-3zeta and the functional interaction of 14-3-3zeta with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14-3-3.

    FEBS letters 2006;580;1;305-10

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

    Khanna H, Hurd TW, Lillo C, Shu X, Parapuram SK, He S, Akimoto M, Wright AF, Margolis B, Williams DS and Swaroop A

    Department of Ophthalmology & Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105, USA.

    Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

    Funded by: NEI NIH HHS: EY07003, EY07961, EY12598, EY13408, F31 EY007003, P30 EY007003, P30 EY012598, R01 EY007961, R01 EY013408; NIDDK NIH HHS: DK069605, R01 DK069605

    The Journal of biological chemistry 2005;280;39;33580-7

  • BCR kinase phosphorylates 14-3-3 Tau on residue 233.

    Clokie SJ, Cheung KY, Mackie S, Marquez R, Peden AH and Aitken A

    School of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, UK.

    The breakpoint cluster region protein, BCR, has protein kinase activity that can auto- and trans-phosphorylate serine, threonine and tyrosine residues. BCR has been implicated in chronic myelogenous leukaemia as well as important signalling pathways, and as such its interaction with 14-3-3 is of major interest. 14-3-3tau and zeta isoforms have been shown previously to be phosphorylated in vitro and in vivo by BCR kinase on serine and threonine residue(s) but site(s) were not determined. Phosphorylation of 14-3-3 isoforms at distinct sites is an important mode of regulation that negatively affects interaction with Raf kinase and Bax, and potentially influences the dimerization of 14-3-3. In this study we have further characterized the BCR-14-3-3 interaction and have identified the site phosphorylated by BCR. We show here that BCR interacts with at least five isoforms of 14-3-3 in vivo and phosphorylates 14-3-3tau on Ser233 and to a lesser extent 14-3-3zeta on Thr233. We have previously shown that these two isoforms are also phosphorylated at this site by casein kinase 1, which, in contrast to BCR, preferentially phosphorylates 14-3-3zeta.

    The FEBS journal 2005;272;15;3767-76

  • The cyclin-dependent kinase 11 interacts with 14-3-3 proteins.

    Feng Y, Qi W, Martinez J and Nelson MA

    Department of Pathology, University of Arizona, 1501 N Campbell Avenue, Tucson, AZ 85724, USA. yfeng@email.arizona.edu

    Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.

    Funded by: NCI NIH HHS: CA 107510, CA 70145

    Biochemical and biophysical research communications 2005;331;4;1503-9

  • Phosphorylation of grb10 regulates its interaction with 14-3-3.

    Urschel S, Bassermann F, Bai RY, Münch S, Peschel C and Duyster J

    Department of Internal Medicine III, Laboratory of Leukemogenesis, Technical University of Munich, 81675 Munich, Germany.

    Grb10 is a member of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. Grb10 expression levels can influence Akt activity, and Grb10 may act as an adapter involved in the relocalization of Akt to the cell membrane. Here we identified 14-3-3 as a binding partner of Grb10 by employing a yeast two-hybrid screen. The 14-3-3.Grb10 interaction requires phosphorylation of Grb10, and only the phosphorylated form of Grb10 co-immunoprecipitates with endogenous 14-3-3. We could identify a putative phosphorylation site in Grb10, which is located in a classical 14-3-3 binding motif, RSVSEN. Mutation of this site in Grb10 diminished binding to 14-3-3. Thus, Grb10 exists in two different states of phosphorylation and complexes with 14-3-3 when phosphorylated on serine 428. We provide evidence that Akt directly binds Grb10 and is able to phosphorylate Grb10 in an in vitro kinase assay. Based on these findings, we propose a regulatory circuitry involving a phosphorylation-regulated complex formation of Grb10 with 14-3-3 and Akt.

    The Journal of biological chemistry 2005;280;17;16987-93

  • JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage.

    Yoshida K, Yamaguchi T, Natsume T, Kufe D and Miki Y

    Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan. yos.mgen@mri.tmd.ac.jp

    The ubiquitously expressed c-Abl tyrosine kinase localizes to the cytoplasm and nucleus. Nuclear c-Abl is activated by diverse genotoxic agents and induces apoptosis; however, the mechanisms that are responsible for nuclear targeting of c-Abl remain unclear. Here, we show that cytoplasmic c-Abl is targeted to the nucleus in the DNA damage response. The results show that c-Abl is sequestered into the cytoplasm by binding to 14-3-3 proteins. Phosphorylation of c-Abl on Thr 735 functions as a site for direct binding to 14-3-3 proteins. We also show that, in response to DNA damage, activation of the c-Jun N-terminal kinase (Jnk) induces phosphorylation of 14-3-3 proteins and their release from c-Abl. Together with these results, expression of an unphosphorylated 14-3-3 mutant attenuates DNA-damage-induced nuclear import of c-Abl and apoptosis. These findings indicate that 14-3-3 proteins are pivotal regulators of intracellular c-Abl localization and of the apoptotic response to genotoxic stress.

    Funded by: NCI NIH HHS: CA29431, CA98628

    Nature cell biology 2005;7;3;278-85

  • Vpr protein of human immunodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex formation with Cdc25C: implications for cell cycle arrest.

    Kino T, Gragerov A, Valentin A, Tsopanomihalou M, Ilyina-Gragerova G, Erwin-Cohen R, Chrousos GP and Pavlakis GN

    Human Retrovirus Section, Center for Cancer Research, National Cancer Institute-Frederick, Bldg. 535, Rm. 210, Frederick, MD 21702-1201, USA.

    Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G(2)/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3sigma. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G(2)/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.

    Journal of virology 2005;79;5;2780-7

  • Nucleolar proteome dynamics.

    Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI and Mann M

    Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, Denmark.

    The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

    Funded by: Wellcome Trust: 073980

    Nature 2005;433;7021;77-83

  • Association of 14-3-3 epsilon gene haplotype with completed suicide in Japanese.

    Yanagi M, Shirakawa O, Kitamura N, Okamura K, Sakurai K, Nishiguchi N, Hashimoto T, Nushida H, Ueno Y, Kanbe D, Kawamura M, Araki K, Nawa H and Maeda K

    Division of Psychiatry and Neurology, Department of Environmental Health and Safety, Faculty of Medical Sciences, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho Chuo-Ku, Kobe 650-0017, Japan.

    Genetic factors have been suggested to be involved in suicide. Although some genetic factors, such as serotonergic transduction, have been associated with suicide, the results are inconsistent. There is a possibility that various signaling anomalies are involved in the biological vulnerability to suicide. We carried out a genome-wide gene-expression study in the brains of suicide victims using DNA microarrays;14-3-3 epsilon, which is related to neurogenesis, was one of the genes upregulated in the brains of suicide victims in the microarray analysis. This was confirmed by Western blot analysis. To examine the possibility of the involvement of 14-3-3 epsilon in the pathogenesis of suicide, we investigated the association of the 14-3-3 epsilon gene and completed suicide. We used three high-frequency SNPs (rs1532976, rs3752826, and rs9393) and found a significant association of two alleles (rs1532976 and rs3752826) with completed suicide (p < 0.05). Moreover, the distribution of haplotype revealed a more significant difference between completed suicide and controls (p=0.0005). This finding suggests that 14-3-3 epsilon is a potential suicide susceptibility gene and implies that dysregulation of neurogenesis may be involved in suicide.

    Journal of human genetics 2005;50;4;210-6

  • Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites.

    Nguyen TA, Takemoto LJ and Takemoto DJ

    Department of Biochemistry and Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.

    We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

    Funded by: NEI NIH HHS: EY02932, EY13421

    The Journal of biological chemistry 2004;279;50;52714-25

  • Interactions between extracellular signal-regulated protein kinase 1, 14-3-3epsilon, and heat shock factor 1 during stress.

    Wang X, Grammatikakis N, Siganou A, Stevenson MA and Calderwood SK

    Dana-Farber Cancer Institute and Division of Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Cytoprotection during the heat shock response is a complex phenomenon involving multiple inducible mechanisms. We have examined the interaction of two key molecular components in the response, heat shock transcription factor 1 (HSF1) and extracellular signal regulated protein kinase (ERK). Whereas both HSF1 and ERK are required to protect cells against apoptosis, ERK activation is paradoxically antagonistic to trans-activation of hsp promoters by HSF1 and HSP accumulation during heat shock. We have found that the two pathways interact directly and that heat shock causes the physical association of ERK1 with HSF1, an interaction that promotes the kinase activity of ERK in heat-shocked cells. ERK activation results in the recruitment of the phosphoserine binding protein 14-3-3epsilon in a manner dependent on previous HSF1 phosphorylation by ERK. The effects of 14-3-3epsilon binding on HSF1 were complex, however, depending on extracellular conditions, in that HSF1-14-3-3 binding at 37 degrees C led to the cytoplasmic sequestration and repression of HSF1, whereas heat shock overrode these effects and caused quantitative nuclear localization of HSF1. Although the effects of 14-3-3epsilon binding to HSF1 were overridden acutely by stress, during recovery from heat shock, 14-3-3epsilon association again led to enhanced cytoplasmic localization of HSF1, implicating a role for ERK/14-3-3epsilon in HSF1 deactivation in recovering cells. Association of HSF1 with ERK and 14-3-3epsilon during heat shock may thus modulate the amplitude of the response and lead to efficient termination of HSP expression on resumption of growth conditions.

    Funded by: NCI NIH HHS: CA31303, CA47407, CA50642

    The Journal of biological chemistry 2004;279;47;49460-9

  • SMRT derepression by the IkappaB kinase alpha: a prerequisite to NF-kappaB transcription and survival.

    Hoberg JE, Yeung F and Mayo MW

    Department of Biochemistry and Molecular Genetics, The University of Virginia, Charlottesville, VA 22908, USA.

    Understanding how signaling cascades stimulate chromatin-remodeling events through derepression is one of the foremost questions in the transcription field. Here, we demonstrate that NF-kappaB transcription requires IKKalpha to phosphorylate SMRT on chromatin, stimulating the exchange of corepressor for coactivator complexes. IKKalpha-induced phosphorylation coincides with a loss of chromatin-associated SMRT and HDAC3 and with nuclear export of the SMRT corepressor, events required for expression of the NF-kappaB-regulated cIAP-2 and IL-8 genes. Although SMRT derepression corresponds with the recruitment of TBL1/TBLR1, this complex alone is insufficient to relieve repression. Using a nonphosphorylatable SMRT protein, we demonstrate that IKKalpha-induced phosphorylation is required to recruit 14-3-3epsilon and Ubc5 for SMRT derepression. Failure of IKKalpha to stimulate the removal of SMRT from chromatin inhibits the recruitment of NF-kappaB to promoters, blocking transcription and sensitizing cells to apoptosis. Our work provides evidence that IKKalpha orchestrates SMRT derepression, a prerequisite for NF-kappaB transcription and survival.

    Funded by: NCI NIH HHS: CA095644, CA104397, CA110552, CA78595

    Molecular cell 2004;16;2;245-55

  • Regulation of Dyrk1A kinase activity by 14-3-3.

    Kim D, Won J, Shin DW, Kang J, Kim YJ, Choi SY, Hwang MK, Jeong BW, Kim GS, Joe CO, Chung SH and Song WJ

    Division of CNS, Hanwha Chemical R&D Center, Daejon 305-345, Republic of Korea.

    Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.

    Biochemical and biophysical research communications 2004;323;2;499-504

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

    Diviani D, Abuin L, Cotecchia S and Pansier L

    Département de Pharmacologie et de Toxicologie, Faculté de Médecine, Lausanne, Switzerland. Dario.diviani@ipharm.unil.ch

    A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

    The EMBO journal 2004;23;14;2811-20

  • Human immunodeficiency virus type-1 accessory protein Vpr: a causative agent of the AIDS-related insulin resistance/lipodystrophy syndrome?

    Kino T and Chrousos GP

    Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1583, USA. kinot@mail.nih.gov

    Recent advances in the development of three different types of antiviral drugs, the nucleotide and non-nucleotide analogues acting as reverse transcriptase inhibitors (NRTIs) and the nonpeptidic viral protease inhibitors (PI), and their introduction in the management of patients with AIDS, either alone or in combination, have dramatically improved the clinical course of the disease and prolonged life expectancy in patients with AIDS. The increase in life expectancy in association with the long-term use of the above antiviral agents, however, have generated novel morbidities and complications. Central among them is the quite common AIDS-related insulin resistance and lipodystrophy syndrome, which is characterized by a striking phenotype and marked metabolic disturbances. To look for the pathologic causes of this particular syndrome, we focused on one of the HIV-1 accessory proteins, Vpr, which has multiple functions, such as virion incorporation, nuclear translocation of the HIV-1 preintegration complex, nucleo-cytoplasmic shuttling, transcriptional activation, and induction of apoptosis. Vpr may also act like a hormone, which is secreted into the extracellular space and affects the function of distant organs. Vpr functions as a coactivator of the glucocorticoid receptor and potentiates the action of glucocorticoid hormones, thereby inducing tissue glucocorticoid hypersensitivity. Vpr also arrests host cells at the G2/M phase of the cell cycle by interacting with novel 14-3-3 proteins. Vpr facilitates the interaction of 14-3-3 and its partner protein Cdc25C, which is critical for the transition of G2/M checkpoint in the cell cycle, and suppresses its activity by segregating it into the cytoplasm. The same Vpr protein also suppresses the association of 14-3-3 with other partner molecules, the Foxo transcription factors. Since the Foxo proteins function as negative transcription factors for insulin, Vpr may cause resistance of tissues to insulin. Through these two newly identified functions of Vpr, namely, coactivation of glucocorticoid receptor activity and inhibition of insulin effects on Foxo proteins, Vpr may participate in the development of AIDS-related insulin resistance/lipodystrophy syndrome.

    Annals of the New York Academy of Sciences 2004;1024;153-67

  • CD81 associates with 14-3-3 in a redox-regulated palmitoylation-dependent manner.

    Clark KL, Oelke A, Johnson ME, Eilert KD, Simpson PC and Todd SC

    Program in Molecular, Cellular and Developmental Biology, Division of Biology, 239C Chalmers Hall, Kansas State University, Manhattan, KS 66506, USA. c6vl@hotmail.com

    As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N- and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.

    Funded by: NCRR NIH HHS: RR 16475

    The Journal of biological chemistry 2004;279;19;19401-6

  • 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1).

    Sekimoto T, Fukumoto M and Yoneda Y

    Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Yamada-oka, Suita, Osaka, Japan.

    p27(Kip1) (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin alpha3 and alpha5, but not alpha1, transported p27 into the nucleus in conjunction with importin beta, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin alpha5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin alpha5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin alpha binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin alpha/beta-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.

    The EMBO journal 2004;23;9;1934-42

  • p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor.

    Zenke FT, Krendel M, DerMardirossian C, King CC, Bohl BP and Bokoch GM

    Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

    GEF-H1 is a guanine nucleotide exchange factor for Rho whose activity is regulated through a cycle of microtubule binding and release. Here we identify a region in the carboxyl terminus of GEF-H1 that is important for suppression of its guanine nucleotide exchange activity by microtubules. This portion of the protein includes a coiled-coil motif, a proline-rich motif that may interact with Src homology 3 domain-containing proteins, and a potential binding site for 14-3-3 proteins. We identify GEF-H1 as a binding target and substrate for p21-activated kinase 1 (PAK1), an effector of Rac and Cdc42 GTPases, using an affinity-based screen and localize a PAK1 phosphorylation site to the inhibitory carboxyl-terminal region of GEF-H1. We show that phosphorylation of GEF-H1 at Ser(885) by PAK1 induces 14-3-3 binding to the exchange factor and relocation of 14-3-3 to microtubules. Phosphorylation of GEF-H1 by PAK may be involved in regulation of GEF-H1 activity and may serve to coordinate Rho-, Rac-, and Cdc42-mediated signaling pathways.

    The Journal of biological chemistry 2004;279;18;18392-400

  • Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins.

    Subramanian RR, Zhang H, Wang H, Ichijo H, Miyashita T and Fu H

    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.

    Funded by: NIGMS NIH HHS: GM53165, GM60033

    Experimental cell research 2004;294;2;581-91

  • Partner molecules of accessory protein Vpr of the human immunodeficiency virus type 1.

    Kino T and Pavlakis GN

    Human Retrovirus Section, Center for Basic Research, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, USA.

    Vpr (Viral protein-R) of the Human Immunodeficiency Virus type-1 is a 14-kDa virion-associated protein, conserved in HIV-1, -2 and the Simian Immunodeficiency Virus (SIV). Vpr is incorporated into the virion, travels to the nucleus, and has multiple activities including promoter activation, cell cycle arrest at the G2/M transition and apoptosis induction. Through these activities, Vpr is thought to influence not only viral replication but also numerous host cell functions. These functions may be categorized in three groups depending on the domains of Vpr that support them: (1) functions mediated by the amino terminal portion of Vpr, like virion packaging; (2) functions mediated by the carboxyl terminal portion such as cell cycle arrest; and (3) functions that depend on central alpha-helical structures such as transcriptional activation, apoptosis and subcellular shuttling. Association of these activities to specific regions of the Vpr molecule appears to correlate to the host/viral molecules that interact with corresponding portion of Vpr. They include Gag, host transcription factors/coactivators such as SP1, the glucocorticoid receptor, p300/CREB-binding protein and TFIIB, apoptotic adenine nucleotide translocator, cyclophilin A and 14-3-3 proteins. The properties of Vpr molecule has made it difficult to assess its function and determine the true cellular interactors. Further studies on Vpr function are needed to fully assess the function of this important early regulatory molecule of HIV and other lentiviruses.

    DNA and cell biology 2004;23;4;193-205

  • 14-3-3beta binds to big mitogen-activated protein kinase 1 (BMK1/ERK5) and regulates BMK1 function.

    Zheng Q, Yin G, Yan C, Cavet M and Berk BC

    Department of Medicine, University of Rochester School of Medicine and Dentistry, Aab Institute of Biomedical Sciences, Rochester, New York 14642, USA.

    Big mitogen-activated kinase 1 (BMK1/ERK5) is a member of the MAPK family activated by growth factors that mediates cell growth and survival. Previous data show that BMK1 can be activated by steady laminar flow and is atheroprotective by preventing endothelial cells from undergoing apoptosis. The primary structure of BMK1 is distinct from other MAPK members by virtue of a unique long C-tail, suggesting specific mechanisms of regulation. To characterize regulatory mechanisms for BMK1 function, we identified binding proteins by yeast two-hybrid analysis. Among these proteins, the scaffolding protein 14-3-3 was identified. BMK1 bound to 14-3-3beta in vitro and in vivo as demonstrated by glutathione S-transferase (GST)-14-3-3beta fusion protein pull-down assays and coimmunoprecipitation. Phosphorylation of BMK1 was most likely required for this interaction. GST-14-3-3beta pull-down assays using truncated constructs of BMK1 and site-directed BMK1 mutants demonstrated that the interaction requires serine 486 within the C terminus of BMK1. BMK1 bound to 14-3-3beta basally, and the interaction was greatly abrogated when BMK1 was activated. The interaction of 14-3-3beta and BMK1 inhibited kinase activities stimulated by constitutively active (CA)-MEK5 and epidermal growth factor. Mutation of serine 486 (BMK1-S486A) prevented the interaction with 14-3-3beta and enhanced BMK1 activity upon epidermal growth factor stimulation. These data demonstrate an inhibitory function for 14-3-3beta binding to BMK1 and show that serine 486 phosphorylation represents a novel regulatory mechanism for BMK1.

    The Journal of biological chemistry 2004;279;10;8787-91

  • A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

    Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B and Superti-Furga G

    Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. tewis.bouwmeester@cellzome.com

    Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

    Nature cell biology 2004;6;2;97-105

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding.

    Chen MS, Ryan CE and Piwnica-Worms H

    Howard Hughes Medical Institute, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.

    The order and fidelity of cell cycle events in mammals is intimately linked to the integrity of the Chk1 kinase-Cdc25A phosphatase pathway. Chk1 phosphorylation targets Cdc25A for destruction and, as shown here, inhibits interactions between Cdc25A and its mitotic substrate cyclin B1-Cdk1. Phosphorylation of Cdc25A on serine 178 and threonine 507 facilitates 14-3-3 binding, and Chk1 phosphorylates both residues in vitro. Mutation of T507 to alanine (T507A) enhanced the biological activity of Cdc25A. Cdc25A(T507A) was more efficient in binding to cyclin B1, activating cyclin B1-Cdk1, and promoting premature entry into mitosis. We propose that the Chk1/Cdc25A/14-3-3 pathway functions to prevent cells from entering into mitosis prior to replicating their genomes to ensure the fidelity of the cell division process.

    Molecular and cellular biology 2003;23;21;7488-97

  • Regulation of poly(A) polymerase by 14-3-3epsilon.

    Kim H, Lee JH and Lee Y

    Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    Poly(A) polymerase (PAP) is a key enzyme responsible for the addition of the poly(A) at the 3' end of pre-mRNA. The C-terminal region of mammalian PAP carries target sites for protein-protein interaction with the 25 kDa subunit of cleavage factor I and with splicing factors U1A and U2AF65. We used a yeast two-hybrid screen to identify 14-3-3epsilon as an additional protein binding to the C-terminal region of PAP. Interaction between PAP and 14-3-3epsilon was confirmed by both in vitro and in vivo binding assays. This interaction is dependent on PAP phosphorylation. Deletion analysis of PAP suggests that PAP contains multiple binding sites for 14-3-3epsilon. The binding of 14-3-3epsilon to PAP inhibits the polyadenylation activity of PAP in vitro, and overexpression of 14-3-3epsilon leads to a shorter poly(A) mRNA tail in vivo. In addition, the interaction between PAP and 14-3-3epsilon redistributes PAP within the cell by increasing its cytoplasmic localization. These data suggest that 14-3-3epsilon is involved in regulating both the activity and the nuclear/ cytoplasmic partitioning of PAP through the phosphorylation-dependent interaction.

    The EMBO journal 2003;22;19;5208-19

  • Regulation of molecular chaperone gene transcription involves the serine phosphorylation, 14-3-3 epsilon binding, and cytoplasmic sequestration of heat shock factor 1.

    Wang X, Grammatikakis N, Siganou A and Calderwood SK

    Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3 epsilon. Binding of HSF1 to 14-3-3 epsilon occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3 epsilon requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3 epsilon results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3 epsilon, suggesting that CRM1/14-3-3 epsilon directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3 epsilon pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation.

    Funded by: NCI NIH HHS: CA31303, CA47407, CA50642, CA77465, R01 CA047407, R01 CA077465

    Molecular and cellular biology 2003;23;17;6013-26

  • The MSP receptor regulates alpha6beta4 and alpha3beta1 integrins via 14-3-3 proteins in keratinocyte migration.

    Santoro MM, Gaudino G and Marchisio PC

    Department of Medical Sciences, University of Piemonte Orientale "A. Avogadro", 28100, Novara, Italy. msantoro@med.unipmn.it

    Growth factors, integrins, and the extracellular matrix (ECM) are known to play key roles in epidermal wound healing, although the interplay between these proteins is not fully understood. We show that growth factor macrophage stimulating protein (MSP)- and its receptor Ron-mediated PI3K activation in keratinocytes induces phosphorylation of both Ron and alpha6beta4 integrin at specific 14-3-3 binding sites. Consequently, a Ron/alpha6beta4 complex formed via 14-3-3 binding displaces alpha6beta4 from its location at hemidesmosomes (structures supporting cell adhesion) and relocalizes it to lamellipodia. Concomitant activation of alpha3beta1 and keratinocyte spreading/migration on laminin-5 occurs. Further, MSP-dependent beta4 tyrosine phosphorylation evokes p38 and NF-kappaB signaling required for keratinocyte wound closure. Based on these results, we propose a mechanism based on MSP-Ron-dependent phosphorylation and 14-3-3 association, whereby the function of alpha6beta4 switches from a mechanical adhesive device into a signaling component, and might be critically involved in human epidermal wound healing.

    Developmental cell 2003;5;2;257-71

  • 14-3-3epsilon is important for neuronal migration by binding to NUDEL: a molecular explanation for Miller-Dieker syndrome.

    Toyo-oka K, Shionoya A, Gambello MJ, Cardoso C, Leventer R, Ward HL, Ayala R, Tsai LH, Dobyns W, Ledbetter D, Hirotsune S and Wynshaw-Boris A

    Department of Pediatrics, UCSD Cancer Center, University of California, San Diego School of Medicine, 9500 Gilman Drive, Mailstop 0627, La Jolla, California 92093-0627, USA.

    Heterozygous deletions of 17p13.3 result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mutations in PAFAH1B1 (the gene encoding LIS1) are responsible for ILS and contribute to MDS, but the genetic causes of the greater severity of MDS are unknown. Here, we show that the gene encoding 14-3-3epsilon (YWHAE), one of a family of ubiquitous phosphoserine/threonine-binding proteins, is always deleted in individuals with MDS. Mice deficient in Ywhae have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Pafah1b1. Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. 14-3-3epsilon binds to CDK5/p35-phosphorylated NUDEL and this binding maintains NUDEL phosphorylation. Similar to LIS1, deficiency of 14-3-3epsilon results in mislocalization of NUDEL and LIS1, consistent with reduction of cytoplasmic dynein function. These results establish a crucial role for 14-3-3epsilon in neuronal development by sustaining the effects of CDK5 phosphorylation and provide a molecular explanation for the differences in severity of human neuronal migration defects with 17p13.3 deletions.

    Nature genetics 2003;34;3;274-85

  • Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

    Casenghi M, Meraldi P, Weinhart U, Duncan PI, Körner R and Nigg EA

    Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152, Martinsried, Germany.

    In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

    Developmental cell 2003;5;1;113-25

  • Ten years on: mediation of cell death by the common neurotrophin receptor p75(NTR).

    Rabizadeh S and Bredesen DE

    The Buck Institute for Age Research, 8001 Redwood Blvd, Novato, CA 94945-1400, USA. srabizadeh@buckinstitute.org

    The common neurotrophin receptor p75(NTR) remains one of the most enigmatic of the tumor necrosis factor receptor (TNFR) superfamily: on the one hand, it displays a death domain and has been shown to be capable of mediating programmed cell death (PCD) upon ligand binding; on the other hand, its death domain is of type II (unlike that of Fas or TNFR I), and it has also been shown to be capable of mediating cell death in response to the withdrawal of ligand. Thus, p75(NTR) may function as a death receptor-similar to Fas or TNFR I-or a dependence receptor-similar to deleted in colorectal cancer (DCC) or uncoordinated gene-5 homologues 1-3 (UNC5H1-3). Here, we review the data relating to the mediation of PCD by p75(NTR), and suggest that one reasonable model for the apparently paradoxical effects of p75(NTR) is that this receptor functions as a "quality control" in that it is capable of mediating PCD in at least four situations: (1). withdrawal of neurotrophins; (2). exposure to mismatched neurotrophins; (3). exposure to unprocessed neurotrophins; and (4). exposure of inappropriately immature cells to neurotrophins. Results to date suggest that these functions are mediated through different underlying mechanisms, and that their respective signaling pathways are cell type and co-receptor dependent.

    Cytokine & growth factor reviews 2003;14;3-4;225-39

  • Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis.

    Won J, Kim DY, La M, Kim D, Meadows GG and Joe CO

    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea.

    The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).

    The Journal of biological chemistry 2003;278;21;19347-51

  • Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.

    Chen HK, Fernandez-Funez P, Acevedo SF, Lam YC, Kaytor MD, Fernandez MH, Aitken A, Skoulakis EM, Orr HT, Botas J and Zoghbi HY

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

    Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but the mechanism underlying this accumulation has been unclear. We have discovered that the 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing its normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. Our finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention.

    Funded by: NICHD NIH HHS: HD24064; NINDS NIH HHS: NS22920, NS27699, NS42179

    Cell 2003;113;4;457-68

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.

    Cardoso C, Leventer RJ, Ward HL, Toyo-Oka K, Chung J, Gross A, Martin CL, Allanson J, Pilz DT, Olney AH, Mutchinick OM, Hirotsune S, Wynshaw-Boris A, Dobyns WB and Ledbetter DH

    Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA.

    Deletions of 17p13.3, including the LIS1 gene, result in the brain malformation lissencephaly, which is characterized by reduced gyration and cortical thickening; however, the phenotype can vary from isolated lissencephaly sequence (ILS) to Miller-Dieker syndrome (MDS). At the clinical level, these two phenotypes can be differentiated by the presence of significant dysmorphic facial features and a more severe grade of lissencephaly in MDS. Previous work has suggested that children with MDS have a larger deletion than those with ILS, but the precise boundaries of the MDS critical region and causative genes other than LIS1 have never been fully determined. We have completed a physical and transcriptional map of the 17p13.3 region from LIS1 to the telomere. Using fluorescence in situ hybridization, we have mapped the deletion size in 19 children with ILS, 11 children with MDS, and 4 children with 17p13.3 deletions not involving LIS1. We show that the critical region that differentiates ILS from MDS at the molecular level can be reduced to 400 kb. Using somatic cell hybrids from selected patients, we have identified eight genes that are consistently deleted in patients classified as having MDS. In addition, deletion of the genes CRK and 14-3-3 epsilon delineates patients with the most severe lissencephaly grade. On the basis of recent functional data and the creation of a mouse model suggesting a role for 14-3-3 epsilon in cortical development, we suggest that deletion of one or both of these genes in combination with deletion of LIS1 may contribute to the more severe form of lissencephaly seen only in patients with MDS.

    Funded by: NICHD NIH HHS: R01 HD36715-03; NINDS NIH HHS: P01 NS039404, P01 NS39404

    American journal of human genetics 2003;72;4;918-30

  • Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo.

    Chaudhri M, Scarabel M and Aitken A

    National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

    The 14-3-3 protein family associates with many proteins involved in intracellular signalling. In many cases, there is a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of 14-3-3 dimer formation may therefore influence which of the interacting proteins could be brought together. We have analysed the pattern of dimer formation for two of the most abundant isoforms of 14-3-3, epsilon ( epsilon ) and gamma (gamma), following their stable expression. This revealed a distinct preference for particular dimer combinations that is largely independent of cellular conditions. gamma 14-3-3 occurred as homodimers and also formed heterodimers, mainly with epsilon 14-3-3 (In PC12 and Cos cells). The epsilon isoform formed heterodimers with 14-3-3 beta, gamma, zeta, and eta, but no homodimers were detected. The two 14-3-3 homologues, BMH1 and BMH2 from Saccharomyces cerevisiae, were mainly heterodimers.

    Biochemical and biophysical research communications 2003;300;3;679-85

  • 14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s).

    Liu MY, Cai S, Espejo A, Bedford MT and Walker CL

    Department of Carcinogenesis, Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.

    Tuberin, the product of the tuberous sclerosis complex 2 tumor suppressor gene, is a phosphoprotein that negatively regulates phosphatidylinositol 3'-kinase signaling downstream of Akt. Several high stringency 14-3-3 binding sites that overlapped with Akt phosphorylation sites were identified in tuberin in silico. Recognition of tuberin by an alpha-14-3-3 binding site-specific antibody correlated with mitogen-induced phosphorylation of tuberin and recognition of tuberin by an alpha-Akt phosphorylation substrate antibody. Recognition of tuberin by both antibodies was blocked by inhibiting phosphatidylinositol 3'-kinase activity. Using a protein domain microarray, a tuberin peptide containing Ser(939) demonstrated phospho-specific binding to 14-3-3. Glutathione S-transferase pull-down assays with 14-3-3 fusion proteins revealed that all seven 14-3-3 isoforms (beta, gamma, zeta, epsilon, tau, eta, and sigma) could bind tuberin, and this interaction was abrogated by competition with phosphorylated but not unphosphorylated Ser(939) tuberin peptide. Tuberin also coimmunoprecipitated with 14-3-3, confirming the interaction between endogenous 14-3-3 and tuberin. These data establish the presence of functional and overlapping 14-3-3 and Akt recognition site(s) in tuberin.

    Funded by: NCI NIH HHS: CA63613; NIDDK NIH HHS: DK62268; NIEHS NIH HHS: ES07784, ES08263

    Cancer research 2002;62;22;6475-80

  • Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3.

    Rajan S, Preisig-Müller R, Wischmeyer E, Nehring R, Hanley PJ, Renigunta V, Musset B, Schlichthörl G, Derst C, Karschin A and Daut J

    Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany.

    The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.

    The Journal of physiology 2002;545;1;13-26

  • Identification and characterization of the interaction between tuberin and 14-3-3zeta.

    Nellist M, Goedbloed MA, de Winter C, Verhaaf B, Jankie A, Reuser AJ, van den Ouweland AM, van der Sluijs P and Halley DJ

    Department of Clinical Genetics, Erasmus Medisch Centrum, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. nellist@kgen.fgg.eur.nl

    Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.

    The Journal of biological chemistry 2002;277;42;39417-24

  • Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells.

    Graeser R, Gannon J, Poon RY, Dubois T, Aitken A and Hunt T

    ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK. r.graeser@proqinase.com

    PCTAIRE-1 is a CDK-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Bacterially expressed PCTAIRE is completely inactive as a protein kinase, but is a very good substrate for protein kinase A (PKA), which phosphorylates a total of four sites in the N-terminus of PCTAIRE-1. Phosphorylation of one of these sites, Ser119, generates a 14-3-3 binding site, which is functional in vitro as well as in vivo. Mutation of another PKA site, Ser153, to an alanine residue generated an activated kinase in transfected mammalian cells. This activity was comparable to that of CDK5 activated by a bacterially expressed, truncated version of p35(nck), p21. Gel filtration analysis of a brain extract suggested that monomeric PCTAIRE-1 was the active species, implying that PCTAIRE-1 may not be a true CDK, in that it does not require a partner (cyclin-like) subunit for kinase activity. Finally, we found that various forms of PCTAIRE-1 transfected into neuroblastoma cell lines could either promote or inhibit neurite outgrowth, suggesting a potential role for the PCTAIRE-1 gene product in the control of neurite outgrowth.

    Journal of cell science 2002;115;Pt 17;3479-90

  • Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex.

    Takahashi M, Yamagiwa A, Nishimura T, Mukai H and Ono Y

    Biosignal Research Center, Kobe University, Japan.

    Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.

    Molecular biology of the cell 2002;13;9;3235-45

  • Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization.

    Fujita N, Sato S, Katayama K and Tsuruo T

    Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.

    In many human cancers, the cyclin-dependent kinase inhibitor p27(Kip1) is expressed at low or undetectable levels. The decreased p27(Kip1) expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G(1) arrest, in part by up-regulating p27(Kip1). However, the role of Akt-dependent phosphorylation in p27(Kip1) regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27(Kip1). Screening p27(Kip1) phosphorylation sites identified the COOH-terminal Thr(198) residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27(Kip1) through Thr(198) only when it was phosphorylated by Akt. Although Akt also phosphorylated p27(Kip1) at Ser(10) and Thr(187), these two sites were not involved in the binding to 14-3-3 proteins. p27(Kip1) phosphorylated at Thr(198) exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27(Kip1) and cytoplasmic localization.

    The Journal of biological chemistry 2002;277;32;28706-13

  • RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain.

    Niu J, Scheschonka A, Druey KM, Davis A, Reed E, Kolenko V, Bodnar R, Voyno-Yasenetskaya T, Du X, Kehrl J and Dulin NO

    Department of Pharmacology, University of Illinois at Chicago College of Medicine, Chicago, IL 60612, U.S.A.

    RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the G alpha subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser(264) was then identified as the 14-3-3-binding site of RGS3. The S(264)A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind G alpha(q). Signalling studies showed that the S(264)A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3.

    The Biochemical journal 2002;365;Pt 3;677-84

  • Insulin receptor substrate 4 associates with the protein IRAS.

    Sano H, Liu SC, Lane WS, Piletz JE and Lienhard GE

    Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

    The insulin receptor substrates (IRSs) are key components in signaling from the insulin receptor, and consequently any proteins that interact with them are expected to participate in insulin signaling. In this study we have searched for proteins that interact with IRS-4 by identifying the proteins that coimmunoprecipitated with IRS-4 from human embryonic kidney 293 cells by microsequencing through mass spectrometry. A group of proteins was found. These included phosphatidylinositol 3-kinase, a protein previously identified as an IRS-4 interactor, and several proteins for which there was no previous evidence of IRS-4 association. One of these proteins, named IRAS, that had been found earlier in another context was examined in detail. The results from the overexpression of IRAS, where its amount was about the same as that of IRS-4, indicated that IRAS associated directly with IRS-4 and showed that the increased complexation of IRS-4 with IRAS did not alter the insulin-stimulated tyrosine phosphorylation of IRS-4 or the association of IRS-4 with phosphatidylinositol 3-kinase or Grb2. On the other hand, overexpression of IRAS enhanced IRS-4-dependent insulin stimulation of the extracellularly regulated kinase. The domains of IRAS and IRS-4 responsible for the association of these two proteins were identified, and it was shown that IRAS also associates with IRS-1, IRS-2, and IRS-3.

    Funded by: NIMH NIH HHS: MH49248, R01 MH049248

    The Journal of biological chemistry 2002;277;22;19439-47

  • 14-3-3 amplifies and prolongs adrenergic stimulation of HERG K+ channel activity.

    Kagan A, Melman YF, Krumerman A and McDonald TV

    Section of Molecular Cardiology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

    Acute stress provokes lethal cardiac arrhythmias in the hereditary long QT syndrome. Here we provide a novel molecular mechanism linking beta-adrenergic signaling and altered human ether-a-go-go related gene (HERG) channel activity. Stress stimulates beta-adrenergic receptors, leading to cAMP elevations that can regulate HERG K+ channels both directly and via phosphorylation by cAMP-dependent protein kinase (PKA). We show that HERG associates with 14-3-3epsilon to potentiate cAMP/PKA effects upon HERG. The binding of 14-3-3 occurs simultaneously at the N- and C-termini of the HERG channel. 14-3-3 accelerates and enhances HERG activation, an effect that requires PKA phosphorylation of HERG and dimerization of 14-3-3. The interaction also stabilizes the lifetime of the PKA-phosphorylated state of the channel by shielding the phosphates from cellular phosphatases. The net result is a prolongation of the effect of adrenergic stimulation upon HERG activity. Thus, 14-3-3 interactions with HERG may provide a unique mechanism for plasticity in the control of membrane excitability and cardiac rhythm.

    Funded by: AHRQ HHS: R01 HS57388

    The EMBO journal 2002;21;8;1889-98

  • 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport.

    Brunet A, Kanai F, Stehn J, Xu J, Sarbassova D, Frangioni JV, Dalal SN, DeCaprio JA, Greenberg ME and Yaffe MB

    Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal alpha-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

    Funded by: NICHD NIH HHS: HD18655, HD24926, P30 HD018655; NIGMS NIH HHS: GM60594, R01 GM060594; Wellcome Trust

    The Journal of cell biology 2002;156;5;817-28

  • The RAS effector RIN1 directly competes with RAF and is regulated by 14-3-3 proteins.

    Wang Y, Waldron RT, Dhaka A, Patel A, Riley MM, Rozengurt E and Colicelli J

    Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California 90095, USA.

    Activation of RAS proteins can lead to multiple outcomes by virtue of regulated signal traffic through alternate effector pathways. We demonstrate that the RAS effector protein RIN1 binds to activated RAS with an affinity (K(d), 22 nM) similar to that observed for RAF1. At concentrations close to their equilibrium dissociation constant values, RIN1 and RAF1 compete directly for RAS binding. RIN1 was also observed to inhibit cellular transformation by activated mutant RAS. This distinguishes RIN1 from other RAS effectors, which are transformation enhancing. Blockade of transformation was mediated by the RAS binding domain but required membrane localization. RIN1 recognizes endogenous RAS following transient activation by epidermal growth factor, and a portion of RIN1 fractionates to the cell membrane in a manner consistent with a reversible interaction. RIN1 also binds to 14-3-3 proteins through a sequence including serine 351. Mutation of this residue abolished the 14-3-3 binding capacity of RIN1 and led to more efficient blockade of RAS-mediated transformation. The mutant protein, RIN1(S351A), showed a shift in localization to the plasma membrane. Serine 351 is a substrate for protein kinase D (PKD [also known as PKCmu]) in vitro and in vivo. These data suggest that the normal localization and function of RIN1, as well as its ability to compete with RAF, are regulated in part by 14-3-3 binding, which in turn is controlled by PKD phosphorylation.

    Funded by: NCI NIH HHS: CA 56301, R01 CA056301; NIDDK NIH HHS: DK 55003, KO1 DK 02834, R01 DK055003

    Molecular and cellular biology 2002;22;3;916-26

  • Identification of frequent G(2) checkpoint impairment and a homozygous deletion of 14-3-3epsilon at 17p13.3 in small cell lung cancers.

    Konishi H, Nakagawa T, Harano T, Mizuno K, Saito H, Masuda A, Matsuda H, Osada H and Takahashi T

    Division of Molecular Oncology, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan.

    Accumulating evidence suggests that a coordinately controlled G(2) checkpoint prevents cells with damaged DNA from entering mitosis, thus playing an important role in the maintenance of chromosomal integrity. In the study presented here, we identified a homozygous deletion of the 14-3-3epsilon gene, which resides within a previously identified, commonly deleted region at 17p13.3 in lung cancers, in two small cell lung cancer cell lines that originate from distinct metastatic sites of the same patients. The introduction of 14-3-3epsilon induced significantly restored G(2) checkpoint responses, which resulted in the reduction of mitotic cells as well as of aberrant mitotic figures in the X-ray-irradiated 14-3-3epsilon-null small cell lung cancer cell line. Interestingly, we also found that the G(2) checkpoint response is frequently impaired to various degrees in a large fraction of small cell lung cancer cell lines. These findings suggest the possible involvement of the perturbed G(2) checkpoint in the pathogenesis of this aggressive form of human lung cancers.

    Cancer research 2002;62;1;271-6

  • Identification of a novel interaction of 14-3-3 with p190RhoGEF.

    Zhai J, Lin H, Shamim M, Schlaepfer WW and Cañete-Soler R

    Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104, USA.

    Activation of Rho GTPases by guanine nucleotide exchange factors (GEFs) mediates a broad range of cytoskeletal alterations that determine cell shape. In the nervous system, Rho GTPases are essential for establishing highly asymmetrical neuronal forms and may fine-tune the shape of dendrites in differentiated neurons. p190RhoGEF is a brain-enriched, RhoA-specific GEF whose highly interactive C-terminal domain provides potential linkage to multiple pathways in the cell. In the present study, a yeast two-hybrid screen was used to identify 14-3-3eta and 14-3-3epsilon as additional binding partners of p190RhoGEF. Interactions between p190RhoGEF and 14-3-3eta were confirmed biochemically and by colocalization of the respective proteins when fused to fluorescent markers and transfected in neuronal cells. We also mapped a unique phosphorylation-independent binding site (I(1370)QAIQNL) in p190RhoGEF. Deletion of the binding site abolished interactions in vitro as well as the ability of 14-3-3eta to alter the cytoplasmic aggregation of p190RhoGEF in cotransfected cells. The findings suggest a potential role for 14-3-3 in modulating p190RhoGEF activity or in linking p190RhoGEF to the activities of other pathways in the neuron.

    The Journal of biological chemistry 2001;276;44;41318-24

  • HIV-1 Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe) through a pathway involving regulatory and catalytic subunits of PP2A and acting on both Wee1 and Cdc25.

    Elder RT, Yu M, Chen M, Zhu X, Yanagida M and Zhao Y

    Children's Memorial Institute for Education and Research, Children's Memorial Hospital, Chicago, Illinois 60614, USA.

    Viral protein R (Vpr) of human immunodeficiency virus type 1 induces G2 arrest in cells from distantly related eukaryotes including human and fission yeast through inhibitory phosphorylation of tyrosine 15 (Tyr15) on Cdc2. Since the DNA damage and DNA replication checkpoints also induce G2 arrest through phosphorylation of Tyr15, it seemed possible that Vpr induces G2 arrest through the checkpoint pathways. However, Vpr does not use either the early or the late checkpoint genes that are required for G2 arrest in response to DNA damage or inhibition of DNA synthesis indicating that Vpr induces G2 arrest by an alternative pathway. It was found that protein phosphatase 2A (PP2A) plays an important role in the induction of G2 arrest by Vpr since mutations in genes coding for a regulatory or catalytic subunit of PP2A reduce Vpr-induced G2 arrest. Vpr was also found to upregulate PP2A, supporting a model in which Vpr activates the PP2A holoenzyme to induce G2 arrest. PP2A is known to interact genetically in fission yeast with the Wee1 kinase and Cdc25 phosphatase that act on Tyr15 of Cdc2. Both Wee1 and Cdc25 play a role in Vpr-induced G2 arrest since a wee1 deletion reduces Vpr-induced G2 arrest and a direct in vivo assay shows that Vpr inhibits Cdc25. Additional support for both Wee1 and Cdc25 playing a role in Vpr-induced G2 arrest comes from a genetic screen, which identified genes whose overexpression affects Vpr-induced G2 arrest. For this genetic screen, a strain was constructed in which cell killing by Vpr was nearly eliminated while the effect of Vpr on the cell cycle was clearly indicated by an increase in cell length. Overexpression of the wos2 gene, an inhibitor of Wee1, suppresses Vpr-induced G2 arrest while overexpression of rad25, an inhibitor of Cdc25, enhances Vpr-induced G2 arrest. These two genes may be part of the uncharacterized pathway for Vpr-induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25.

    Funded by: NIAID NIH HHS: 1R29-AI-40891-01

    Virology 2001;287;2;359-70

  • Histone deacetylase 4 possesses intrinsic nuclear import and export signals.

    Wang AH and Yang XJ

    Molecular Oncology Group, Department of Medicine, Royal Victoria Hospital, McGill University Health Center, 687 Pine Avenue, Montréal, Quebec H3A 1A1, Canada.

    Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.

    Molecular and cellular biology 2001;21;17;5992-6005

  • Differential localization of HDAC4 orchestrates muscle differentiation.

    Miska EA, Langley E, Wolf D, Karlsson C, Pines J and Kouzarides T

    Wellcome/CRC Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.

    The class II histone deacetylases HDAC4 and HDAC5 interact specifically with the myogenic MEF2 transcription factor and repress its activity. Here we show that HDAC4 is cytoplasmic during myoblast differentiation, but relocates to the nucleus once fusion has occurred. Inappropriate nuclear entry of HDAC4 following overexpression suppresses the myogenic programme as well as MEF2-dependent transcription. Activation of the Ca(2+)/calmodulin signalling pathway via constitutively active CaMKIV prevents nuclear entry of HDAC4 and HDAC4-mediated inhibition of differentiation. Consistent with a role of phosphorylation in HDAC4 cytoplasmic localisation, HDAC4 binds to 14-3-3 proteins in a phosphorylation-dependent manner. Together these data establish a role for HDAC4 in muscle differentiation. Recently, HDAC5 has also been implicated in muscle differentiation. However, despite the functional similarities of HDAC4 and HDAC5, their intracellular localisations are opposed, suggesting a distinct role for these enzymes during muscle differentiation.

    Nucleic acids research 2001;29;16;3439-47

  • 14-3-3 is involved in p75 neurotrophin receptor-mediated signal transduction.

    Kimura MT, Irie S, Shoji-Hoshino S, Mukai J, Nadano D, Oshimura M and Sato TA

    Molecular Oncology Laboratory, Tsukuba Institute, RIKEN (Institute of Physical and Chemical Research), Ibaraki 305-0074, Japan.

    The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.

    Funded by: NIGMS NIH HHS: R01 GM55147

    The Journal of biological chemistry 2001;276;20;17291-300

  • Conserved role for 14-3-3epsilon downstream of type I TGFbeta receptors.

    McGonigle S, Beall MJ, Feeney EL and Pearce EJ

    Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 14853-6401, Ithaca, NY, USA.

    Schistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor beta (TGFbeta) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S. mansoni 14-3-3epsilon. The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3epsilon also binds to TbetaRI, the human type I TGFbeta receptor. 14-3-3epsilon enhances TGFbeta-mediated signaling by TbetaRI and is the first TbetaRI-interacting non-Smad protein identified that positively regulates this receptor. The interaction of 14-3-3epsilon with schistosome and human TbetaRI suggests a conserved, but previously unappreciated, role for this protein in TGFbeta signaling pathways.

    Funded by: NIAID NIH HHS: AI39085

    FEBS letters 2001;490;1-2;65-9

  • Role of 14-3-3epsilon, c-Myc/Max, and Akt phosphorylation in HIV-1 gp 120-induced mesangial cell proliferation.

    Kapasi AA, Fan S and Singhal PC

    Department of Medicine, Long Island Jewish Medical Center, The Long Island Campus for Albert Einstein College of Medicine, New Hyde Park, New York 11040, USA.

    Focal glomerulosclerosis (FGS) is the predominant glomerular lesion in patients with human immunodeficiency virus (HIV)-associated nephropathy. Initial mesangial cell hyperplasia and subsequent hypoplasia are common features of FGS. In the present study we evaluated the effect of HIV-1 glycoprotein (gp) 120 on human mesangial cell (HMC) growth. HIV-1 gp 120 stimulated HMC proliferation at lower concentrations, whereas it suppressed cell proliferation at higher concentrations. In parallel to the modulation of cell growth, gp 120 at low concentrations resulted in an increase in the expression of c-Myc, Max, and 14-3-3epsilon proteins and phosphorylation of ATP-dependent tyrosine kinases (Akt) at Ser(473). However, the expression of these proteins decreased with increasing concentrations of gp 120. Furthermore, gp 120 also exhibited a dose-dependent inhibition of Akt phosphorylation at Ser-473 without any significant alteration of Akt expression. Little or no effects of gp 120 were observed on the expression of extracellular signal-regulated kinase (ERK), phospho-ERK, Bcl-2, and Bax proteins. At a higher concentration, gp 120 not only promoted HMC apoptosis but also enhanced expression of Fas and FasL. These results suggest that HIV-1 gp 120 induces alterations in conflicting survival signaling pathways that contribute to the potential dual effects of gp 120 in promoting or inhibiting HMC proliferation.

    Funded by: NIDA NIH HHS: R01 DA-12111

    American journal of physiology. Renal physiology 2001;280;2;F333-42

  • Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5.

    McKinsey TA, Zhang CL and Olson EN

    Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.

    Skeletal muscle differentiation is controlled by interactions between myocyte enhancer factor-2 (MEF2) and myogenic basic helix-loop-helix transcription factors. Association of MEF2 with histone deacetylases (HDAC) -4 and -5 results in repression of MEF2 target genes and inhibition of myogenesis. Calcium/calmodulin-dependent protein kinase (CaMK) signaling promotes myogenesis by disrupting MEF2-HDAC complexes and stimulating HDAC nuclear export. To further define the mechanisms that confer CaMK responsiveness to HDAC4 and -5, we performed yeast two-hybrid screens to identify HDAC-interacting factors. These screens revealed interactions between HDAC4 and members of the 14-3-3 family of proteins, which function as signal-dependent intracellular chaperones. HDAC4 binds constitutively to 14-3-3 in yeast and mammalian cells, whereas HDAC5 binding to 14-3-3 is largely dependent on CaMK signaling. CaMK phosphorylates serines -259 and -498 in HDAC5, which subsequently serve as docking sites for 14-3-3. Our studies suggest that 14-3-3 binding to HDAC5 is required for CaMK-dependent disruption of MEF2-HDAC complexes and nuclear export of HDAC5, and implicate 14-3-3 as a signal-dependent regulator of muscle cell differentiation.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;26;14400-5

  • TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.

    Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC and Yaffe MB

    Division of Signal Transduction, Department of Medicine and Department of Surgery, Beth Israel Deaconess Medical Center, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.

    Funded by: NHLBI NIH HHS: HL03601; NIGMS NIH HHS: GM56203, GM59281, R01 GM056203, R01 GM059281

    The EMBO journal 2000;19;24;6778-91

  • The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

    Mayor T, Stierhof YD, Tanaka K, Fry AM and Nigg EA

    Department of Molecular Biology, Sciences II, University of Geneva, CH-1211 Geneva, Switzerland.

    Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

    The Journal of cell biology 2000;151;4;837-46

  • Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo.

    Tzivion G, Luo ZJ and Avruch J

    Diabetes Unit, the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

    14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.

    The Journal of biological chemistry 2000;275;38;29772-8

  • Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization.

    Grozinger CM and Schreiber SL

    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.

    Transcription is controlled in part by the dynamic acetylation and deacetylation of histone proteins. The latter process is mediated by histone deacetylases (HDACs). Previous analysis of the regulation of HDAC activity in transcription has focused primarily on the recruitment of HDAC proteins to specific promoters or chromosomal domains by association with DNA-binding proteins. To characterize the cellular function of the recently identified HDAC4 and HDAC5 proteins, complexes were isolated by immunoprecipitation. Both HDACs were found to interact with14-3-3 proteins at three phosphorylation sites. The association of 14-3-3 with HDAC4 and HDAC5 results in the sequestration of these proteins in the cytoplasm. Loss of this interaction allows HDAC4 and HDAC5 to translocate to the nucleus, interact with HDAC3, and repress gene expression. Regulation of the cellular localization of HDAC4 and HDAC5 by 14-3-3 represents a mechanism for controlling the transcriptional activity of these class II HDAC proteins.

    Funded by: NIGMS NIH HHS: GM38627, R01 GM038627, R37 GM038627

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;14;7835-40

  • Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon.

    Kurz EU, Leader KB, Kroll DJ, Clark M and Gieseler F

    Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Denver, Colorado 80262, USA.

    Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.

    Funded by: NCI NIH HHS: CA-76201

    The Journal of biological chemistry 2000;275;18;13948-54

  • Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase.

    Mils V, Baldin V, Goubin F, Pinta I, Papin C, Waye M, Eychene A and Ducommun B

    LBCMCP - CNRS, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse cedex, France.

    CDC25 dual-specificity phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In human cells, CDC25A and C are involved in the control of G1/S and G2/M respectively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidence for an interaction between CDC25 phosphatases and members of the 14-3-3 protein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that phosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phosphatases and 14-3-3 proteins. We show that in the two-hybrid assay all three splice variants of CDC25B interact similarly and strongly with 14-3-3eta, beta and zeta proteins, but poorly with epsilon and Theta. In vitro, CDC25B interacts at a low level with 14-3-3beta, epsilon, zeta, eta, and Theta isoforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a specific, strong interaction between CDC25B and 14-3-3zeta and eta isoforms is revealed by a deletion of 288 residues in the amino-terminal region of CDC25B. This interaction requires the integrity of Ser 323, although it is independent of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is different from that with CDC25C. We propose that, in addition to a low affinity binding site that is available for all 14-3-3 isoforms, post-translational modification of CDC25B in vivo exposes a high-affinity binding site that is specific for the zeta and eta14-3-3 isoforms.

    Oncogene 2000;19;10;1257-65

  • Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1.

    Aoki H, Hayashi J, Moriyama M, Arakawa Y and Hino O

    Department of Experimental Pathology, Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan.

    Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core-14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.

    Journal of virology 2000;74;4;1736-41

  • The kinesin-like motor protein KIF1C occurs in intact cells as a dimer and associates with proteins of the 14-3-3 family.

    Dorner C, Ullrich A, Häring HU and Lammers R

    Medical Clinic IV, University of Tübingen, Tübingen, 72076 Germany.

    Proteins of the kinesin superfamily are regulated in their motor activity as well as in their ability to bind to their cargo by carboxyl-terminal associating proteins and phosphorylation. KIF1C, a recently identified member of the KIF1/Unc104 family, was shown to be involved in the retrograde vesicle transport from the Golgi-apparatus to the endoplasmic reticulum. In a yeast two-hybrid screen using the carboxyl-terminal 350 amino acids of KIF1C as a bait, we identified as binding proteins 14-3-3 beta, gamma, epsilon, and zeta. In addition, a clone encoding the carboxyl-terminal 290 amino acids of KIF1C was found, indicating a potential for KIF1C to dimerize. Subsequent transient overexpression experiments showed that KIF1C can dimerize efficiently. However, in untransfected cells, only a small portion of KIF1C was detected as a dimer. The association of 14-3-3 proteins with KIF1C could be confirmed in transient expression systems and in untransfected cells and was dependent on the phosphorylation of serine 1092 located in a consensus binding sequence for 14-3-3 ligands. Serine 1092 was a substrate for the protein kinase casein kinase II in vitro, and inhibition of casein kinase II in cells diminished the association of KIF1C with 14-3-3gamma. Our data thus suggest that KIF1C can form dimers and is associated with proteins of the 14-3-3 family.

    The Journal of biological chemistry 1999;274;47;33654-60

  • Phosphorylation-dependent association of the Ras-related GTP-binding protein Rem with 14-3-3 proteins.

    Finlin BS and Andres DA

    Department of Biochemistry, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky, 40536-0084, USA.

    Rem belongs to a subfamily of Ras-related GTPases that includes Rad, Gem, and Kir. These proteins are unique among the Ras superfamily since their expression is under transcriptional regulation and they contain distinct amino and carboxyl termini. To gain insight into the cellular function of Rem, we have undertaken an expression screen using a mouse embryo cDNA library to identify Rem-interacting proteins and find that Rem interacts with a series of 14-3-3 isoforms (epsilon, eta, theta, and zeta). Immunoprecipitation studies demonstrate an interaction that is independent of the nucleotide state of Rem. Rem is phosphorylated in vivo, and binding of Rem to 14-3-3zeta is abolished by pretreating Rem with protein phosphatase 1. Thus, the association of Rem and 14-3-3zeta is phosphorylation-dependent. Examination of the interaction between 14-3-3zeta and various Rem deletion mutants mapped a critical binding site to the C-terminus of Rem. Finally, we demonstrate the interaction of Rad but not the newly identified Rem2 protein with 14-3-3 proteins. These results suggest that 14-3-3 may allow the recruitment of distinct proteins that participate in Rem-mediated signal transduction pathways.

    Funded by: NEI NIH HHS: EY11231

    Archives of biochemistry and biophysics 1999;368;2;401-12

  • Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex.

    Pan S, Sehnke PC, Ferl RJ and Gurley WB

    Program of Plant Molecular and Cellular Biology, Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700, USA.

    The 14-3-3 family of multifunctional proteins is highly conserved among animals, plants, and yeast. Several studies have shown that these proteins are associated with a G-box DNA binding complex and are present in the nucleus in several plant and animal species. In this study, 14-3-3 proteins are shown to bind the TATA box binding protein (TBP), transcription factor IIB (TFIIB), and the human TBP-associated factor hTAF(II)32 in vitro but not hTAF(II)55. The interactions with TBP and TFIIB were highly specific, requiring amino acid residues in the box 1 domain of the 14-3-3 protein. These interactions do not require formation of the 14-3-3 dimer and are not dependent on known 14-3-3 recognition motifs containing phosphoserine. The 14-3-3-TFIIB interaction appears to occur within the same domain of TFIIB that binds the human herpes simplex virus transcriptional activator VP16, because VP16 and 14-3-3 were able to compete for interaction with TFIIB in vitro. In a plant transient expression system, 14-3-3 was able to activate GAL4-dependent beta-glucuronidase reporter gene expression at low levels when translationally fused with the GAL4 DNA binding domain. The in vitro binding with general transcription factors TBP and TFIIB together with its nuclear location provide evidence supporting a role for 14-3-3 proteins as transcriptional activators or coactivators when part of a DNA binding complex.

    The Plant cell 1999;11;8;1591-602

  • alpha-Synuclein shares physical and functional homology with 14-3-3 proteins.

    Ostrerova N, Petrucelli L, Farrer M, Mehta N, Choi P, Hardy J and Wolozin B

    Department of Pharmacology, Loyola University Medical Center, Maywood, Illinois 60153, USA.

    alpha-Synuclein has been implicated in the pathophysiology of many neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease. Mutations in alpha-synuclein cause some cases of familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998). In addition, many neurodegenerative diseases show accumulation of alpha-synuclein in dystrophic neurites and in Lewy bodies (Spillantini et al., 1998). Here, we show that alpha-synuclein shares physical and functional homology with 14-3-3 proteins, which are a family of ubiquitous cytoplasmic chaperones. Regions of alpha-synuclein and 14-3-3 proteins share over 40% homology. In addition, alpha-synuclein binds to 14-3-3 proteins, as well as some proteins known to associate with 14-3-3, including protein kinase C, BAD, and extracellular regulated kinase, but not Raf-1. We also show that overexpression of alpha-synuclein inhibits protein kinase C activity. The association of alpha-synuclein with BAD and inhibition of protein kinase C suggests that increased expression of alpha-synuclein could be harmful. Consistent with this hypothesis, we observed that overexpression of wild-type alpha-synuclein is toxic, and overexpression of alpha-synuclein containing the A53T or A30P mutations exhibits even greater toxicity. The activity and binding profile of alpha-synuclein suggests that it might act as a protein chaperone and that accumulation of alpha-synuclein could contribute to cell death in neurodegenerative diseases.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;14;5782-91

  • Homo- and heterodimerization of synapsins.

    Hosaka M and Südhof TC

    Center for Basic Neuroscience and Department of Molecular Genetics, Howard Hughes Medical Institute, The University of Texas Southwestern Medical School, Dallas, Texas 75235, USA.

    In vertebrates, synapsins constitute a family of synaptic vesicle proteins encoded by three genes. Synapsins contain a central ATP-binding domain, the C-domain, that is highly homologous between synapsins and evolutionarily conserved in invertebrates. The crystal structure of the C-domain from synapsin I revealed that it constitutes a large (>300 amino acids), independently folded domain that forms a tight dimer with or without bound ATP. We now show that the C-domains of all synapsins form homodimers, and that in addition, C-domains from different synapsins associate into heterodimers. This conclusion is based on four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins; 2) quantitative yeast two-hybrid protein-protein binding assays demonstrated pairwise strong interactions between all synapsins; 3) immunoprecipitations from transfected COS cells confirmed that synapsin II heteromultimerizes with synapsins I and III in intact cells, and similar results were obtained with bacterial expression systems; and 4) quantification of the synapsin III level in synapsin I/II double knockout mice showed that the level of synapsin III is decreased by 50%, indicating that heteromultimerization of synapsin III with synapsins I or II occurs in vivo and is required for protein stabilization. These data suggest that synapsins coat the surface of synaptic vesicles as homo- and heterodimers in which the C-domains of the various subunits have distinct regulatory properties and are flanked by variable C-terminal sequences. The data also imply that synapsin III does not compensate for the loss of synapsins I and II in the double knockout mice.

    The Journal of biological chemistry 1999;274;24;16747-53

  • Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site.

    Dalal SN, Schweitzer CM, Gan J and DeCaprio JA

    Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, USA.

    cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (amino acids 201 to 258) in cdc25C that was required for the cytoplasmic localization of cdc25C. This region contained a specific binding site for 14-3-3 proteins, and mutations in cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic localization of cdc25C during interphase. cdc25C proteins that do not contain a binding site for 14-3-3 proteins showed a pancellular localization and an increased ability to induce premature chromosome condensation. The cytoplasmic localization of cdc25C was not altered by gamma irradiation or treatment with the nuclear export inhibitor leptomycin B. These results suggest that 14-3-3 proteins may negatively regulate cdc25C function by sequestering cdc25C in the cytoplasm.

    Funded by: NCI NIH HHS: CA-50661, CA-63113, P01 CA050661, R01 CA063113

    Molecular and cellular biology 1999;19;6;4465-79

  • In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation.

    Luk SC, Ngai SM, Tsui SK, Fung KP, Lee CY and Waye MM

    Department of Biochemistry, Chinese University of Hong Kong, Shatin, N.T.

    Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin. In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent. Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation.

    Journal of cellular biochemistry 1999;73;1;31-5

  • Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins.

    Ku NO, Liao J and Omary MB

    VA Palo Alto Health Care System, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304, USA.

    Members of the 14-3-3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.

    Funded by: NIDDK NIH HHS: DK38707, DK47918, DK52951

    The EMBO journal 1998;17;7;1892-906

  • 14-3-3 proteins interact with specific MEK kinases.

    Fanger GR, Widmann C, Porter AC, Sather S, Johnson GL and Vaillancourt RR

    Program in Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

    MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.

    Funded by: NIDDK NIH HHS: DK37871, DK48845; NIGMS NIH HHS: GM18643-01; ...

    The Journal of biological chemistry 1998;273;6;3476-83

  • The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

    Nagata Ki, Puls A, Futter C, Aspenstrom P, Schaefer E, Nakata T, Hirokawa N and Hall A

    MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.

    The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

    The EMBO journal 1998;17;1;149-58

  • Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.

    Hsu SY, Kaipia A, Zhu L and Hsueh AJ

    Department of Gynecology and Obstetrics, Stanford University Medical School, California 94305-5317, USA.

    Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.

    Funded by: NICHD NIH HHS: HD31566

    Molecular endocrinology (Baltimore, Md.) 1997;11;12;1858-67

  • 14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain.

    Ogihara T, Isobe T, Ichimura T, Taoka M, Funaki M, Sakoda H, Onishi Y, Inukai K, Anai M, Fukushima Y, Kikuchi M, Yazaki Y, Oka Y and Asano T

    Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan.

    Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with 32P-labeled recombinant IRS-1 and obtained two isoforms (epsilon and zeta) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.

    The Journal of biological chemistry 1997;272;40;25267-74

  • Protein binding and signaling properties of RIN1 suggest a unique effector function.

    Han L, Wong D, Dhaka A, Afar D, White M, Xie W, Herschman H, Witte O and Colicelli J

    Department of Biological Chemistry, Molecular Genetics, and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

    Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the "effector domain" of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL-SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.

    Funded by: NCI NIH HHS: CA53867, CA56301, R01 CA056301, R01 CA071443; NIGMS NIH HHS: GM24787

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;10;4954-9

  • 14-3-3 (epsilon) interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner.

    Craparo A, Freund R and Gustafson TA

    Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    The 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways. Here, using two-hybrid assays and in vitro assays of protein interaction, we show that the epsilon isoform of 14-3-3 interacts with the insulin-like growth factor I receptor (IGFIR) and with insulin receptor substrate I (IRS-1), but not with the insulin receptor (IR). Coprecipitation studies demonstrated an IGFI-dependent in vitro interaction between 14-3-3-glutathione S-transferase proteins and the IGFIR. In similar studies no interaction of 14-3-3 with the IR was observed. We present evidence to suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR. Specifically, peptide competition studies combined with mutational analysis suggested that the 14-3-3 interaction was dependent upon phosphorylation of IGFIR serine residues 1272 and/or 1283, a region which has been implicated in IGFIR-dependent transformation. Phosphorylation of these serines appears to be dependent upon prior IGFIR activation since no interaction of 14-3-3 was observed with a kinase-inactive IGFIR in the two-hybrid assay nor was any in vitro interaction with unstimulated IGFIR derived from mammalian cells. We show that the interaction of 14-3-3 with IRS-1 also appears to be phosphoserine-dependent. Interestingly, 14-3-3 appears to interact with IRS-1 before and after hormonal stimulation. In summary, our data suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR and within the central domain of IRS-1. The potential functional roles which 14-3-3 may play in IGFIR and IRS-1-mediated signaling remain to be elucidated.

    Funded by: NIDDK NIH HHS: DK44093, DK50602; NIGMS NIH HHS: GM08181; ...

    The Journal of biological chemistry 1997;272;17;11663-9

  • Assignment of the human 14-3-3 epsilon isoform (YWHAE) to human chromosome 17p13 by in situ hybridization.

    Luk SC, Garcia-Barcelo M, Tsui SK, Fung KP, Lee CY and Waye MM

    Department of Biochemistry, Basic Medical Science Building, The Chinese University of Hong Kong, Shatin NT, Hong Kong.

    Cytogenetics and cell genetics 1997;78;2;105-6

  • 14-3-3 proteins associate with A20 in an isoform-specific manner and function both as chaperone and adapter molecules.

    Vincenz C and Dixit VM

    Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    A20, a novel zinc finger protein, is an inhibitor of tumor necrosis factor-induced apoptosis. The mechanism by which A20 exerts its protective effect is currently unknown. Several isoforms of the 14-3-3 proteins were found to interact with A20 in a yeast two-hybrid screen. A20 bound several 14-3-3 isoforms in vitro. Moreover, transfected A20 was found to preferentially bind the endogenous eta14-3-3 isoform, whereas the beta/zeta isoforms co-immunoprecipitated much less efficiently, and epsilon14-3-3 had an intermediate affinity. Importantly, c-Raf, a previously described 14-3-3-interacting protein, also preferentially bound the eta isoform. The cellular localization and subcellular fractionation of A20 was dramatically altered by co-transfected 14-3-3, providing the first experimental evidence for the notion that 14-3-3 can function as a chaperone. Furthermore, c-Raf and A20 co-immunoprecipitated in a 14-3-3-dependent manner, suggesting that 14-3-3 can function as a bridging or adapter molecule.

    Funded by: NCI NIH HHS: CA61348

    The Journal of biological chemistry 1996;271;33;20029-34

  • 14-3-3 epsilon has no homology to LIS1 and lies telomeric to it on chromosome 17p13.3 outside the Miller-Dieker syndrome chromosome region.

    Chong SS, Tanigami A, Roschke AV and Ledbetter DH

    National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Previously, we isolated several cDNA clones of the LIS1 gene implicated in Miller-Dieker syndrome. Analysis of the 5' end of one of the clones (8-1), which was originally thought to represent the 5' end of LIS1, indicates a striking similarity to mouse 14-3-3 epsilon. We have isolated a full-length cDNA of human 14-3-3 epsilon, for which sequence analysis reveals a strong nucleotide conservation with mouse 14-3-3 epsilon in both translated and untranslated regions (UTRs). Additionally, the predicted peptides of human, sheep, rat, and mouse 14-3-3 epsilon are identical. Using a 205-bp fragment common to LIS1 (8-1) and 14-3-3 epsilon as probe on adult and fetal multiple-tissue Northern blots, a -2-kb transcript is detected, identical to the pattern observed with a full-length 14-3-3 epsilon cDNA probe. LIS1-specific transcripts of approximately 7.5 and approximately 5 kb are not detected by the 0.2-kb probe, indicating that the similarity between the 5' sequence of LIS1 (8-1) and the 3' UTR of 14-3-3 epsilon is not the result of shared homology between the two genes. Instead, clone 8-1 is a chimera of 14-3-3 epsilon and LIS1 partial cDNAs, and therefore its 5' sequence does not represent the LIS1 5' end. Interestingly, we have mapped the 14-3-3 epsilon gene to the same chromosomal sub-band as LIS1 (17p13.3). However, 14-3-3 epsilon lies telomeric to LIS1 and outside the Miller-Dieker syndrome chromosome region but in a region frequently deleted in several types of cancer, and is a reasonable candidate tumor suppressor gene.

    Genome research 1996;6;8;735-41

  • Function of 14-3-3 proteins.

    Jin DY, Lyu MS, Kozak CA and Jeang KT

    Nature 1996;382;6589;308

  • 14-3-3 proteins associate with cdc25 phosphatases.

    Conklin DS, Galaktionov K and Beach D

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, NY 11724, USA.

    The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening. 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery.

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;17;7892-6

  • Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins.

    Jones DH, Ley S and Aitken A

    Division of Protein Structure, National Institute for Medical Research, London, UK.

    14-3-3 proteins play a role in many cellular functions: they bind to and regulate several proteins which are critical for cell proliferation and differentiation. 14-3-3 proteins exist as dimers, and in this study we have shown that diverse 14-3-3 proteins can form both homo- and heterodimers in vitro (by cross-linking studies) and in vivo (by coimmunoprecipitation and Western blot analysis); this interaction is mediated solely through the N-terminal domain of the proteins. The composition of 14-3-3 dimers within a cell may play a key part in the role of this family of proteins as modulators or adapters which facilitate the interaction of distinct components of signalling pathways.

    FEBS letters 1995;368;1;55-8

  • Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    Golsteyn RM, Mundt KE, Fry AM and Nigg EA

    Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

    The Journal of cell biology 1995;129;6;1617-28

  • [Anti-brain antibodies in serum and cerebrospinal fluid following cranio-cerebral trauma].

    Detlav IE

    The author evaluated the precipitating antibrain antibodies by the Hoigne' method in the blood serum of 292 patients and in the CSF of 194 patients with acute brain traumas. Accordingly 52 and 21 patients were studied in the remote period. Antibodies were revealed more frequently and for a longer period of time following severe damages and less frequently in mild forms. They could also be revealed in patients with residual symptoms of brain trauma. Antibodies were revealed in the CSF earlier, more frequently and for a longer period of time than in the blood sera. These data speak in favour of a probable local formation of antibrain antibodies.

    Zhurnal nevropatologii i psikhiatrii imeni S.S. Korsakova (Moscow, Russia : 1952) 1976;76;3;344-8

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000040 G2C Homo sapiens Pocklington H9 Human orthologues of cluster 9 (mouse) from Pocklington et al (2006) 6
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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