G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide
G00000149 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000030833 (Vega human gene)
ENSG00000128245 (Ensembl human gene)
7533 (Entrez Gene)
162 (G2Cdb plasticity & disease)
YWHAH (GeneCards)
113508 (OMIM)
Marker Symbol
HGNC:12853 (HGNC)
Protein Sequence
Q04917 (UniProt)

Literature (87)

Pubmed - other

  • Family-based association of YWHAH in psychotic bipolar disorder.

    Grover D, Verma R, Goes FS, Mahon PL, Gershon ES, McMahon FJ, Potash JB, NIMH Genetics Initiative Bipolar Disorder Collaborative, Bipolar Disorder Phenome Group, Gershon ES, McMahon FJ and Potash JB

    Department of Psychiatry, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.

    YWHAH is a positional and functional candidate gene for both schizophrenia and bipolar disorder (BP). This gene has been previously shown to be associated with both disorders, and the chromosome location (22q12.3) has been repeatedly implicated in linkage studies for these disorders. It codes for the eta subtype of the 14-3-3 protein family, is expressed mainly in brain, and is involved in HPA axis regulation. We investigated the association of YWHAH with BP in a large sample, consisting of 1211 subjects from 318 nuclear families including 554 affected offspring. We tested for association with the standard BP phenotype as well as subtypes defined by psychotic and mood-incongruent features. We genotyped five tag SNPs and the (GCCTGCA)(n) polymorphic locus present in this gene. Using a family-based association test, we found that rs2246704 was associated with BP (OR 1.31, P = 0.03) and psychotic BP (OR = 1.66, P = 0.002). The polymorphic repeat and two other SNPs were also modestly associated with psychotic BP. We have provided additional evidence for association of variants in YWHAH with major mental illness. Additional association analyses of larger sample sets will be required to clarify the role of YWHAH in schizophrenia and BP. The use of clinical sub-phenotypes such as psychotic features or other potential schizophrenia/BP overlap variables including cognitive abnormalities and poor functioning might shed further light on the potential subtypes of illness most closely associated with genetic variation in YWHAH.

    Funded by: Intramural NIH HHS: Z01 MH002810-05; NIMH NIH HHS: 1Z01MH002810-01, R01 MH-042243, R01 MH-061613, R01 MH042243, R01 MH059533, R01 MH059534, R01 MH059535, R01 MH059545, R01 MH059548, R01 MH059553, R01 MH059556, R01 MH059567, R01 MH060068, R01 MH061613, R01 MH59533, R01 MH59535, R01 MH59545, R01 MH59567, R01MH059534, R01MH59553, R01MH60068, U01 MH46274, U01 MH46280, U01 MH46282, Z01 MH002810

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;7;977-83

  • 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

  • Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis.

    Martins-de-Souza D, Gattaz WF, Schmitt A, Rewerts C, Marangoni S, Novello JC, Maccarrone G, Turck CW and Dias-Neto E

    Laboratório de Neurociências, Faculdade de Medicina da USP, Instituto de Psiquiatria, Universidade de São Paulo, Rua Dr. Ovídio Pires de Campos, No 785, s/n Consolação, São Paulo, SP, CEP 05403-010, Brazil. danms90@gmail.com

    Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

    Journal of neural transmission (Vienna, Austria : 1996) 2009;116;3;275-89

  • Evaluating new candidate SNPs as low penetrance risk factors in sporadic breast cancer: a two-stage Spanish case-control study.

    Vega A, Salas A, Milne RL, Carracedo B, Ribas G, Ruibal A, de León AC, González-Hernández A, Benítez J and Carracedo A

    Fundación Pública Galega de Medicina Xenómica SERGAS, CIBERER, Santiago de Compostela, Galicia, Spain.

    Objectives: A polygenic model has been proposed in order to explain the genetic susceptibility to sporadic breast cancer. According to this model, common population variants would be responsible for low to modest effects on the risk of developing the disease. We have carried out a high-throughput SNP genotyping project in order to shed some light on the complex genetic aetiology of non-familial breast cancer.

    Methods: Ninety-one genes have been selected because of their implications in several candidate cell pathways for breast cancer. A total of 640 SNPs in these genes were genotyped in a series of 450 consecutive cases and 448 controls from mainland Spain. Promising SNPs were then studied in an independent series of 294 cases and 299 controls from the Canary Islands.

    Results: In the first case-control series we identified 25 SNPs with P-values below 0.05 (under a 1 df Chi-square test), five of them with P-values below 0.01 (best=0.0008). In the stage 2 Canary Islands series, odd ratios (OR) for two SNPs in HUS1 were in a consistent direction.

    Conclusions: SNPs located at the gene HUS1 are good candidates for further investigation in independent association studies and functional assays.

    Gynecologic oncology 2009;112;1;210-4

  • 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

  • A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.

    Gloeckner CJ, Boldt K, Schumacher A, Roepman R and Ueffing M

    Institute of Human Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany.

    Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.

    Proteomics 2007;7;23;4228-34

  • Detection of high levels of 2 specific isoforms of 14-3-3 proteins in synovial fluid from patients with joint inflammation.

    Kilani RT, Maksymowych WP, Aitken A, Boire G, St-Pierre Y, Li Y and Ghahary A

    Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.

    Objective: To investigate whether 14-3-3 proteins were detectable in synovial fluid (SF) of patients with inflamed joints, and if so, what isoform(s); and to examine whether there was a correlation between the levels of these proteins and those of MMP-1 and MMP-3 in the same samples.

    Methods: In general, 2 sets of synovial and serum samples were analyzed. The first set of 17 SF -samples from patients with inflamed joints were analyzed for 14-3-3 eta isoform by Western blot. The second set of 12 matching serum and SF samples were analyzed for 14-3-3 eta, gamma, MMP-1, and MMP-3 by the same procedure. The MMP-1 stimulatory effect of various concentrations of 14-3-3 eta in cultured fibroblasts was then evaluated.

    Results: We found that of the seven 14-3-3 isoforms tested (beta, gamma, epsilon, eta, sigma, Theta, and zeta), the levels of only 2 isoforms, eta and gamma, were easily detectable in SF samples from patients with inflammatory joint diseases. The levels of these proteins were significantly higher in inflammatory SF and serum samples relative to controls. The values of these proteins correlated strongly with the levels of MMP-1 and MMP-3, 2 biomarkers for rheumatoid arthritis, detected in sera. Further, the level of 14-3-3 eta was significantly higher in a pool of 12 serum samples from patients with inflammatory joint disease than those from healthy individuals.

    Conclusion: Detection of only 2 (14-3-3 eta and gamma) out of 7 different isoforms in SF suggests they are specific to the site of inflammation, and that distinguishes them from barely detectable levels of these isoforms found in normal serum. The MMP-1 stimulatory effect of the eta isoform explains its correlation with MMP-1 levels seen in these samples.

    The Journal of rheumatology 2007;34;8;1650-7

  • Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-3-3 protein binding and inhibits phosphatase-catalyzed inactivation.

    Palmer D, Jimmo SL, Raymond DR, Wilson LS, Carter RL and Maurice DH

    Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario K7L 3N6, Canada.

    Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B.

    The Journal of biological chemistry 2007;282;13;9411-9

  • 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

  • 14-3-3sigma is a p37 AUF1-binding protein that facilitates AUF1 transport and AU-rich mRNA decay.

    He C and Schneider R

    Department of Microbiology, New York University School of Medicine, New York, NY, USA.

    Short-lived cytokine mRNAs contain an AU-rich destabilizing element (ARE). AUF1 proteins bind the ARE, undergo shuttling, and promote cytoplasmic ARE-mRNA decay through a poorly understood mechanism. We therefore identified AUF1-interacting proteins that may play a role in ARE-mRNA decay. We used mass-spectrometry to identify 14-3-3sigma protein as an AUF1-interacting protein. 14-3-3sigma binds selectively and strongly to p37 AUF1 and to a lesser extent to the p40 isoform, the two isoforms most strongly associated with ARE-mRNA decay, but not to the two larger isoforms, p42 and p45. The 14-3-3sigma interaction site on p37 was mapped to a region found only in the two smaller AUF1 isoforms and which overlaps a putative nuclear localization signal (NLS). Stable overexpression of 14-3-3sigma significantly increased cytoplasmic accumulation of p37 AUF1 and reduced the steady-state level and half-life of a reporter ARE-mRNA. siRNA silencing of AUF1 eliminated the effect of 14-3-3sigma overexpression. 14-3-3sigma therefore binds to p37 AUF1, retains it in the cytoplasm probably by masking its NLS, and enhances rapid turnover of ARE-mRNAs.

    Funded by: NIGMS NIH HHS: GM 60428, R01 GM060428

    The EMBO journal 2006;25;16;3823-31

  • 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

  • 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

  • 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

  • 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

  • The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein.

    Namkoong H, Shin SM, Kim HK, Ha SA, Cho GW, Hur SY, Kim TE and Kim JW

    Molecular Genetic Laboratory, College of Medicine, The Catholic University of Korea, Seoul 137-040, Korea. nkhong1@hanmail.net

    Background: Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of our study was to identify an unique gene that shows cancer-associated expression, and characterizes its function related to human carcinogenesis.

    Methods: We used the differential display (DD) RT-PCR method using normal cervical, cervical cancer, metastatic cervical tissues, and cervical cancer cell lines to identify genes overexpressed in cervical cancers and identified gremlin 1 which was overexpressed in cervical cancers. We determined expression levels of gremlin 1 using Northern blot analysis and immunohistochemical study in various types of human normal and cancer tissues. To understand the tumorigenesis pathway of identified gremlin 1 protein, we performed a yeast two-hybrid screen, GST pull down assay, and immunoprecipitation to identify gremlin 1 interacting proteins.

    Results: DDRT-PCR analysis revealed that gremlin 1 was overexpressed in uterine cervical cancer. We also identified a human gremlin 1 that was overexpressed in various human tumors including carcinomas of the lung, ovary, kidney, breast, colon, pancreas, and sarcoma. PIG-2-transfected HEK 293 cells exhibited growth stimulation and increased telomerase activity. Gremlin 1 interacted with homo sapiens tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (14-3-3 eta; YWHAH). YWHAH protein binding site for gremlin 1 was located between residues 61-80 and gremlin 1 binding site for YWHAH was found to be located between residues 1 to 67.

    Conclusion: Gremlin 1 may play an oncogenic role especially in carcinomas of the uterine cervix, lung, ovary, kidney, breast, colon, pancreas, and sarcoma. Over-expressed gremlin 1 functions by interaction with YWHAH. Therefore, Gremlin 1 and its binding protein YWHAH could be good targets for developing diagnostic and therapeutic strategies against human cancers.

    BMC cancer 2006;6;74

  • 14-3-3eta is a novel regulator of parkin ubiquitin ligase.

    Sato S, Chiba T, Sakata E, Kato K, Mizuno Y, Hattori N and Tanaka K

    Department of Neurology, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan.

    Mutation of the parkin gene, which encodes an E3 ubiquitin-protein ligase, is the major cause of autosomal recessive juvenile parkinsonism (ARJP). Although various substrates for parkin have been identified, the mechanisms that regulate the ubiquitin ligase activity of parkin are poorly understood. Here we report that 14-3-3eta, a chaperone-like protein present abundantly in neurons, could bind to parkin and negatively regulate its ubiquitin ligase activity. Furthermore, 14-3-3eta could bind to the linker region of parkin but not parkin with ARJP-causing R42P, K161N, and T240R mutations. Intriguingly, alpha-synuclein (alpha-SN), another familial Parkinson's disease (PD) gene product, abrogated the 14-3-3eta-induced suppression of parkin activity. alpha-SN could bind tightly to 14-3-3eta and consequently sequester it from the parkin-14-3-3eta complex. PD-causing A30P and A53T mutants of alpha-SN could not bind 14-3-3eta, and failed to activate parkin. Our findings indicate that 14-3-3eta is a regulator that functionally links parkin and alpha-SN. The alpha-SN-positive and 14-3-3eta-negative control of parkin activity sheds new light on the pathophysiological roles of parkin.

    The EMBO journal 2006;25;1;211-21

  • [Association study between NPY and YWHAH gene polymorphisms and schizophrenia].

    Wang HS, Duan SW, Xing QH, Du J, Li XW, Xu YF, Zhang ZZ, Wang YJ, Feng GY and He L

    Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 200030, China. hongsheng@sjtu.edu.cn

    A case-control study was carried out on a sample of 583 cases vs. 372 controls in the Chinese Han population, investigating several published polymorphisms in the YWHAH and NPY genes, which reported to be associated with schizophrenia. The polymorphism -134 (GCCTGCA)2-4, in the YWHAH was not analyzed for the failure of amplification, and the polymorphism T1128C in the NPY is not existent in the samples. The analysis was then emphasized on the variants -485C > T(NPY) and G753A(YWHAH). However, no significant differences of allele frequencies (with P values of 0.696 and 0.743, OR values of 1.041 and 0.962 respectively) or genotype frequencies (with P value of 0.45 and 0.75, chi2 = 1.51 and 0.58 respectively) among the matched groups were found. No sex-dependent effect was found either. Also,the analysis of the relative risk between the genotypes of the two genes indicates that the two genes could not cooperate with each other to add the risk of disease (P > 0.05). The results suggest that the polymorphisms - 485C > T (NPY) and G753A (YWHAH) are unlikely to be linked with genetic susceptibility to schizophrenia in the Chinese Han population.

    Yi chuan xue bao = Acta genetica Sinica 2005;32;12;1235-40

  • 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

  • Role of 14-3-3 eta as a positive regulator of the glucocorticoid receptor transcriptional activation.

    Kim YS, Jang SW, Sung HJ, Lee HJ, Kim IS, Na DS and Ko J

    School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul 136-701, South Korea.

    The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily, mediates the effects of glucocorticoids. It is known that 14-3-3 family proteins interact with GR and regulate its transcriptional activity. They also bind to several molecules and influence many cellular events by altering their subcellular localization and/or acting as a chaperone. Recently, it has been proposed that ligand-activated degradation of GR occurs via the ubiquitin-proteasomal degradation pathway and that inhibition of proteasomal activity induces up-regulation of GR and enhances the transcriptional activity of GR. To examine the function of 14-3-3eta in the glucocorticoid-dependent signal pathway, we studied the regulatory role of 14-3-3eta in ligand-induced GR transcriptional activation. 14-3-3eta Enhanced the transcriptional activity of GR, and the levels of GR were higher in cells transfected with the 14-3-3eta expression vector in response to glucocorticoid. The GR level increased in both cytosol and nucleus, and endogenous GR was also elevated by 14-3-3eta in HeLa cells. 14-3-3eta Inhibited ligand-induced down-regulation of GR. Proteasomal inhibition did not induce any synergistic effect on the 14-3-3eta-induced increase in GR in response to glucocorticoid, and inhibition of translation did not block elevation of GR by 14-3-3eta, indicating that 14-3-3eta induces stabilization of GR. These results suggest that 14-3-3eta functions as a positive regulator in the glucocorticoid signal pathway by blocking the degradation of GR and inducing an elevation of GR, thus enhancing the transcriptional activity of GR.

    Endocrinology 2005;146;7;3133-40

  • 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

  • Akt and 14-3-3eta regulate Miz1 to control cell-cycle arrest after DNA damage.

    Wanzel M, Kleine-Kohlbrecher D, Herold S, Hock A, Berns K, Park J, Hemmings B and Eilers M

    Institute for Molecular Biology and Tumor Research, University of Marburg, Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany.

    The transcription factor Miz1 is required for DNA-damage-induced cell-cycle arrest. We have now identified 14-3-3eta as a gene that inhibits Miz1 function through interaction with its DNA binding domain. Binding of 14-3-3eta to Miz1 depends on phosphorylation by Akt and regulates the recovery of cells from arrest after DNA damage. Miz1 has two functions in response to DNA damage: first, it is required for upregulation of a large group of genes, a function that is regulated by c-Myc, but not by 14-3-3eta; second, Miz1 represses the expression of many genes in response to DNA damage in an Akt- and 14-3-3eta-regulated manner.

    Nature cell biology 2005;7;1;30-41

  • 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

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • 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

  • Disruption of 14-3-3 binding does not impair Protein 4.1B growth suppression.

    Robb VA, Li W and Gutmann DH

    Department of Neurology, Washington University School of Medicine, Box 8111, 660 S. Euclid Avenue, St Louis, MO 63110, USA.

    Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely unknown. Collaborative studies from our laboratory demonstrated a direct association of 14-3-3 with the meningioma tumor suppressor Protein 4.1B, which was not observed with other members of the Protein 4.1 family, including the NF2 meningioma tumor suppressor, merlin/schwannomin. Given the role of 14-3-3 in the regulation of cell proliferation and apoptosis, we sought to determine the functional significance of 14-3-3 binding to Protein 4.1B growth suppression. Based on comparative binding studies performed with additional members of the Protein 4.1 family, we generated specific missense mutations within the minimal growth suppressor fragment of Protein 4.1B (DAL-1, differentially expressed in adenocarcinoma of the lung). Complementary in vitro GST affinity chromatography and in vivo interaction experiments demonstrated that the F359Y mutation abrogated binding to 14-3-3, but did not impair DAL-1 binding to other known Protein 4.1B interacting proteins. Similar to wild-type DAL-1, the expression of the F359Y DAL-1 14-3-3-binding mutant resulted in reduced Protein 4.1B-deficient IOMM-Lee and CH157-MN meningioma cell line colony formation. Moreover, similar to wild-type DAL-1, the stable expression of the DAL-1 F359Y mutant significantly reduced cell proliferation in independently isolated IOMM-Lee clones, as assessed by thymidine incorporation. Collectively, these results suggest that binding to 14-3-3 is not essential for the growth suppressor function of Protein 4.1B in meningiomas.

    Funded by: NCI NIH HHS: 1-F32-CA-097816-01; NINDS NIH HHS: NS35848, NS41520

    Oncogene 2004;23;20;3589-96

  • 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

  • Comprehensive proteomic analysis of human Par protein complexes reveals an interconnected protein network.

    Brajenovic M, Joberty G, Küster B, Bouwmeester T and Drewes G

    Cellzome AG, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

    The polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3zeta and eta (Par-5), Par-6a, -b, -c, and PKClambda (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex "modules" formed around MARK4 (Par-1), Par-3.Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1.Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity.

    The Journal of biological chemistry 2004;279;13;12804-11

  • 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

  • A genome annotation-driven approach to cloning the human ORFeome.

    Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y, Huckle EJ, Beare DM and Dunham I

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.

    Genome biology 2004;5;10;R84

  • Identification of proteins that interact with the central coiled-coil region of the human protein kinase NEK1.

    Surpili MJ, Delben TM and Kobarg J

    Centro de Biologia Molecular Estrutural (CEBIME), Laboratório Nacional de Luz Síncrotron (LNLS), Rua Giuseppe Máximo Scolfaro 10.000, CP 6192, 13084-971 Campinas, SP, Brazil.

    NEK protein kinases are evolutionarily conserved kinases structurally related to the Aspergillus nidulans mitotic regulator NIMA. At least nine members of the NEK family in vertebrates have been described to date, but for most of them the interacting protein partners are unknown. The pleiotropic deleterious effects and the formation of kidney cysts caused by NEK1 mutation in mice emphasize its involvement in the regulation of diverse cellular processes and in the etiology of polycystic kidney disease (PKD), respectively. Here we report the identification of proteins that interacted with the human NEK1 protein kinase in a yeast two-hybrid screen of a human fetal brain cDNA library, using the catalytic and regulatory domains of NEK1 separately as baits. These proteins are known to take part either in the development of PKD, in the double-strand DNA break repair at the G2/M transition phase of the cell cycle, or in neural cell development. The proteins involved in PKD include the motor protein KIF3A and the proteins tuberin and alpha-catulin. Mapping studies of the human NEK1 regulatory domain (NRD) indicated a strong interaction of most of the proteins retrieved from the library with putative coiled coils located in the central region of NRD. Our results give further support to the previous observation that NEK1 is of functional importance for the etiology of PKD.

    Biochemistry 2003;42;51;15369-76

  • Rim, a component of the presynaptic active zone and modulator of exocytosis, binds 14-3-3 through its N terminus.

    Sun L, Bittner MA and Holz RW

    Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.

    Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.

    Funded by: NIDDK NIH HHS: R01-DK50177

    The Journal of biological chemistry 2003;278;40;38301-9

  • 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

  • Protein 14-3-3sigma interacts with and favors cytoplasmic subcellular localization of the glucocorticoid receptor, acting as a negative regulator of the glucocorticoid signaling pathway.

    Kino T, Souvatzoglou E, De Martino MU, Tsopanomihalu M, Wan Y and Chrousos GP

    Pediatric and Reproductive Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA. kinot@mail.nih.gov

    The glucocorticoid receptor (GR) alpha interacts with the highly conserved 14-3-3 family proteins. The latter bind phosphorylated serine/threonine residues of "partner" molecules and influence many signal transduction events by altering their subcellular localization and/or protecting them from proteolysis. To examine the physiologic role of 14-3-3 on the glucocorticoid-signaling pathway, we studied the nucleocytoplasmic shuttling and transactivation properties of GRalpha in a cell line replete with or devoid of 14-3-3sigma. We found that endogenous 14-3-3sigma helped localize green fluorescent protein-fused GRalpha in the cytoplasm in the absence of ligand and potentiated its nuclear export after ligand withdrawal. 14-3-3sigma also suppressed the transcriptional activity of GRalpha on a glucocorticoid-responsive promoter. Disruption of the classic nuclear export signal of 14-3-3sigma inactivated its ability to influence the nucleocytoplasmic trafficking and transactivation activity of GRalpha, whereas introduction of a mutation inactivating the binding activity of 14-3-3sigma to some of its partner proteins did not. 14-3-3sigma bound the ligand-binding domain of GRalpha through its COOH-terminal portion, in a partially ligand-dependent fashion, while it did not interact with "ligand-binding domain" of GRbeta at all. These results suggest that 14-3-3sigma functions as a negative regulator in the glucocorticoid signaling pathway, possibly by shifting the subcellular localization/circulation of this receptor toward the cytoplasm through its nuclear export signal. Since 14-3-3 proteins play significant roles in numerous cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these actions might indirectly influence the transcriptional activity of GRalpha. Conversely, through its 14-3-3 protein interactions, GRalpha may influence these processes.

    The Journal of biological chemistry 2003;278;28;25651-6

  • 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

  • Interaction of the parathyroid hormone receptor with the 14-3-3 protein.

    Tazawa H, Takahashi S and Zilliacus J

    Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden.

    The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development. 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization. We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell. 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used. Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding. The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR. The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling.

    Biochimica et biophysica acta 2003;1620;1-3;32-8

  • 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 protein is a component of Lewy bodies in Parkinson's disease-mutation analysis and association studies of 14-3-3 eta.

    Ubl A, Berg D, Holzmann C, Krüger R, Berger K, Arzberger T, Bornemann A and Riess O

    Department of Medical Genetics, Children's Hospital, University Rostock, Rembrandt Str. 16/17, Germany.

    Mutations in alpha-synuclein have been identified in some rare families with autosomal dominant Parkinson's disease (PD). The synuclein gene family shares physical and functional homology with 14-3-3 proteins and binds to 14-3-3 proteins and to its ligands. We therefore investigated whether 14-3-3 proteins are also involved in the pathogenesis of PD. Here we demonstrate that 14-3-3 proteins are colocalized with Lewy bodies in PD. We investigated the 14-3-3 eta (YWHAH) gene by mutation analysis and association studies as it maps to human chromosome 22q12.1-q13.1, a region which has been recently implicated in PD and carried out immunohistochemical studies of Lewy bodies with two different 14-3-3 eta antibodies. In 358 sporadic and familial PD patients, disease causing mutations were not identified. Furthermore, association studies with intragenic polymorphisms do not provide evidence for an involvement of 14-3-3 eta in the pathogenesis of PD. In accordance with these findings, there was no staining of substantia nigra Lewy bodies with antibodies specific for the 14-3-3 eta subunit.

    Brain research. Molecular brain research 2002;108;1-2;33-9

  • 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

  • MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

    Lim R, Winteringham LN, Williams JH, McCulloch RK, Ingley E, Tiao JY, Lalonde JP, Tsai S, Tilbrook PA, Sun Y, Wu X, Morris SW and Klinken SP

    Laboratory for Cancer Medicine, Medical Research Foundation, Royal Perth Hospital, Western Australian Institute for Medical Research, Rear 50 Murray Street, Perth, WA 6000, Australia.

    A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

    Funded by: NCI NIH HHS: CA 21765, CA 76301

    The Journal of biological chemistry 2002;277;43;40997-1008

  • 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 kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3.

    Sato S, Fujita N and Tsuruo T

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

    3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitro through the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3 in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241.

    The Journal of biological chemistry 2002;277;42;39360-7

  • 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

  • The 4.1/ezrin/radixin/moesin domain of the DAL-1/Protein 4.1B tumour suppressor interacts with 14-3-3 proteins.

    Yu T, Robb VA, Singh V, Gutmann DH and Newsham IF

    David and Doreen Hermelin Laboratory of Molecular Oncogenetics, Department of Neurosurgery and Hermelin Brain Tumor Center, Henry Ford Hospital, E&R Bldg. Rm. 3096, 2799 W. Grand Blvd. Detroit, MI 48202, U.S.A.

    The Protein 4.1 family contains at least two members that function as tumour suppressors, the neurofibromatosis 2 gene product merlin and the recently identified differentially expressed in adenocarcinoma of the lung (DAL-1)/Protein 4.1B molecule. DAL-1/Protein 4.1B loss is observed in a variety of tumours, including breast and lung cancers as well as meningiomas. We have previously demonstrated that DAL-1/Protein 4.1B interacts with some but not all merlin-binding proteins, raising the possibility that DAL-1/Protein 4.1B associates with additional unique proteins specific to its function as a negative growth regulator. Using yeast two-hybrid interaction cloning, we identified three 14-3-3 isoforms, beta, gamma and eta, to be DAL-1/Protein 4.1B-binding proteins. These interactions were verified by using glutathione S-transferase affinity chromatography in vitro and co-immunoprecipitation in vivo. The interaction of 14-3-3 with DAL-1/Protein 4.1B was specific, as 14-3-3 did not bind to the related Protein 4.1 family members merlin, ezrin or radixin. The DAL-1/Protein 4.1B domain that mediates 14-3-3 binding was mapped to residues Pro(244) and Leu(280) within the 4.1/ezrin/radixin/moesin domain. The identification of this novel DAL-1/Protein 4.1B-interacting protein represents the first step towards elucidating its potentially unique mechanism of action.

    Funded by: NCI NIH HHS: CA77730; NINDS NIH HHS: NS/CA41520

    The Biochemical journal 2002;365;Pt 3;783-9

  • Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms.

    Johnson BA, Stehn JR, Yaffe MB and Blackwell TK

    Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The immediate early gene tristetraprolin (TTP) is induced transiently in many cell types by numerous extracellular stimuli. TTP encodes a zinc finger protein that can bind and destabilize mRNAs that encode tumor necrosis factor-alpha (TNFalpha) and other cytokines. We hypothesize that TTP also has a broader role in growth factor-responsive pathways. In support of this model, we have previously determined that TTP induces apoptosis through the mitochondrial pathway, analogously to certain oncogenes and other immediate-early genes, and that TTP sensitizes cells to the pro-apoptotic signals of TNFalpha. In this study, we show that TTP and the related proteins TIS11b and TIS11d bind specifically to 14-3-3 proteins and that individual 14-3-3 isoforms preferentially bind to different phosphorylated TTP species. 14-3-3 binding does not appear to inhibit or promote induction of apoptosis by TTP but is one of multiple mechanisms that localize TTP to the cytoplasm. Our results provide the first example of 14-3-3 interacting functionally with an RNA binding protein and binding in vivo to a Type II 14-3-3 binding site. They also suggest that 14-3-3 binding is part of a complex network of stimuli and interactions that regulate TTP function.

    Funded by: NCI NIH HHS: CA84418; NIGMS NIH HHS: GM60594

    The Journal of biological chemistry 2002;277;20;18029-36

  • Phosphorylation-dependent interaction of kinesin light chain 2 and the 14-3-3 protein.

    Ichimura T, Wakamiya-Tsuruta A, Itagaki C, Taoka M, Hayano T, Natsume T and Isobe T

    Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan. ichimura@mail.comp.metro-u.ac.jp

    The protein 14-3-3 is a key regulator in a cell signaling pathway mediated by protein phosphorylation. To identify the cellular targets of this protein systematically, we have employed a proteomic approach: protein components pulled down from PC12 cells stably expressing a myc-tagged 14-3-3eta isoform were analyzed by means of SDS-PAGE and mass spectrometry. This procedure allowed us to identify more than 30 proteins that include various known and unknown targets of the 14-3-3 protein. Among them are several proteins in the membrane traffic pathway, such as the heavy and light chains (KHC/KIF5B and KLC2) of conventional kinesin, a heterotetrameric mechanochemical motor involved in the ATP-dependent movement of vesicles and organelles along microtubules. Subsequent analysis showed that 14-3-3 directly binds to kinesin heterodimers through interaction with KLC2 and that this interaction is dependent on the phosphorylation of KLC2. Studies on the interaction between 14-3-3 and KLC2 variants expressed in cultured cells coupled with mass spectrometric analysis proved that Ser575 is the site of phosphorylation in KLC2 that is responsible for the in vivo interaction with the 14-3-3 protein. These data add KLC2 to the growing list of 14-3-3 targets, and suggest a role of 14-3-3 in the phosphorylation-regulated cellular transport of vesicles and organelles.

    Biochemistry 2002;41;17;5566-72

  • 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

  • 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

  • The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization.

    Jeanclos EM, Lin L, Treuil MW, Rao J, DeCoster MA and Anand R

    Neuroscience Center of Excellence and Department of Neurology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.

    By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.

    Funded by: NINDS NIH HHS: NS33625

    The Journal of biological chemistry 2001;276;30;28281-90

  • Regulation of glucocorticoid receptor activity by 14--3-3-dependent intracellular relocalization of the corepressor RIP140.

    Zilliacus J, Holter E, Wakui H, Tazawa H, Treuter E and Gustafsson JA

    Department of Medical Nutrition, Karolinska Institutet Novum, S-141 86 Huddinge, Sweden. johanna.zilliacus@mednut.ki.se

    Proteins belonging to the 14--3-3 family interact with various regulatory proteins involved in cellular signaling, cell cycle regulation, or apoptosis. 14--3-3 proteins have been suggested to act by regulating the cytoplasmic/nuclear localization of their target proteins or by acting as molecular scaffolds or chaperones. We have previously shown that overexpression of 14--3-3 enhances the transcriptional activity of the glucocorticoid receptor (GR), which is a member of the nuclear receptor family. In this study, we show that 14--3-3 interacts with the nuclear receptor corepressor RIP140. In transfection assays, RIP140 antagonizes 14--3-3- enhanced GR transactivation. Using colocalization studies we demonstrate that 14--3-3 can export RIP140 out of the nucleus and, interestingly, can also change its intranuclear localization. Moreover, we also observed that 14--3-3 can bind various other nuclear receptors and cofactors. In summary, our findings suggest that 14--3-3-mediated intracellular relocalization of the GR corepressor RIP140 might be a novel mechanism to enhance glucocorticoid responsiveness of target genes. They furthermore indicate a more general role for 14--3-3 protein by influencing the nuclear availability of nuclear receptor-associated cofactors.

    Molecular endocrinology (Baltimore, Md.) 2001;15;4;501-11

  • Systematic screening of the 14-3-3 eta (eta) chain gene for polymorphic variants and case-control analysis in schizophrenia.

    Bell R, Munro J, Russ C, Powell JF, Bruinvels A, Kerwin RW and Collier DA

    Section of Genetics, Department of Psychological Medicine, The Institute of Psychiatry, London, United Kingdom.

    The neuronal protein 14-3-3 eta is a candidate gene for schizophrenia because it maps chromosome 22q12, a region implicated in the disease by linkage analysis, and is involved in brain development. We systematically screened this gene for polymorphic variants by comparison of public EST sequence data (five cDNAs and 72 ESTs, 21,155 bp of sequence) in parallel with single-stranded conformational polymorphism analysis, and we compared these methods by using a simple power calculation. Twelve potential polymorphisms were identified from EST sequence comparison, and two of these (a 5'-VNTR and 753G/A) were confirmed by SSCP analysis and sequencing. Three additional infrequent polymorphisms (-408T/G; 177 C/G; and 989 A/G) were found by SSCP only. We next examined these variants for association with schizophrenia. One variant in untranslated region of exon 1 (-408 T/G) was found to occur more frequently in the schizophrenic subjects (8%) than the controls (3%; P = 0.01). After fivefold correction of the P value for multiple testing, marginal association was found. Haplotype analysis of pairs of polymorphisms provided no evidence for association of this gene with schizophrenia in the population studied. Am. J. Med. Genet. (Neuropsychiatr. Genet. ) 96:736-743, 2000.

    American journal of medical genetics 2000;96;6;736-43

  • 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

  • 14-3-3 isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation.

    Van Der Hoeven PC, Van Der Wal JC, Ruurs P, Van Dijk MC and Van Blitterswijk J

    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

    14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -θ were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.

    The Biochemical journal 2000;345 Pt 2;297-306

  • The DNA sequence of human chromosome 22.

    Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Bruskiewich R, Beare DM, Clamp M, Smink LJ, Ainscough R, Almeida JP, Babbage A, Bagguley C, Bailey J, Barlow K, Bates KN, Beasley O, Bird CP, Blakey S, Bridgeman AM, Buck D, Burgess J, Burrill WD, O'Brien KP et al.

    Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. id1@sanger.ac.uk

    Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

    Nature 1999;402;6761;489-95

  • 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

  • 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

  • 14-3-3 protein eta chain gene (YWHAH) polymorphism and its genetic association with schizophrenia.

    Toyooka K, Muratake T, Tanaka T, Igarashi S, Watanabe H, Takeuchi H, Hayashi S, Maeda M, Takahashi M, Tsuji S, Kumanishi T and Takahashi Y

    National Saigata Hospital, Ogata, Japan.

    Recent genetic analyses have suggested a linkage between schizophrenia and the chromosomal region 22q12-q13. 14-3-3 protein, abundant in the brain, mediates interactions between diverse molecules of biological activities; its gene was recently mapped to chromosome 22q12.1-q13.1. We therefore investigated allele frequencies of a variable number of tandem repeat (VNTR) in the 5'-noncoding region of the 14-3-3 eta chain gene in controls and schizophrenics. The frequencies of the two-repeat allele were significantly higher (P < 0.05) in the schizophrenics, and particularly in those with onset before age 22 (early-onset schizophrenics, P < 0.02), than in the controls. The odds ratio was significantly increased in the early-onset schizophrenics homozygous for the two-repeat allele (OR = 3.3, 95% CI = 1.1-9.7). The 14-3-3 eta chain gene is a potential susceptibility gene for schizophrenia, and particularly for early-onset schizophrenia.

    American journal of medical genetics 1999;88;2;164-7

  • Characterization of brain PCTAIRE-1 kinase immunoreactivity and its interactions with p11 and 14-3-3 proteins.

    Le Bouffant F, Capdevielle J, Guillemot JC and Sladeczek F

    CNRS UPR 2212, Institut Alfred Fessard, Gif-sur-Yvette, France.

    An antibody directed against the C-terminal part of PCTAIRE-1 recognized three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant PCTAIRE-1 and interacted, like recombinant PCTAIRE-1, with p11 and 14-3-3 proteins. Combination of p11 or 14-3-3 affinity resins with immunoprecipitation and peptide elution allowed us to obtain a purified full-length PCTAIRE-1 preparation having significant kinase activity. These results suggest that PCTAIRE-1 is an active kinase in brain. The catalytic core region of PCTAIRE-1 which is common for all cyclin-dependent kinases, does not interact with p11 and 14-3-3 proteins in the two-hybrid assay. Full interaction with p11 and 14-3-3 proteins requires both, the N-terminal and C-terminal ends of PCTAIRE-1, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of PCTAIRE-1, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24-1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. Our results allow us to attribute these widely distributed cDNA sequences to an existing 26-kDa protein and to localize a gene within two recently published genomic sequences.

    European journal of biochemistry 1998;257;1;112-20

  • Association of the TLX-2 homeodomain and 14-3-3eta signaling proteins.

    Tang SJ, Suen TC, McInnes RR and Buchwald M

    Department of Genetics, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

    Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitro and in vivo, 14-3-3eta is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3eta shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus. Tlx-2 and 14-3-3eta are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3eta as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that the in vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3eta.

    The Journal of biological chemistry 1998;273;39;25356-63

  • A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

    Megidish T, Cooper J, Zhang L, Fu H and Hakomori S

    Pacific Northwest Research Institute, Seattle, Washington 98122 and Department of Pathobiology, University of Washington, Seattle, Washington 98195, USA.

    Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent phosphorylation of specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or induce conformational changes that alter 14-3-3 association with other kinases involved in signal transduction.

    Funded by: NCI NIH HHS: CA42505, CA54786; NIGMS NIH HHS: GM53165

    The Journal of biological chemistry 1998;273;34;21834-45

  • 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

  • The structural basis for 14-3-3:phosphopeptide binding specificity.

    Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, Gamblin SJ, Smerdon SJ and Cantley LC

    Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.

    The 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Using phosphoserine-oriented peptide libraries to probe all mammalian and yeast 14-3-3s, we identified two different binding motifs, RSXpSXP and RXY/FXpSXP, present in nearly all known 14-3-3 binding proteins. The crystal structure of 14-3-3zeta complexed with the phosphoserine motif in polyoma middle-T was determined to 2.6 A resolution. The bound peptide is in an extended conformation, with a tight turn created by the pS +2 Pro in a cis conformation. Sites of peptide-protein interaction in the complex rationalize the peptide library results. Finally, we show that the 14-3-3 dimer binds tightly to single molecules containing tandem repeats of phosphoserine motifs, implicating bidentate association as a signaling mechanism with molecules such as Raf, BAD, and Cbl.

    Funded by: NIGMS NIH HHS: GM56203, R01 GM056203

    Cell 1997;91;7;961-71

  • 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

  • A20 inhibits NF-kappaB activation independently of binding to 14-3-3 proteins.

    De Valck D, Heyninck K, Van Criekinge W, Vandenabeele P, Fiers W and Beyaert R

    Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, University of Ghent, K. L. Ledeganckstraat 35, Ghent, B-9000, Belgium.

    The A20 protein, which belongs to a class of Cys2/Cys2 zinc finger proteins, has been characterized as an inhibitor of NF-kappaB activation. In order to clarify its molecular mechanism of action, the yeast two-hybrid system was used to screen for interacting proteins. We report that different isoforms of 14-3-3 proteins, viz. eta and zeta, are able to bind A20, involving the 14-3-3-binding motif RSKSDP located between zinc fingers 3 and 4. However, A20 mutants that no longer associated with 14-3-3 proteins could still fully inhibit NF-kappaB activation induced by tumor necrosis factor, interleukin-1beta or phorbol 12-myristate 13-acetate, thus excluding a crucial role for 14-3-3 interaction in this A20 function.

    Biochemical and biophysical research communications 1997;238;2;590-4

  • Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.

    Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS and Piwnica-Worms H

    Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

    Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.

    Funded by: NIAID NIH HHS: AI34094; NIGMS NIH HHS: GM18428, GM47017

    Science (New York, N.Y.) 1997;277;5331;1501-5

  • The Cdk-like protein PCTAIRE-1 from mouse brain associates with p11 and 14-3-3 proteins.

    Sladeczek F, Camonis JH, Burnol AF and Le Bouffant F

    CNRS UPR 2212, Institut Alfred Fessard, Gif-sur-Yvette.

    PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk)-like class of proteins, and is localized mainly in the mammalian brain. Using the yeast two-hybrid system we screened a mouse brain cDNA library with PCTAIRE-1 as bait, and isolated several clones coding for the mouse homologs of the following proteins: p11 (also known as calpactin I light chain) and the eta, theta (also known as tau) and zeta isoforms of 14-3-3 proteins. We confirmed that these four proteins interact with PCTAIRE-1 by demonstrating the biochemical interactions using the pure recombinant proteins. The fact that 14-3-3 proteins are known to interact with many other intracellular proteins (such as C-kinase, Raf, Bcr, P13-kinase) and p11 with annexin II (a major pp60(v-src) and C-kinase substrate) suggests that PCTAIRE-1 might be part of multiple signal transduction cascades and cellular protein networks.

    Molecular & general genetics : MGG 1997;254;5;571-7

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823

    Genome research 1997;7;4;353-8

  • Interaction of the ligand-activated glucocorticoid receptor with the 14-3-3 eta protein.

    Wakui H, Wright AP, Gustafsson J and Zilliacus J

    Department of Biosciences, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden.

    The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3eta protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.

    The Journal of biological chemistry 1997;272;13;8153-6

  • 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

  • Structural organization and chromosomal assignment of the human 14-3-3 eta chain gene (YWHAH).

    Muratake T, Hayashi S, Ichikawa T, Kumanishi T, Ichimura Y, Kuwano R, Isobe T, Wang Y, Minoshima S, Shimizu N and Takahashi Y

    National Saigata Hospital, Niigata Prefecture, Ogata, Nakakubiki, 949-31, Japan.

    14-3-3 protein, a brain-specific protein, is thought to be a multifunctional protein involved in the activation of tyrosine and tryptophan hydroxylases, the inhibition or activation of protein kinase C, and the activation of signal transduction. The human 14-3-3 eta chain gene was isolated and its structure was determined. It is composed of two exons separated by one long intron (approximately 8 kb) and spans about 10 kb. A transcription initiation site was identified by a combination of S1 nuclease mapping, primer extension analysis, and RACE methods. In the 5'-flanking region, we found four GC box sequences, four anti-GC box sequences, a TATA box-like sequence, CAAT box-like sequences, a C/EBP element, two AP-2 sequences, an AP-3 sequence, an Oct-6-like sequence, six E boxes, and a CRE sequence. FISH with DNA probes of the human 14-3-3 eta chain gene mapped the 14-3-3 eta chain gene to chromosome 22q12.1-q13.1.

    Genomics 1996;36;1;63-9

  • Identification of signalling proteins interacting with B-Raf in the yeast two-hybrid system.

    Papin C, Denouel A, Calothy G and Eychène A

    Unité Mixte de Recherche 146 du CNRS, Institut Curie, Orsay, France.

    Recent studies suggested the existence of Ras/B-Raf/ MEK-1 complexes and a critical role for B-Raf in regulating the MAP kinase/ERKs signalling pathway. We report, here, that both Ras and MEK-1 proteins interact physically with B-Raf proteins in the yeast two-hybrid system. In addition, by screening a mouse brain cDNA library, we isolated additional B-Raf interacting proteins. These include three members of the 14-3-3 proteins family (eta, theta and zeta) and the MEK-2 protein. We also show that c-Raf-1, previously reported to interact with beta and zeta 14-3-3 proteins, also interacts with eta and theta 14-3-3 proteins in the two-hybrid system. By using different portions of the B-Raf protein, we mapped the regions of the protein involved in these interactions. Specifically, we have characterized B-Raf specific sequences required for an efficient interaction with MEK proteins. We show that, consequently, B-Raf interacts with MEK-1 and MEK-2 with a better affinity than does c-Raf-1, thus strengthening the notion that B-Raf is a stronger MEK activator than c-Raf-l. Our results also suggest that a MEK specific sequence, not present in MAP kinase kinases which are not activated by members of the Raf family, is required for the interaction with Raf proteins.

    Oncogene 1996;12;10;2213-21

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Assignment of the human genes encoding 14,3-3 Eta (YWHAH) to 22q12, 14-3-3 zeta (YWHAZ) to 2p25.1-p25.2, and 14-3-3 beta (YWHAB) to 20q13.1 by in situ hybridization.

    Tommerup N and Leffers H

    Danish Center for Human Genome Research, John F. Kennedy Institute, Glostrup, Denmark.

    Genomics 1996;33;1;149-50

  • Identification of the site of interaction of the 14-3-3 protein with phosphorylated tryptophan hydroxylase.

    Ichimura T, Uchiyama J, Kunihiro O, Ito M, Horigome T, Omata S, Shinkai F, Kaji H and Isobe T

    Department of Biochemistry, Faculty of Science, Niigata University, Japan.

    The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

    The Journal of biological chemistry 1995;270;48;28515-8

  • The effect on methamphetamine on the mRNA level for 14.3.3 eta chain in the human cultured cells.

    Muratake T, Hayashi S, Ichimura Y, Morii K, Kuwano R, Ichikawa T, Kumanishi T, Isobe T, Watanabe M, Kondo H et al.

    National Saigata Hospital, Niigata Prefecture, Japan.

    14.3.3 protein, a brain-specific protein, is an activator of tyrosine and tryptophan hydroxylases, key enzymes for biosynthesis of dopamine and serotonin. In this article, we describe cloning of cDNA for human brain 14.3.3 eta chain and expression of 14.3.3 eta chain mRNA in some human cultured cells. The cloned cDNA is 1730 bp long and contains 191 bp of a 5'-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3'-noncoding region, containing three polyadenylation signals. This cDNA encoded a polypeptide of 246 amino acids (M(r) 28,196). Furthermore, using in situ hybridization histochemistry, the expression of mRNA for this protein was examined in the rat central nervous system. In situ hybridization histochemistry indicated that 14.3.3 eta chain mRNA is detected not only in the monoamine-synthetic neurons, but also in other neurons in the discrete nuclei, which synthesize neither cathecholamine nor serotonin. Northern blot analysis demonstrated that the addition of methamphetamine into the cultured medium increased the mRNA level for 14.3.3 eta chain in U-251 cells, but did not increase that of GFAP.

    Molecular neurobiology 1995;11;1-3;223-30

  • 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

  • The human and bovine 14-3-3 eta protein mRNAs are highly conserved in both their translated and untranslated regions.

    Swanson KD, Dhar MS and Joshi JG

    Department of Biochemistry, University of Tennessee, Knoxville 37996.

    14-3-3 proteins form a highly conserved protein family whose members have been shown to activate tyrosine and tryptophan hydroxylases, inhibit protein kinase C and possess phospholipase A2 activity in vitro. We have isolated and analyzed a 14-3-3 protein cDNA clone (H14-3-3) from a human fetal brain cDNA library and found it to possess a high level of sequence identity with the bovine 14-3-3 eta protein cDNA in both the translated and untranslated regions, suggesting the presence of cis-regulatory elements in the untranslated regions of these mRNAs. The proteins encoded by these two cDNAs are 98.4% identical. Two different sized RNA species, approx. 1.9 and 3.5 kb in size that are expressed in a variety of tissues hybridize with this cDNA. However, only the 1.9 kb RNA is detected in the fetal brain. Northern blot analysis of poly(A)+ RNA isolated from eight different human tissues shows that 14-3-3 protein mRNAs are expressed in many tissues in the body. In agreement with previous reports, the highest abundance of RNA hybridizing with this cDNA is seen in the brain.

    Biochimica et biophysica acta 1993;1216;1;145-8

  • cDNA clones contain autonomous replication activity.

    Wu C, Friedlander P, Lamoureux C, Zannis-Hadjopoulos M and Price GB

    McGill Cancer Center, McGill University, Montréal, Canada.

    We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication. Subpopulations of transcripts represented in a cDNA library of human embryo lung fibroblasts (IMR90) were examined for their ability to support autonomous replication after transfection into human cells (HeLa). Two of three cDNA clones (343, 363) containing 'O'-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replication. One of these cDNA clones, 343, is enriched by selection for poly(A)+ RNA. In contrast, none of five Alu-containing transcripts was capable of autonomous replication in human cells. However, six out of ten cDNA clones contained neither 'O'-family or Alu homologous sequences and were as efficient as the cDNA clones containing 'O'-family sequences in replicating autonomously in human cells. cDNA clones, from an oligo-d(T)-primed library of human poly(A)+ enriched RNA, contain a significant proportion of independent clones that can also support autonomous replication of bacterial plasmids in human cells. cDNA clone 343 was observed to contain in a 448 bp EcoRI-HincII fragment, yeast ARS consensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and structural characteristics often associated with many prokaryotic, viral and eukaryotic origins. Sequence analysis of seven other cDNA clones (from non-'O'-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences. Two NOA clones (NOA4 and -5) did not contain any representations of ARS and SAR consensus sequences, suggesting that these two features may not be essential for autonomous replication activity in mammalian cells.

    Biochimica et biophysica acta 1993;1174;3;241-57

  • Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

    Leffers H, Madsen P, Rasmussen HH, Honoré B, Andersen AH, Walbum E, Vandekerckhove J and Celis JE

    Institute of Medical Biochemistry, Danish Centre for Human Genome Research, Aarhus University.

    We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

    Journal of molecular biology 1993;231;4;982-98

  • cDNA cloning and chromosome assignment of the gene for human brain 14-3-3 protein eta chain.

    Ichimura-Ohshima Y, Morii K, Ichimura T, Araki K, Takahashi Y, Isobe T, Minoshima S, Fukuyama R, Shimizu N and Kuwano R

    Research Laboratory for Molecular Genetics, Niigata University, Japan.

    We present the nucleotide sequence of a cDNA clone of mRNA encoding human 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases and an endogenous inhibitor of protein kinase C. The 1,730-nucleotide sequence of the cloned cDNA contains 191 bp of a 5'-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3'-noncoding region containing three canonical polyadenylation signals. The 14-3-3 protein eta chain cDNA encoded a polypeptide of 246 amino acids with a predicted molecular weight 28,196. The predicted amino acid sequence of human 14-3-3 protein eta was highly homologous to that of previously reported bovine and rat 14-3-3 proteins with only two amino acid differences. The sequence carries structural features as putative regions responsible for activation of tyrosine and tryptophan hydroxylases and for inhibition of Ca2+/phospholipid-dependent protein kinase C. Northern blot analysis demonstrated widespread expression of the 14-3-3 protein eta chain in cultured cell lines derived from various human tumors. These findings suggest the conservative functions of the 14-3-3 protein among species. Spot blot hybridization analysis with flow-sorted chromosomes showed that the human 14-3-3 protein eta chain gene is assigned to chromosome 22.

    Journal of neuroscience research 1992;31;4;600-5

  • Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases.

    Ichimura T, Isobe T, Okuyama T, Takahashi N, Araki K, Kuwano R and Takahashi Y

    Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Japan.

    The 14-3-3 protein is a family of acidic proteins present exclusively in the brain and is believed to have a function in monoamine biosynthesis because of its ability to activate tyrosine hydroxylase and tryptophan hydroxylase in the presence of Ca2+/calmodulin-dependent protein kinase type II. In this study, we resolved bovine brain 14-3-3 protein into seven polypeptide components by means of reversed-phase chromatography and determined the amino acid sequence of one of these components (eta chain) by cloning its cDNA from a bovine cerebellum cDNA library. The eta-chain mRNA is 1.8 kilobases long and encodes a polypeptide of 246 amino acids and Mr 28,221. Computer-assisted analysis of the sequence indicates that the eta chain exhibits no internal sequence repeats, nor does it have significant sequence similarity to other proteins with known amino acid sequence. However, the eta chain appears to consist of two structural regions that are distinguishable in their clearly different charge characteristics: the almost neutral amino-terminal region and the strongly acidic carboxyl-terminal region. The structural features of the eta chain and the domain organization of tyrosine and tryptophan hydroxylases suggest that the 14-3-3 protein binds to the regulatory domain of the phosphorylated hydroxylases through its acidic carboxyl-terminal region and activates the hydroxylases by inducing an active conformation.

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;19;7084-8

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
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
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
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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