G2Cdb::Gene report

Gene id
G00000184
Gene symbol
Pdpk1 (MGI)
Species
Mus musculus
Description
3-phosphoinositide dependent protein kinase 1
Orthologue
G00001433 (Homo sapiens)

Databases (11)

Curated Gene
OTTMUSG00000016280 (Vega mouse gene)
Gene
ENSMUSG00000024122 (Ensembl mouse gene)
18607 (Entrez Gene)
75 (G2Cdb plasticity & disease)
Gene Expression
NM_011062 (Allen Brain Atlas)
g01192 (BGEM)
18607 (Genepaint)
pdpk1 (gensat)
Literature
605213 (OMIM)
Marker Symbol
MGI:1338068 (MGI)
Protein Sequence
Q9Z2A0 (UniProt)

Synonyms (2)

  • Pdk1
  • Pkb kinase

Literature (95)

Pubmed - other

  • APOA-1 is a novel marker of erythroid cell maturation from hematopoietic stem cells in mice and humans.

    Inoue T, Sugiyama D, Kurita R, Oikawa T, Kulkeaw K, Kawano H, Miura Y, Okada M, Suehiro Y, Takahashi A, Marumoto T, Inoue H, Komatsu N and Tani K

    Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. yokotomo@bioreg.kyushu-u.ac.jp

    The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.

    Stem cell reviews 2011;7;1;43-52

  • Protein kinase B controls transcriptional programs that direct cytotoxic T cell fate but is dispensable for T cell metabolism.

    Macintyre AN, Finlay D, Preston G, Sinclair LV, Waugh CM, Tamas P, Feijoo C, Okkenhaug K and Cantrell DA

    College of Life Sciences, Division of Cell Biology & Immunology, University of Dundee, Scotland, UK.

    In cytotoxic T cells (CTL), Akt, also known as protein kinase B, is activated by the T cell antigen receptor (TCR) and the cytokine interleukin 2 (IL-2). Akt can control cell metabolism in many cell types but whether this role is important for CTL function has not been determined. Here we have shown that Akt does not mediate IL-2- or TCR-induced cell metabolic responses; rather, this role is assumed by other Akt-related kinases. There is, however, a nonredundant role for sustained and strong activation of Akt in CTL to coordinate the TCR- and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector versus memory and naive T cells. Akt is thus dispensable for metabolism, but the strength and duration of Akt activity dictates the CTL transcriptional program and determines CTL fate.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/C509890/1, BBS/E/B/0000C236; Wellcome Trust: 065975, 065975/Z/01/A

    Immunity 2011;34;2;224-36

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells.

    Park SG, Mathur R, Long M, Hosh N, Hao L, Hayden MS and Ghosh S

    Department of Microbiology & Immunology, Columbia University, College of Physicians & Surgeons, New York, NY 10032, USA.

    Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in T cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α(+) T cell receptor (TCR)γδ(+) T cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ(+) T cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ(+) T cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ(+) T cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ(+) T cells by Treg cells maintains enteric immune tolerance.

    Funded by: NIAID NIH HHS: R01 AI059440, R01 AI068977, R01 AI068977-01, R01 AI068977-02, R01 AI068977-03, R01 AI068977-04, R01 AI068977-05, R01 AI068977-06, R01-AI59440

    Immunity 2010;33;5;791-803

  • Temporal differences in the dependency on phosphoinositide-dependent kinase 1 distinguish the development of invariant Valpha14 NKT cells and conventional T cells.

    Finlay DK, Kelly AP, Clarke R, Sinclair LV, Deak M, Alessi DR and Cantrell DA

    Division of Cell Biology and Immunology, University of Dundee, Dundee, United Kingdom.

    This study uses two independent genetic strategies to explore the requirement for phosphoinositide-dependent kinase-1 (PDK1) in the development of mature T cell populations from CD4/CD8 double-positive thymocytes. The data show that CD4/CD8 double-positive thymocytes that do not express PDK1 or express a catalytically inactive PDK1 mutant fail to produce mature invariant Vα14 NKT cells but can differentiate to conventional CD4, CD8, or regulatory T cell subsets in the thymus. The PDK1 requirement for Vα14 NKT cell development reflects that these cells require the PDK1 substrate protein kinase B to meet the metabolic demands for proliferative expansion in response to IL-15 or AgR stimulation. There is also constitutive PDK1 signaling in conventional α/β T cells that is not required for lineage commitment of these cells but fine-tunes the expression of coreceptors and adhesion molecules. Also, although PDK1 is dispensable for thymic development of conventional α/β T cells, peripheral cells are reduced substantially. This reflects a PDK1 requirement for lymphopenia-induced proliferation, a process necessary for initial population of the peripheral T cell niche in neonatal mice. PDK1 is thus indispensable for T cell developmental programs, but the timing of the PDK1 requirement is unique to different T cell subpopulations.

    Funded by: Medical Research Council: MC_U127015387; Wellcome Trust: 065975, 065975/Z/01/A0

    Journal of immunology (Baltimore, Md. : 1950) 2010;185;10;5973-82

  • Maternal phosphatidylinositol 3-kinase signalling is crucial for embryonic genome activation and preimplantation embryogenesis.

    Zheng W, Gorre N, Shen Y, Noda T, Ogawa W, Lundin E and Liu K

    Department of Medical Biochemistry & Biophysics, Umeå University, Umeå, Sweden.

    Maternal effect factors derived from oocytes are important for sustaining early embryonic development before the major wave of embryonic genome activation (EGA). In this study, we report a two-cell-stage arrest of embryos lacking maternal 3-phosphoinositide-dependent protein kinase 1 as a result of suppressed EGA. Concurrent deletion of maternal Pten completely rescued the suppressed EGA and embryonic progression through restored AKT signalling, which fully restored the fertility of double-mutant females. Our study identifies maternal phosphatidylinositol 3-kinase signalling as a new maternal effect factor that regulates EGA and preimplantation embryogenesis in mice.

    EMBO reports 2010;11;11;890-5

  • Regulation of 3-phosphoinositide-dependent protein kinase 1 activity by homodimerization in live cells.

    Masters TA, Calleja V, Armoogum DA, Marsh RJ, Applebee CJ, Laguerre M, Bain AJ and Larijani B

    Cell Biophysics Laboratory, Cancer Research UK, London WC2A 3LY, UK.

    3-Phosphoinositide-dependent kinase 1 (PDK1) plays a central role in regulating the activity of protein kinases that are essential for signaling; however, how PDK1 itself is regulated is largely unknown. We found that homodimerization of PDK1 is a spatially and temporally regulated mechanism for controlling PDK1 activity. We used Förster resonance energy transfer monitored by fluorescence lifetime imaging microscopy to observe PDK1 homodimerization in live cells. A pleckstrin homology (PH) domain-dependent, basal dimeric association of PDK1 was increased upon cell stimulation with growth factors; this association was prevented by a phosphatidylinositol 3-kinase inhibitor and by a mutation in, or a complete deletion of, the PH domain of PDK1. The distinct spatial distribution of PDK1 homodimers relative to that of heterodimers of PDK1 and protein kinase B (PKB), and the ability of monomeric mutants of PDK1 to phosphorylate PKB, suggested that the monomer was the active conformation. Mutation of the autophosphorylation residue threonine-513 to glutamate, which was predicted to destabilize the homodimer interface, enhanced the interaction between PDK1 and PKB and the activity of PKB. Through in vitro, time-resolved fluorescence intensity and anisotropy measurements, combined with existing crystal structures and computational molecular modeling, we determined the geometrical arrangement of the PDK1 homodimer. With this approach, we calculated the size of the population of PDK1 dimers in cells. This description of a previously uncharacterized regulatory mechanism for the activation of PDK1 offers possibilities for controlling PDK1 activity therapeutically.

    Funded by: Cancer Research UK

    Science signaling 2010;3;145;ra78

  • Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).

    Mouton V, Toussaint L, Vertommen D, Gueuning MA, Maisin L, Havaux X, Sanchez-Canedo C, Bertrand L, Dequiedt F, Hemmings BA, Hue L and Rider MH

    Université catholique de Louvain and de Duve Institute, 75 Avenue Hippocrate, Brussels, Belgium.

    On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBβ-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBα/β siRNA or PKBα siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBα.

    The Biochemical journal 2010;431;2;267-75

  • Phosphoinositide-dependent kinase 1 provides negative feedback inhibition to Toll-like receptor-mediated NF-kappaB activation in macrophages.

    Chaurasia B, Mauer J, Koch L, Goldau J, Kock AS and Brüning JC

    Institute for Genetics, Department of Mouse Genetics and Metabolism, University of Cologne, Cologne, Germany.

    Phosphoinositide-dependent kinase 1 (PDK-1) represents an important signaling component in the phosphatidylinositol 3-kinase (PI3K) pathway, which plays an essential role in controlling a coordinated innate immune response. Here, we show that mice with conditional disruption of PDK-1 specifically in myeloid lineage cells (PDK-1(Deltamyel) mice) show enhanced susceptibility to lipopolysaccharide (LPS)-induced septic shock accompanied by exaggerated liver failure. Furthermore, primary macrophages derived from PDK-1(Deltamyel) mice lack LPS- and Pam3CSK4-stimulated AKT activity but exhibit increased mRNA expression and release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Moreover, LPS- and Pam3CSK4-stimulated primary macrophages exhibit enhanced phosphorylation and degradation of IkappaBalpha. While immediate upstream Toll-like receptor 4 (TLR-4)-induced signaling, including IL-1 receptor (IL-1R)-associated protein kinase (IRAK) phosphorylation, is unaltered in the absence of PDK-1, macrophages from PDK-1(Deltamyel) mice exhibit prolonged ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF-6) in response to LPS stimulation. These experiments reveal a novel PDK-1-dependent negative feedback inhibition of TLR-induced NF-kappaB activation in macrophages in vivo.

    Molecular and cellular biology 2010;30;17;4354-66

  • PDK1 plays a critical role in regulating cardiac function in mice and human.

    Di RM, Feng QT, Chang Z, Luan Q, Zhang YY, Huang J, Li XL and Yang ZZ

    Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China.

    Background: PDK1 is an essential protein kinase that plays a critical role in mammalian development. Mouse lacking PDK1 leads to multiple abnormalities and embryonic lethality at E9.5. To elucidate the role of PDK1 in the heart, we investigated the cardiac phenotype of mice that lack PDK1 in the heart in different growth periods and the alteration of PDK1 signaling in human failing heart.

    Methods: We employed Cre/loxP system to generate PDK1(flox/flox): α-MHC-Cre mice, which specifically deleted PDK1 in cardiac muscle at birth, and tamoxifen-inducible heart-specific PDK1 knockout mice (PDK1(flox/flox):MerCreMer mice), in which PDK1 was deleted in myocardium in response to the treatment with tamoxifen. Transmural myocardial tissues from human failing hearts and normal hearts were sampled from the left ventricular apex to analyze the activity of PDK1/Akt signaling pathways by Western blotting.

    Results: PDK1(flox/flox): α-MHC-Cre mice died of heart failure at 5 and 10 weeks old. PDK1(flox/flox) -MerCreMer mice died of heart failure from 5 to 21 weeks after the initiation of tamoxifen treatment at 8 weeks old. We found that expression levels of PDK1 in human failing heart tissues were significantly decreased compared with control hearts.

    Conclusion: Our results suggest that PDK1 signaling network takes part in regulating cardiac viability and function in mice, and may be also involved in human heart failure disease.

    Chinese medical journal 2010;123;17;2358-63

  • PDK1 regulates vascular remodeling and promotes epithelial-mesenchymal transition in cardiac development.

    Feng Q, Di R, Tao F, Chang Z, Lu S, Fan W, Shan C, Li X and Yang Z

    Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.

    One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.

    Molecular and cellular biology 2010;30;14;3711-21

  • Activation of the cardiac mTOR/p70(S6K) pathway by leucine requires PDK1 and correlates with PRAS40 phosphorylation.

    Sanchez Canedo C, Demeulder B, Ginion A, Bayascas JR, Balligand JL, Alessi DR, Vanoverschelde JL, Beauloye C, Hue L and Bertrand L

    Université catholique de Louvain, Brussels, Belgium.

    Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70(S6K)) axis in various organs. Insulin proceeds via the canonical association of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB/Akt). The signaling involved in leucine effect, although known to implicate a PI3K mechanism independent of PKB/Akt, is more poorly understood. In this study, we investigated whether PDK1 could also participate in the events leading to mTOR/p70(S6K) activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70(S6K) activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70(S6K) activity induced by insulin and leucine correlated with changes in phosphorylation of Thr(389), the mTOR phosphorylation site on p70(S6K), and of Ser(2448) on mTOR, both related to mTOR activity. Leucine also triggered phosphorylation of the proline-rich Akt/PKB substrate of 40 kDa (PRAS40), a new pivotal mTOR regulator. In PDK1 knockout hearts, leucine, similarly to insulin, failed to induce the phosphorylation of mTOR and p70(S6K), leading to the absence of p70(S6K) activation. The loss of leucine effect in absence of PDK1 correlated with the lack of PRAS40 phosphorylation. Moreover, the introduction in PDK1 of the L155E mutation, which is known to preserve the insulin-induced and PKB/Akt-dependent phosphorylation of mTOR/p70(S6K), suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70(S6K). We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70(S6K) pathway requires PDK1 in a way that differs from that of insulin.

    American journal of physiology. Endocrinology and metabolism 2010;298;4;E761-9

  • PDK-1/FoxO1 pathway in POMC neurons regulates Pomc expression and food intake.

    Iskandar K, Cao Y, Hayashi Y, Nakata M, Takano E, Yada T, Zhang C, Ogawa W, Oki M, Chua S, Itoh H, Noda T, Kasuga M and Nakae J

    International Center for Medical Research and Treatment, Kobe University Graduate school of Medicine, Japan.

    Both insulin and leptin signaling converge on phosphatidylinositol 3-OH kinase [PI(3)K]/3-phosphoinositide-dependent protein kinase-1 (PDK-1)/protein kinase B (PKB, also known as Akt) in proopiomelanocortin (POMC) neurons. Forkhead box-containing protein-O1 (FoxO1) is inactivated in a PI(3)K-dependent manner. However, the interrelationship between PI(3)K/PDK-1/Akt and FoxO1, and the chronic effects of the overexpression of FoxO1 in POMC neurons on energy homeostasis has not been elucidated. To determine the extent to which PDK-1 and FoxO1 signaling in POMC neurons was responsible for energy homeostasis, we generated POMC neuron-specific Pdk1 knockout mice (POMCPdk1(-/-)) and mice selectively expressing a constitutively nuclear (CN)FoxO1 or transactivation-defective (Delta256)FoxO1 in POMC neurons (CNFoxO1(POMC) or Delta256FoxO1(POMC)). POMCPdk1(-/-) mice showed increased food intake and body weight accompanied by decreased expression of Pomc gene. The CNFoxO1(POMC) mice exhibited mild obesity and hyperphagia compared with POMCPdk1(-/-) mice. Although expression of the CNFoxO1 made POMCPdk1(-/-) mice more obese due to excessive suppression of Pomc gene, overexpression of Delta256FoxO1 in POMC neurons had no effects on metabolic phenotypes and Pomc expression levels of POMCPdk1(-/-) mice. These data suggest a requirement for PDK-1 and FoxO1 in transcriptional regulation of Pomc and food intake.

    American journal of physiology. Endocrinology and metabolism 2010;298;4;E787-98

  • Initiation of neuronal differentiation requires PI3-kinase/TOR signalling in the vertebrate neural tube.

    Fishwick KJ, Li RA, Halley P, Deng P and Storey KG

    Neural Development Group, Division of Cell & Developmental Biology, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK.

    Regulated neuron production within the vertebrate nervous system relies on input from multiple signalling pathways. Work in the Drosophila retina has demonstrated that PI3-kinase and downstream TOR signalling regulate the timing of photoreceptor differentiation; however, the function of such signals during vertebrate neurogenesis is not well understood. Here we show that mutant mice lacking PKB activity downstream of PDK1, the master kinase of the PI3-kinase pathway, exhibit deficient neuron production. We further demonstrate expression of PI3-kinase signalling components and active PKB and TOR signalling in the chick spinal cord, an early site of neurogenesis. Neuron production was also attenuated in the chick neural tube following exposure to small molecule inhibitors of PI3-kinase (LY294002) or TOR (Rapamycin) activity. Furthermore, Rapamycin repressed expression of early neuronal differentiation genes, such as Ngn2, but did not inhibit expression of Sox1B genes characteristic of proliferating neural progenitors. In addition, some cells expressing an early neuronal marker were mis-localised at the ventricular surface in the presence of Rapamycin and remained aberrantly within the cell cycle. These findings suggest that TOR signalling is necessary to initiate neuronal differentiation and that it may facilitate coordination of cell cycle and differentiation programmes. In contrast, stimulating PI3-kinase signalling did not increase neuron production, suggesting that such activity is simply permissive for vertebrate neurogenesis.

    Funded by: Medical Research Council: G0600234, G0600234(78011)

    Developmental biology 2010;338;2;215-25

  • Tsc/mTORC1 signaling in oocytes governs the quiescence and activation of primordial follicles.

    Adhikari D, Zheng W, Shen Y, Gorre N, Hämäläinen T, Cooney AJ, Huhtaniemi I, Lan ZJ and Liu K

    1Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

    To maintain the female reproductive lifespan, the majority of ovarian primordial follicles are preserved in a quiescent state in order to provide ova for later reproductive life. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Here we provide genetic evidence to show that the tumor suppressor tuberous sclerosis complex 1 (Tsc1), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the quiescence of primordial follicles. In mutant mice lacking the Tsc1 gene in oocytes, the entire pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in the oocyte, ending up with follicular depletion in early adulthood and causing premature ovarian failure (POF). We further show that maintenance of the quiescence of primordial follicles requires synergistic, collaborative functioning of both Tsc and PTEN (phosphatase and tensin homolog deleted on chromosome 10) and that these two molecules suppress follicular activation through distinct ways. Our results suggest that Tsc/mTORC1 signaling and PTEN/PI3K (phosphatidylinositol 3 kinase) signaling synergistically regulate the dormancy and activation of primordial follicles, and together ensure the proper length of female reproductive life. Deregulation of these signaling pathways in oocytes results in pathological conditions of the ovary, including POF and infertility.

    Human molecular genetics 2010;19;3;397-410

  • The fibroblast growth factor receptor substrate 3 adapter is a developmentally regulated microtubule-associated protein expressed in migrating and differentiated neurons.

    Hryciw T, MacDonald JI, Phillips R, Seah C, Pasternak S and Meakin SO

    Molecular Brain Research Group, Robarts Research Institute, London, Ontario, Canada.

    Fibroblast growth factor (FGF) mediated signaling is essential to many aspects of neural development. Activated FGF receptors signal primarily through the FGF receptor substrate (Frs) adapters, which include Frs2/Frs2alpha and Frs3/Frs2beta. While some studies suggest that Frs3 can compensate for the loss of Frs2 in transfected cells, the lack of an effective Frs3 specific antibody has prevented efforts to determine the role(s) of the endogenous protein. To this end, we have generated a Frs3 specific antibody and have characterized the pattern of Frs3 expression in the developing nervous system, its subcellular localization as well as its biochemical properties. We demonstrate that Frs3 is expressed at low levels in the ventricular zone of developing cortex, between E12 and E15, and it co-localizes with nestin and acetylated alpha-tubulin in radial processes in the ventricular/subventricular zones as well as with betaIII tubulin in differentiated cortical neurons. Subcellular fractionation studies demonstrate that endogenous Frs3 is both soluble and plasma membrane associated while Frs3 expressed in 293T cells associates exclusively with lipid rafts. Lastly, we demonstrate that neuronal Frs3 binds microtubules comparable to the microtubule-associated protein, MAP2, while Frs2 does not. Collectively, these data suggest that neuronal Frs3 functions as a novel microtubule binding protein and they provide the first biochemical evidence that neuronal Frs3 is functionally distinct from Frs2/Frs2alpha.

    Journal of neurochemistry 2010;112;4;924-39

  • Reciprocal negative regulation of PDK1 and ASK1 signaling by direct interaction and phosphorylation.

    Seong HA, Jung H, Ichijo H and Ha H

    Department of Biochemistry, Biotechnology Research Institute, School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea.

    Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H(2)O(2), tumor necrosis factor alpha, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser(967), a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H(2)O(2)-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser(394) and Ser(398), indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.

    The Journal of biological chemistry 2010;285;4;2397-414

  • Phosphoinositide-dependent kinase PDK1 in the regulation of Ca2+ entry into mast cells.

    Shumilina E, Zemtsova IM, Heise N, Schmid E, Eichenmüller M, Tyan L, Rexhepaj R and Lang F

    Department of Physiology, University of Tübingen, Tübingen, Germany.

    The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKCδ. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKCδ inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 μM) increased the K(+) channel conductance to similar values in both genotypes. β-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKCδ inhibitor rottlerin increased β-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKCδ and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2010;26;4-5;699-706

  • PI3 kinase and PDK1 in the regulation of the electrogenic intestinal dipeptide transport.

    Rexhepaj R, Rotte A, Pasham V, Gu S, Kempe DS and Lang F

    Department of Physiology, University of Tübingen, 72076 Tübingen, Germany.

    The phosphoinositol 3 kinase (PI3K) and the phosphoinositide dependent kinase (PDK1) stimulate the serum and glucocorticoid inducible kinase (SGK) and protein kinase B (PKB/Akt) isoforms, kinases stimulating a variety of transporters. Most recently, SGK1 was shown to stimulate the peptide transporters PepT1 and PepT2, and to mediate the glucocorticoid stimulation of PepT1. Basal electrogenic intestinal peptide transport was, however, not dependent on the presence of SGK1. The present study explored whether basal electrogenic intestinal peptide transport is dependent on PI3K or PDK1. To this end, peptide transport in intestinal segments was determined utilizing Ussing chamber analysis. Cytosolic pH (pH(i)) was determined by BCECF fluorescence. The luminal addition of 5 mM dipeptide gly-gly induced a current (Ip) across intestinal segments. Ip was significantly decreased in the presence of PI3 kinase inhibitors Wortmannin (1 microM) or LY294002 (50 microM). Exposure of isolated intestinal cells to 5 mM gly-gly was followed by cytosolic acidification (DeltapH(i)), which was significantly blunted by Wortmannin and by LY294002. Both, Ip and DeltapHi were significantly smaller in PDK1 hypomorphic mice (pdk(1flfl)) than in their wild type littermates (pdk1(wt)). In conclusion, PI3K and PDK1 participate in the regulation of basal peptide transport.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2010;25;6;715-22

  • Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes.

    Finlay DK, Sinclair LV, Feijoo C, Waugh CM, Hagenbeek TJ, Spits H and Cantrell DA

    Division of Immunology and Cell Biology, University of Dundee, Dundee DD15EH, Scotland, UK.

    In normal T cell progenitors, phosphoinositide-dependent kinase l (PDK1)-mediated phosphorylation and activation of protein kinase B (PKB) is essential for the phosphorylation and inactivation of Foxo family transcription factors, and also controls T cell growth and proliferation. The current study has characterized the role of PDK1 in the pathology caused by deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN). PDK1 is shown to be essential for lymphomagenesis caused by deletion of PTEN in T cell progenitors. However, PTEN deletion bypasses the normal PDK1-controlled signaling pathways that determine thymocyte growth and proliferation. PDK1 does have important functions in PTEN-null thymocytes, notably to control the PKB-Foxo signaling axis and to direct the repertoire of adhesion and chemokine receptors expressed by PTEN-null T cells. The results thus provide two novel insights concerning pathological signaling caused by PTEN loss in lymphocytes. First, PTEN deletion bypasses the normal PDK1-controlled metabolic checkpoints that determine cell growth and proliferation. Second, PDK1 determines the cohort of chemokine and adhesion receptors expressed by PTEN-null cells, thereby controlling their migratory capacity.

    Funded by: Wellcome Trust: 065975, 065975/Z/01/A

    The Journal of experimental medicine 2009;206;11;2441-54

  • Pdk1 activity controls proliferation, survival, and growth of developing pancreatic cells.

    Westmoreland JJ, Wang Q, Bouzaffour M, Baker SJ and Sosa-Pineda B

    Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

    The formation of adequate masses of endocrine and exocrine pancreatic tissues during embryogenesis is essential to ensure proper nutrition and glucose homeostasis at postnatal stages. We generated mice with pancreas-specific ablation of the 3-phosphoinositide-dependent protein kinase 1 (Pdk1) to investigate how signaling downstream of the phosphatidylinositol-3-OH kinase (PI3K) pathway controls pancreas development. Pdk1-conditional knock-out mice were born with conspicuous pancreas hypoplasia, and within a few weeks, they developed severe hyperglycemia. Our detailed characterization of the mutant embryonic pancreas also revealed distinct temporal, cell type-specific requirements of Pdk1 activity in the control of cell proliferation, cell survival, and cell size during pancreas development. These results thus uncover Pdk1 as a novel, crucial regulator of pancreatic growth during embryogenesis. In addition, we provide evidence that Pdk1 activity is required differently in mature pancreatic cell types, since compensatory proliferation and possible mTORC2 activation occurred in exocrine cells but not in beta cells of the Pdk1-deficient postnatal pancreas.

    Funded by: NCI NIH HHS: R01 CA135554; NIDDK NIH HHS: 5 R01DK060542, R01 DK060542, R01 DK060542-05A1, R01 DK060542-06, R01 DK060542-07

    Developmental biology 2009;334;1;285-98

  • Epinephrine-induced hyperpolarization of pancreatic islet cells is sensitive to PI3K-PDK1 signaling.

    Zhang Y, Shumilina E, Häring HU, Lang F and Ullrich S

    Institute of Physiology, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.

    Epinephrine inhibits insulin release by activation of K(+) channels and subsequent hyperpolarization of pancreatic beta cells. The present study explored whether epinephrine-induced hyperpolarization is modified by phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositide-dependent kinase PDK1. Perforated patch-clamp was performed in islet cells isolated from PDK1 hypomorphic mice (pdk1(fl/fl)), expressing only 20% of PDK1, and in their wild-type littermates. At 16.8mM glucose, the cell membrane was hyperpolarized by epinephrine (1 microM), an effect significantly blunted in pdk1(fl/fl) and abrogated in wild-type cells by inhibition of PI3K with wortmannin (100 nM) or LY294002 (10 microM). The hyperpolarizing effect of epinephrine in pancreatic islet cells is thus sensitive to PI3K and PDK1.

    FEBS letters 2009;583;18;3101-6

  • Selective induction of neocortical GABAergic neurons by the PDK1-Akt pathway through activation of Mash1.

    Oishi K, Watatani K, Itoh Y, Okano H, Guillemot F, Nakajima K and Gotoh Y

    Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    Extracellular stimuli regulate neuronal differentiation and subtype specification during brain development, although the intracellular signaling pathways that mediate these processes remain largely unclear. We now show that the PDK1-Akt pathway regulates differentiation of telencephalic neural precursor cells (NPCs). Active Akt promotes differentiation of NPC into gamma-aminobutyric acid-containing (GABAergic) but not glutamatergic neurons. Disruption of the Pdk1 gene or expression of dominant-negative forms of Akt suppresses insulin-like growth factor (IGF)-1 enhancement of NPC differentiation into neurons in vitro and production of neocortical GABAergic neurons in vivo. Furthermore, active Akt increased the protein levels and transactivation activity of Mash1, a proneural basic helix-loop-helix protein required for the generation of neocortical GABAergic neurons, and Mash1 was required for Akt-induced neuronal differentiation. These results have unveiled an unexpected role of the PDK1-Akt pathway: a key mediator of extracellular signals regulating the production of neocortical GABAergic neurons.

    Funded by: Medical Research Council: MC_U117570528

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;31;13064-9

  • PDK1 signaling in oocytes controls reproductive aging and lifespan by manipulating the survival of primordial follicles.

    Reddy P, Adhikari D, Zheng W, Liang S, Hämäläinen T, Tohonen V, Ogawa W, Noda T, Volarevic S, Huhtaniemi I and Liu K

    Department of Medical Biochemistry and Biophysics, Umeå University SE-901 87, Umeå, Sweden.

    The molecular mechanisms that control reproductive aging and menopausal age in females are poorly understood. Here, we provide genetic evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) signaling in oocytes preserves reproductive lifespan by maintaining the survival of ovarian primordial follicles. In mice lacking the PDK1-encoding gene Pdk1 in oocytes, the majority of primordial follicles are depleted around the onset of sexual maturity, causing premature ovarian failure (POF) during early adulthood. We further showed that suppressed PDK1-Akt-p70 S6 kinase 1 (S6K1)-ribosomal protein S6 (rpS6) signaling in oocytes appears to be responsible for the loss of primordial follicles, and mice lacking the Rps6 gene in oocytes show POF similar to that in Pdk1-deficient mice. In combination with our earlier finding that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in oocytes suppresses follicular activation, we have now pinpointed the molecular network involving phosphatidylinositol 3 kinase (PI3K)/PTEN-PDK1 signaling in oocytes that controls the survival, loss and activation of primordial follicles, which together determine reproductive aging and the length of reproductive life in females. Underactivation or overactivation of this signaling pathway in oocytes is shown to cause pathological conditions in the ovary, including POF and infertility.

    Human molecular genetics 2009;18;15;2813-24

  • Cell type specificity of PI3K signaling in Pdk1- and Pten-deficient brains.

    Chalhoub N, Zhu G, Zhu X and Baker SJ

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

    Loss of PTEN causes unregulated activation of downstream components of phosphatidylinositol 3-kinase (PI3K) signaling, including PDK1, and disrupts normal nervous system development and homeostasis. We tested the contribution of Pdk1 to the abnormalities induced by Pten deletion in the brain. Conditional deletion of Pdk1 caused microcephaly. Combined deletion of Pdk1 and Pten rescued hypertrophy, but not migration defects of Pten-deficient neurons. Pdk1 inactivation induced strikingly different effects on the regulation of phosphorylated Akt in glia versus neurons. Our results show Pdk1-dependent and Pdk1-independent abnormalities in Pten-deficient brains, and demonstrate cell type specific differences in feedback regulation of the ubiquitous PI3K pathway.

    Funded by: NCI NIH HHS: CA096832, CA135554, P01 CA096832, R01 CA135554

    Genes & development 2009;23;14;1619-24

  • PDK1 coordinates survival pathways and beta-adrenergic response in the heart.

    Ito K, Akazawa H, Tamagawa M, Furukawa K, Ogawa W, Yasuda N, Kudo Y, Liao CH, Yamamoto R, Sato T, Molkentin JD, Kasuga M, Noda T, Nakaya H and Komuro I

    Department of Cardiovascular Science and , Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

    The 3-phosphoinositide-dependent kinase-1 (PDK1) plays an important role in the regulation of cellular responses in multiple organs by mediating the phosphoinositide 3-kinase (PI3-K) signaling pathway through activating AGC kinases. Here we defined the role of PDK1 in controlling cardiac homeostasis. Cardiac expression of PDK1 was significantly decreased in murine models of heart failure. Tamoxifen-inducible and heart-specific disruption of Pdk1 in adult mice caused severe and lethal heart failure, which was associated with apoptotic death of cardiomyocytes and beta(1)-adrenergic receptor (AR) down-regulation. Overexpression of Bcl-2 protein prevented cardiomyocyte apoptosis and improved cardiac function. In addition, PDK1-deficient hearts showed enhanced activity of PI3-Kgamma, leading to robust beta(1)-AR internalization by forming complex with beta-AR kinase 1 (betaARK1). Interference of betaARK1/PI3-Kgamma complex formation by transgenic overexpression of phosphoinositide kinase domain normalized beta(1)-AR trafficking and improved cardiac function. Taken together, these results suggest that PDK1 plays a critical role in cardiac homeostasis in vivo by serving as a dual effector for cell survival and beta-adrenergic response.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;21;8689-94

  • HMGB1 is phosphorylated by classical protein kinase C and is secreted by a calcium-dependent mechanism.

    Oh YJ, Youn JH, Ji Y, Lee SE, Lim KJ, Choi JE and Shin JS

    Department of Microbiology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

    High-mobility group box 1 protein (HMGB1) has been studied as a key mediator of inflammatory diseases, including sepsis. Regulating secretion is important in the control of HMGB1-mediated inflammation. Previously, it was shown that HMGB1 needs to be phosphorylated for secretion. In this study, we show that HMGB1 is phosphorylated by the classical protein kinase C (cPKC) and is secreted by a calcium-dependent mechanism. For this study, RAW264.7 cells and human peripheral blood monocytes were treated with PI3K inhibitors wortmannin, LY294002, and ZSTK474, resulting in inhibition of LPS-stimulated HMGB1 secretion, whereas inhibitors of NF-kappaB and MAPKs p38 and ERK showed no inhibition. Akt inhibitor IV and mammalian target of rapamycin inhibitor rapamycin did not inhibit HMGB1 secretion. However, the PKC inhibitors Gö6983 (broad-spectrum PKC), Gö6976 (cPKC), and Ro-31-7549 (cPKC) and phosphoinositide-dependent kinase 1 inhibitor, which results in protein kinase C (PKC) inhibition, inhibited LPS-stimulated HMGB1 secretion. PKC activators, PMA and bryostatin-1, enhanced HMGB1 secretion. In an in vitro kinase assay, HMGB1 was phosphorylated by recombinant cPKC and by purified nuclear cPKC from LPS-stimulated RAW264.7 cells, but not by casein kinase II or cdc2. HMGB1 secretion was also induced by the calcium ionophore A23187 and inhibited by the Ca(2+) chelators BAPTA-AM and EGTA. These findings support a role for Ca(2+)-dependent PKC in HMGB1 secretion. Thus, we propose that cPKC is an effector kinase of HMGB1 phosphorylation in LPS-stimulated monocytes and PI3K-phosphoinositide-dependent kinase 1 may act in concert to control HMGB1 secretion independent of the NF-kappaB, p38, and ERK pathways.

    Journal of immunology (Baltimore, Md. : 1950) 2009;182;9;5800-9

  • The kinase PDK1 integrates T cell antigen receptor and CD28 coreceptor signaling to induce NF-kappaB and activate T cells.

    Park SG, Schulze-Luehrman J, Hayden MS, Hashimoto N, Ogawa W, Kasuga M and Ghosh S

    Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

    In addition to ligation of the T cell antigen receptor (TCR), activation of the CD28 coreceptor by the costimulatory molecule B7 is required for induction of the transcription factor NF-kappaB and robust T cell activation, although the contribution of CD28 to this process remains incompletely understood. We show here that phosphoinositide-dependent kinase 1 (PDK1) is essential for integrating the TCR and CD28 signals. After we deleted PDK1 from T cells, TCR-CD28 signals were unable to induce activation of NF-kappaB or phosphorylation of protein kinase C-theta, although T cell survival and pathways dependent on the kinases p38 and Jnk or the transcription factor NFAT were unaffected. CD28 facilitated NF-kappaB activation by regulating recruitment and phosphorylation of PDK1, which are necessary for efficient binding of PDK1 to protein kinase C-theta and the adaptor CARMA1 and thus for NF-kappaB induction.

    Funded by: NIAID NIH HHS: R01 AI059440-01, R01 AI059440-02, R01 AI059440-03, R01 AI059440-04, R01 AI068977-01, R01 AI068977-02, R01 AI068977-03, R01 AI068977-04, R01-AI59440

    Nature immunology 2009;10;2;158-66

  • Protein kinase C theta (PKCtheta)-dependent phosphorylation of PDK1 at Ser504 and Ser532 contributes to palmitate-induced insulin resistance.

    Wang C, Liu M, Riojas RA, Xin X, Gao Z, Zeng R, Wu J, Dong LQ and Liu F

    Department of Pharmacology, Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78229, USA.

    Clinical, epidemiological, and biochemical studies have highlighted the role of obesity-induced insulin resistance in various metabolic diseases. However, the underlying molecular mechanisms remain to be established. In the present study, we show that palmitate-induced serine phosphorylation of phosphoinositide-dependent protein kinase-1 (PDK1) negatively regulates insulin signaling. PDK1-mediated Akt phosphorylation at Thr308 in the activation loop is reduced in C2C12 myotubes treated with palmitate or overexpressing protein kinase C theta (PKCtheta), a kinase that has been implicated in hyperlipidemia-induced insulin resistance. Palmitate treatment also inhibited platelet-derived growth factor-stimulated Akt phosphorylation, suggesting that the inhibition could occur at a site independent of IRS1/2. The inhibitory effect of palmitate on PDK1 and Akt was diminished in PKCtheta-deficient mouse embryonic fibroblasts (MEFs) by treating C2C12 myotubes with PKCtheta pseudosubstrates. In vivo labeling studies revealed that PDK1 undergoes palmitate-induced phosphorylation at two novel sites, Ser504 and Ser532. Replacing Ser504/532 with alanine disrupted PKCtheta-catalyzed PDK1 phosphorylation in vitro and palmitate-induced PDK1 phosphorylation in cells. PDK1-deficient MEFs transiently expressing PDK1S504A/S532A but not PDK1S504E/S532D showed increased basal and insulin-stimulated Akt phosphorylation at Thr308 when compared with MEFs expressing wild-type PDK1. Taken together, our results identify PDK1 as a novel target in free fatty acid-induced insulin resistance and PKCtheta as the kinase mediating the negative regulation.

    Funded by: NIDDK NIH HHS: R01 DK056166, R01 DK069930, R01 DK080344

    The Journal of biological chemistry 2009;284;4;2038-44

  • The survival pathways phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation.

    Haga S, Ozaki M, Inoue H, Okamoto Y, Ogawa W, Takeda K, Akira S and Todo S

    Department of Surgery, Hokkaido University School of Medicine, Sapporo, Japan.

    Unlabelled: Liver regeneration comprises a series of complicated processes. The current study was designed to investigate the roles of phosphoinositide-dependent protein kinase 1 (PDK1)-associated pathways in liver regeneration after partial hepatectomy (PH) using liver-specific Pdk1-knockout (L-Pdk1KO) and Pdk1/STAT3 double KO (L-DKO) mice. There was no liver regeneration, and 70% PH was lethal in L-Pdk1KO mice. Liver regeneration was severely impaired equally in L-Pdk1KO and L-DKO mice, even after nonlethal 30% PH. There was no cell growth (measured as increase of cell size) after hepatectomy in L-Pdk1KO mice, although the post-PH mitotic response was the same as in controls. As expected, hepatectomy did not induce hepatic Akt-phosphorylation (Thr308) in L-Pdk1KO mice, and post-PH phosphorylation of Akt, mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase (p70(S6K)), and S6 were also reduced. To examine the specific role of PDK1-associated signals, a "pif-pocket" mutant of PDK1, which allows PDK1 only to phosphorylate Akt, was used. Liver regeneration was recovered in L-Pdk1KO mice with a "pif-pocket" mutant of PDK1. This re-activated Akt in L-Pdk1KO mice liver and induced post-PH cell growth, without affecting cell proliferation. Further deletion of STAT3 (L-DKO mice) did not further deteriorate liver regeneration, although this certainly reduced post-PH mitotic response. These findings indicate that PDK1/Akt contribute to liver regeneration by regulating cell size. Regarding phosphatidylinositol-3 kinase (PI3-K), immediate upstream signal of PDK1, activation of PI3-K induced cell proliferation via STAT3 activation in the liver of L-Pdk1KO mice but did not improve impaired liver regeneration. This confirmed the pivotal role of PDK1 in liver regeneration and cell growth.

    Conclusion: PDK1/Akt-mediated responsive cell growth is essential for normal liver regeneration after PH, especially when cell proliferation is impaired.

    Hepatology (Baltimore, Md.) 2009;49;1;204-14

  • miR-375 targets 3'-phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells.

    El Ouaamari A, Baroukh N, Martens GA, Lebrun P, Pipeleers D and van Obberghen E

    Institut National de la Santé et de la Recherche Médicale, U907, Nice, France.

    Objective: MicroRNAs are short, noncoding RNAs that regulate gene expression. We hypothesized that the phosphatidylinositol 3-kinase (PI 3-kinase) cascade known to be important in beta-cell physiology could be regulated by microRNAs. Here, we focused on the pancreas-specific miR-375 as a potential regulator of its predicted target 3'-phosphoinositide-dependent protein kinase-1 (PDK1), and we analyzed its implication in the response of insulin-producing cells to elevation of glucose levels.

    We used insulinoma-1E cells to analyze the effects of miR-375 on PDK1 protein level and downstream signaling using Western blotting, glucose-induced insulin gene expression using quantitative RT-PCR, and DNA synthesis by measuring thymidine incorporation. Moreover, we analyzed the effect of glucose on miR-375 expression in both INS-1E cells and primary rat islets. Finally, miR-375 expression in isolated islets was analyzed in diabetic Goto-Kakizaki (GK) rats.

    Results: We found that miR-375 directly targets PDK1 and reduces its protein level, resulting in decreased glucose-stimulatory action on insulin gene expression and DNA synthesis. Furthermore, glucose leads to a decrease in miR-375 precursor level and a concomitant increase in PDK1 protein. Importantly, regulation of miR-375 expression by glucose occurs in primary rat islets as well. Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets.

    Conclusions: Our findings provide evidence for a role of a pancreatic-specific microRNA, miR-375, in the regulation of PDK1, a key molecule in PI 3-kinase signaling in pancreatic beta-cells. The effects of glucose on miR-375 are compatible with the idea that miR-375 is involved in glucose regulation of insulin gene expression and beta-cell growth.

    Diabetes 2008;57;10;2708-17

  • Pancreatic beta cell mass preserved in heterozygous PDK1 knockout mice.

    Takeda A, Kido Y, Hashimoto N, Noda T and Kasuga M

    Department of Internal Medicine, Division of Diabetes, Metabolism, and Endocrinology, Kobe University Graduate School of Medicine, Kobe 17, Japan.

    We have demonstrated that 3-phosphoinositide-dependent protein kinase 1 (PDK1) contributes to signaling by insulin or insulin-like growth factor-1 (IGF-1) that is responsible for the regulation of both the number and size of pancreatic beta cells in mice. Complete ablation of PDK1 in pancreatic beta cells leads to progressive hyperglycemia as a result of loss of beta cell mass. In this study, we generated heterozygous pancreatic beta cell-specific PDK1 knockout (betaPDK1+/-) mice and fed them a high-fat diet as a model of human type 2 diabetes. The betaPDK1+/- mice exhibited normal glucose tolerance even on a high-fat diet. Further, islet morphology and beta cell mass were normal in betaPDK1+/- mice, and haploinsufficiency of PDK1 did not impair the compensatory hyperplasia of beta cells on a high-fat diet. The phosphorylation and expression of the molecules that are expressed downstream of PDK1 were similar in the islets of the betaPDK1+/- and control mice. Eventually, we concluded that glucose homeostasis and islet mass were maintained in betaPDK1+/- mice.

    The Kobe journal of medical sciences 2008;54;3;E183-90

  • Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells.

    Tamgüney T, Zhang C, Fiedler D, Shokat K and Stokoe D

    UCSF Cancer Research Institute, USA.

    3-phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1(-/-) ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1(-/-) ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.

    Funded by: NCI NIH HHS: R21 CA113976, R21 CA113976-01, R21 CA113976-02, R21CA113976-02

    Experimental cell research 2008;314;11-12;2299-312

  • PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression.

    Nakamura K, Sakaue H, Nishizawa A, Matsuki Y, Gomi H, Watanabe E, Hiramatsua R, Tamamori-Adachi M, Kitajima S, Noda T, Ogawa W and Kasuga M

    Department of Internal Medicine, Division of Diabetes, Metabolism, and Endocrinology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

    PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).

    The Journal of biological chemistry 2008;283;25;17702-11

  • Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance.

    Bayascas JR, Wullschleger S, Sakamoto K, García-Martínez JM, Clacher C, Komander D, van Aalten DM, Boini KM, Lang F, Lipina C, Logie L, Sutherland C, Chudek JA, van Diepen JA, Voshol PJ, Lucocq JM and Alessi DR

    MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.

    PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

    Funded by: Medical Research Council: MC_U127015387, MC_U127088492; Wellcome Trust

    Molecular and cellular biology 2008;28;10;3258-72

  • PDK1 deficiency in POMC-expressing cells reveals FOXO1-dependent and -independent pathways in control of energy homeostasis and stress response.

    Belgardt BF, Husch A, Rother E, Ernst MB, Wunderlich FT, Hampel B, Klöckener T, Alessi D, Kloppenburg P and Brüning JC

    Institute for Genetics, Department of Mouse Genetics and Metabolism, Center for Molecular Medicine Cologne (CMMC), University of Cologne, D-50674 Cologne, Germany.

    Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.

    Funded by: Medical Research Council: MC_U127015387

    Cell metabolism 2008;7;4;291-301

  • Parvovirus interference with intracellular signalling: mechanism of PKCeta activation in MVM-infected A9 fibroblasts.

    Lachmann S, Bär S, Rommelaere J and Nüesch JP

    Program 'Infection and Cancer', Abteilung F010 and Institut National de la Santé et de la Recherche Médicale U701, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

    Autonomous parvoviruses are strongly dependent on the phosphorylation of the major non-structural protein NS1 by members of the protein kinase C (PKC) family. Besides being accompanied with changes in the overall phosphorylation pattern of NS1 and acquiring new modifications at consensus PKC sites, ongoing minute virus of mice (MVM) infections lead to the appearance of new phosphorylated cellular protein species. This prompted us to investigate whether MVM actively interferes with phosphoinositol-dependent kinase (PDK)/PKC signalling. The activity, subcellular localization and phosphorylation status of the protein kinases PDK1, PKCeta and PKClambda were measured in A9 cells in the presence or absence of MVM infection. Parvovirus infection was found to result in activation of both PDK1 and PKCeta, as evidenced by changes in their subcellular distribution and overall (auto)phosphorylation. We show evidence that activation of PKCeta by PDK1 is driven by atypical PKClambda. By modifying the hydrophobic motif of PKCeta, PKClambda appeared to control docking and consecutive phosphorylation of PKCeta's activation-loop by PDK1, a process that was inhibited in vivo in the presence of a dominant-negative PKClambda mutant.

    Cellular microbiology 2008;10;3;755-69

  • 3-phosphoinositide-dependent kinase 1 deficiency perturbs Toll-like receptor signaling events and actin cytoskeleton dynamics in dendritic cells.

    Zaru R, Mollahan P and Watts C

    Division of Cell Biology and Immunology, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.

    The adaptive immune response depends on dendritic cell (DC) activation by microbial products that signal via pattern recognition receptors and activate mitogen-activated protein kinases, NFkappaB and PI3K. The contribution of the AGC kinase family, including protein kinase B, protein kinase C, p90kDa ribosomal S6 kinase, and S6 kinase, has been little investigated because the probable redundancy among their isoforms makes their study difficult. We took advantage of the fact that all these kinases are regulated by the upstream master kinase 3-phosphoinositide-dependent kinase 1 (PDK1). Here we analyze various properties of DC from mice expressing approximately 10% of normal PDK1 (PDK1(fl/-)). DC populations in lymphoid and nonlymphoid tissues appeared normal in PDK1(fl/-) mice, and some in vitro responses to lipopolysaccharide (LPS) such as cytokine production were normal in cultured bone marrow DC. However, LPS-induced expression of class II major histocompatibility complex and CD86 were elevated in PDK1(fl/-) BMDC and PDK1(fl/-) spleen DC produced more interleukin-10 and -12, implying an attenuating role for PDK1. Unexpectedly, PDK1(fl/-) DC had a significantly reduced capacity for LPS-stimulated macropinocytosis and phagocytosis that correlated with a lowered F-actin/G-actin ratio, apparently because of increased actin depolymerization. Several PDK1-regulated kinases, some of which feed into actin regulators, showed reduced activation in PDK1(fl/-) DC. Reintroduction of PDK1 restored S6 kinase activity, increased levels of F-actin, and boosted macropinocytosis thus linking PDK1 and its downstream effectors to the unusual phenotype of PDK1(fl/-) DC.

    Funded by: Medical Research Council: G0100536

    The Journal of biological chemistry 2008;283;2;929-39

  • Phosphatidylinositide dependent kinase deficiency increases anxiety and decreases GABA and serotonin abundance in the amygdala.

    Ackermann TF, Hörtnagl H, Wolfer DP, Colacicco G, Sohr R, Lang F, Hellweg R and Lang UE

    Department of Physiology I, University of Tuebingen, Tuebingen, Germany.

    Pathological anxiety is paralleled by deficits in serotonergic and GABAergic neurotransmission in the amygdala. Conversely, anxiety disorders and depression may be reversed by brain-derived neurotrophic factor (BDNF). BDNF signaling involves Phosphatidylinositol 3-Kinase / 3-phosphoinositide-dependent protein kinase 1 (PI3K/PDK1). We thus hypothesized that impaired function of PDK1 might be associated with increased anxiety and concomitant neurotransmitter changes. Here we used the hypomorphic PDK1(hm) mouse to investigate anxiety behavior in different settings: PDK1(hm) mice differed from Wt littermates PDK1(WT) in several behavioral measures related to anxiety and exploration, namely in the open field, dark-light box, O-maze and startle response. Further we analyzed the brain substrate underlying this phenotype and found significantly decreased GABA, taurine and serotonin concentrations in the amygdala and olfactory bulb of PDK1(hm) mice, while BDNF and nerve growth factor (NGF) concentrations were not significantly different between PDK1(hm) and PDK1(WT) mice. These results suggest that impaired PI3K signaling in the PDK1(hm) mouse reduces concentrations of GABA and serotonin in anxiety related brain regions and can serve as a molecular substrate for behavior indicative for anxious and depressive-like mood states.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2008;22;5-6;735-44

  • Role of PDK1 in regulation of gastric acid secretion.

    Rotte A, Bhandaru M, Ackermann TF, Boini KM and Lang F

    Department of Physiology, University of Tübingen, Tübingen, Germany.

    Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) has previously been shown to enhance basal gastric acid secretion. Signaling of PI-3-kinase includes activation of the phosphoinositide dependent kinase PDK1. We thus hypothesized that PDK1 may influence gastric acid secretion. In the present study gastric acid secretion in mice expressing ñ20 % of PDK1 (pdk1(hm)) was compared to gastric acid secretion in their wild type littermates (pdk1(wt)). According to BCECF-fluorescence cytosolic pH in isolated gastric glands was similar in pdk1(hm) and in pdk1(wt) mice. Na(+)-independent pH recovery (DeltapH/min) following an ammonium pulse, which reflects K(+)/H(+)-ATPase activity, was however, significantly faster in pdk1(hm) than in pdk1(wt) mice. In both genotypes, DeltapH/min was abolished in the presence of K(+)/H(+)-ATPase inhibitor omeprazole (100 microM). Increase of local K(+)-concentrations to 35 mM (replacing Na(+)/NMDG) significantly increased DeltapH/min in both, pdk1(hm) and pdk1(wt) mice, and abrogated the differences between genotypes. Similarly, treatment with 5 microM forskolin as well as stimulation of protein kinase C with phorbolester phorbol 12 myristate 13 acetate (100 nM) enhanced DeltapH/min to almost identical values in pdk1(hm) and in pdk1(wt)mice. Protein kinase A inhibitor H89 (50 nM) decreased DeltapH/min in pdk1(hm) to values similar to those in pdk1(wt). In conclusion, deficient activity of PDK1 leads to a marked increase of gastric acid secretion. The present observations thus disclose a novel element in the regulation of gastric H(+) secretion.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2008;22;5-6;725-34

  • EUCOMM--the European conditional mouse mutagenesis program.

    Friedel RH, Seisenberger C, Kaloff C and Wurst W

    GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.

    Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.

    Briefings in functional genomics & proteomics 2007;6;3;180-5

  • Notch-induced T cell development requires phosphoinositide-dependent kinase 1.

    Kelly AP, Finlay DK, Hinton HJ, Clarke RG, Fiorini E, Radtke F and Cantrell DA

    College of Life Science, Division of Cell Biology & Immunology, MSI/WTB complex, University of Dundee, Dundee, UK.

    Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.

    Funded by: Wellcome Trust: 065975, 60/3116

    The EMBO journal 2007;26;14;3441-50

  • Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D.

    Wood CD, Kelly AP, Matthews SA and Cantrell DA

    Division of Cell Biology and Immunology, College of Life Sciences, MSI/WTB/CIR Complex, University of Dundee, Dow Street, Dundee, United Kingdom.

    Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.

    Funded by: Wellcome Trust: 065975

    FEBS letters 2007;581;18;3494-8

  • Restoration of glucokinase expression in the liver normalizes postprandial glucose disposal in mice with hepatic deficiency of PDK1.

    Okamoto Y, Ogawa W, Nishizawa A, Inoue H, Teshigawara K, Kinoshita S, Matsuki Y, Watanabe E, Hiramatsu R, Sakaue H, Noda T and Kasuga M

    Department of Clinical Molecular Medicine, Division of Diabetes and Digestive and Kidney Diseases, Kobe University Graduate School of Medicine, Kobe, Japan 657-0011.

    Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase-dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes-like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.

    Diabetes 2007;56;4;1000-9

  • Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

    Munton RP, Tweedie-Cullen R, Livingstone-Zatchej M, Weinandy F, Waidelich M, Longo D, Gehrig P, Potthast F, Rutishauser D, Gerrits B, Panse C, Schlapbach R and Mansuy IM

    Brain Research Institute, Medical Faculty of the University of Zürich, Switzerland.

    Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

    Molecular & cellular proteomics : MCP 2007;6;2;283-93

  • 3'Phosphoinositide-dependent kinase-1 is essential for ischemic preconditioning of the myocardium.

    Budas GR, Sukhodub A, Alessi DR and Jovanović A

    Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK.

    Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that are yet to be fully defined. 3'-Phosphoinositide-dependent kinase-1 (PDK1) has been implicated in numerous cellular processes. However, the involvement of PDK1 in preconditioning has yet to be elucidated. Studying PDK1 is not as straightforward as it is for the majority of kinases, due to the lack of a specific inhibitor of PDK1. Therefore, we have taken advantage of PDK1 hypomorphic mutant mice with reduced expression of PDK1 to study the role of PDK1 in preconditioning. Whole heart and single cell models of preconditioning demonstrated that the hearts and cardiac cells from PDK1 hypomorphic mice could not be preconditioned. The cardioprotective effect of PDK1 was not related to the effect that preconditioning has on sarcolemmal membrane action potential as revealed by di-8-ANEPPS, a sarcolemmal-potential sensitive dye, and laser confocal microscopy. In contrast, experiments with JC-1, a mitochondrial membrane potential-sensitive dye, has demonstrated that intact PDK1 levels were required for preconditioning-mediated regulation of mitochondrial membrane potential. Western blotting combined with functional experiments have shown that intact PDK1 levels were required for preconditioning-induced phosphorylation of protein kinase B (PKB), glycogen synthase kinase-3beta (GSK-3beta), and cardioprotection. We conclude that PDK1 mediates preconditioning in the heart by regulating activating PKB-GSK-3beta to regulate mitochondrial but not sarcolemmal membrane potential. 3'Phosphoinositide-dependent kinase-1 (PDK1) is essential for ischemic preconditioning of the myocardium.

    Funded by: Biotechnology and Biological Sciences Research Council: S18744; British Heart Foundation: PG/02/117/14488, PG/04/086/17410, PG/04/096/17627; Medical Research Council: G0400608, G0400608(71317), MC_U127015387; Wellcome Trust: 059528/Z/99/Z/JMW/CP/JF

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;14;2556-8

  • Impaired intestinal and renal glucose transport in PDK-1 hypomorphic mice.

    Artunc F, Rexhepaj R, Völkl H, Grahammer F, Remy C, Sandulache D, Nasir O, Wagner CA, Alessi DR and Lang F

    Dept. of Physiology, Univ. of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.

    The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na+-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing approximately 20% of PDK-1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1hm than of pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1hm compared with pdk1wt mice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.

    American journal of physiology. Regulatory, integrative and comparative physiology 2006;291;5;R1533-8

  • Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse.

    Sandu C, Artunc F, Palmada M, Rexhepaj R, Grahammer F, Hussain A, Yun C, Alessi DR and Lang F

    Department of Physiology I, University of Tübingen, Germany.

    In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.

    Funded by: Medical Research Council: MC_U127015387

    American journal of physiology. Gastrointestinal and liver physiology 2006;291;5;G868-76

  • Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice.

    Rexhepaj R, Grahammer F, Völkl H, Remy C, Wagner CA, Sandulache D, Artunc F, Henke G, Nammi S, Capasso G, Alessi DR and Lang F

    Department of Physiology I, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.

    The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up-regulate a variety of sodium-coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1(hm)) were compared with their wild-type (WT) littermates (pdk1(wt)). Body weight was significantly less in pdk1(hm) than in pdk1(wt) mice. Despite lower body weight of pdk1(hm) mice, food and water intake were similar in pdk1(hm) and pdk1(wt) mice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1(hm) mice than in pdk1(wt) mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfused proximal tubules of pdk1(hm) mice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan was significantly larger in pdk1(hm) than in pdk1(wt) mice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B(0)AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1), and the transporter for cationic amino acids and cystine b(0,+)AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1(hm) mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1-deficient mice.

    Funded by: Medical Research Council: MC_U127015387

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;13;2214-22

  • Antigen receptor regulation of phosphoinositide-dependent kinase 1 pathways during thymocyte development.

    Hinton HJ, Clarke RG and Cantrell DA

    Division of Cell Biology and Immunology, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK.

    Phosphoinositide-dependent kinase 1 (PDK1) is essential for T cell development but little is know about the stimuli that regulate PDK1 signaling in vivo. The thymus contains a heterogeneous mixture of cells at different stages of development making it difficult to use biochemical techniques to examine the activity of PDK1 pathways as thymocytes develop in situ. Herein, we use a single cell assay to quantify activation of the PDK1 target kinase ribosomal S6 kinase 1 (S6K1) in different murine thymocyte subsets immediately ex vivo. This technique allows an assessment of S6K1 activation as thymocytes respond to developmental stimuli in vivo. These studies reveal that only a small percentage of thymocytes show evidence for activation of PDK1 mediated signaling in situ. The thymic subpopulations that contain active PDK1/S6K1 are those known to be responding to signaling by the pre T cell receptor and the mature alpha/beta T cell antigen receptor (TCR). Moreover, loss of antigen receptor signaling in T cell progenitors that cannot rearrange their TCR beta locus prevents in vivo activation of S6K1. The present data identifying antigen receptor signaling as a key activator of PDK1 mediated signaling afford a molecular explanation for the important role of this molecule in T cells.

    Funded by: Wellcome Trust

    FEBS letters 2006;580;25;5845-50

  • Evaluation of approaches to generation of tissue-specific knock-in mice.

    Bayascas JR, Sakamoto K, Armit L, Arthur JS and Alessi DR

    MRC Protein Phosphorylation Unit and School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom. j.bayascas@dundee.ac.uk

    We explored three approaches to create tissue-specific knock-in mice by generating knock-in mice in which a substrate-docking site of the PDK1 protein kinase was ablated in Cre-expressing tissues in a way that prevented activation of one of its substrates, p70 ribosomal S6 kinase (S6K), but not another (protein kinase B (PKB)). Employing two of the approaches, termed the "heterozygous" and "minigene" methods, we generated mice in which Cre-expressing skeletal and cardiac muscle produced the mutant rather than wild type PDK1. Consistent with this, injection of these mice with insulin only induced activation of PKB but not S6K in muscle tissues. We have also demonstrated that insulin-stimulated glucose uptake proceeds normally in knock-in mice, consistent with the notion that PKB mediates this process. In contrast to conditional knock-out of PDK1 in muscle, the knock-in mice did not develop dilated cardiomyopathy, suggesting that PKB plays a key role in protecting mice from heart failure. The third knock-in strategy that was evaluated, termed the "inversion" method, did not proceed with high efficiency. We discuss the merits and disadvantages of each of the conditional knock-in approaches, along with the applications for which they may be most suited, and suggest how they could be further refined.

    Funded by: Medical Research Council: MC_U127015387, MC_U127088492

    The Journal of biological chemistry 2006;281;39;28772-81

  • Fused protein of deltaPKC activation loop and PDK1-interacting fragment (deltaAL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells.

    Seki T, Irie N, Nakamura K, Sakaue H, Ogawa W, Kasuga M, Yamamoto H, Ohmori S, Saito N and Sakai N

    Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.

    To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing deltaAL-PIF, basal phosphorylation level at the activation loop of PKBalpha, deltaPKC and gammaPKC was reduced, compared with that in control cells, suggesting that deltaAL-PIF functions as an inhibitory molecule for PDK1. deltaAL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of deltaAL-PIF on gammaPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing deltaAL-PIF for 3 days. These findings revealed that deltaAL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.

    Genes to cells : devoted to molecular & cellular mechanisms 2006;11;9;1051-70

  • Phosphoinositide-dependent kinase 1 targets protein kinase A in a pathway that regulates interleukin 4.

    Nirula A, Ho M, Phee H, Roose J and Weiss A

    Department of Medicine, The Rosalind Russell Medical Research Center for Arthritis and the Howard Hughes Medical Institute, University of California at San Francisco, CA 94143, USA.

    CD28 plays a critical role in T cell immune responses. Although the kinase Akt has been shown to act downstream of CD28 in T helper (Th)1 cytokine induction, it does not induce Th2 cytokines such as interleukin 4 (IL-4). We recently reported that phosphoinositide-dependent kinase 1 (PDK1) partially corrects the defect in IL-4 production present in CD28-deficient T cells, suggesting that PDK1 regulates IL-4 independently of Akt. We now describe a signaling pathway in which PDK1 targets IL-4 in the murine Th2 cell line D10. PDK1-mediated activation of this pathway is dependent on protein kinase A (PKA) and the nuclear factor of activated T cells (NFAT) P1 transcriptional element in the IL-4 promoter. PDK1 localizes to the immune synapse in a phosphatidylinositol 3-kinase-dependent manner, partially colocalizes with PKA at the synapse, and physically interacts with PKA. In RNA interference knockdown experiments, PDK1 is necessary for phosphorylation of PKA in T cells, as well as for activation of the IL-4 NFAT P1 element by the T cell receptor (TCR) and CD28. Phosphorylation of the critical PKA threonine residue is stimulated by engagement of TCR/CD28 via a PDK1-dependent mechanism. These findings together define a pathway linking the kinases PDK1 and PKA in the induction of the Th2 cytokine IL-4.

    The Journal of experimental medicine 2006;203;7;1733-44

  • Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of loss of beta cell mass.

    Hashimoto N, Kido Y, Uchida T, Asahara S, Shigeyama Y, Matsuda T, Takeda A, Tsuchihashi D, Nishizawa A, Ogawa W, Fujimoto Y, Okamura H, Arden KC, Herrera PL, Noda T and Kasuga M

    Department of Clinical Molecular Medicine, Division of Diabetes and Digestive and Kidney Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

    The total mass of islets of Langerhans is reduced in individuals with type 2 diabetes, possibly contributing to the pathogenesis of this condition. Although the regulation of islet mass is complex, recent studies have suggested the importance of a signaling pathway that includes the insulin or insulin-like growth factor-1 receptors, insulin receptor substrate and phosphatidylinositol (PI) 3-kinase. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a serine-threonine kinase that mediates signaling downstream of PI 3-kinase. Here we show that mice that lack PDK1 specifically in pancreatic beta cells (betaPdk1-/- mice) develop progressive hyperglycemia as a result of a loss of islet mass. The mice show reductions in islet density as well as in the number and size of cells. Haploinsufficiency of the gene for the transcription factor Foxo1 resulted in a marked increase in the number, but not the size, of cells and resulted in the restoration of glucose homeostasis in betaPdk1-/- mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.

    Nature genetics 2006;38;5;589-93

  • Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes.

    Kelly AP, Hinton HJ, Clarke RG and Cantrell DA

    Division of Cell Biology and Immunology, School of Life Sciences, WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

    In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.

    Funded by: Wellcome Trust

    FEBS letters 2006;580;8;2135-40

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

    Nadra K, Anghel SI, Joye E, Tan NS, Basu-Modak S, Trono D, Wahli W and Desvergne B

    Center for Integrative Genomics, University of Lausanne, CH-1015 Lausanne, Switzerland.

    Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

    Molecular and cellular biology 2006;26;8;3266-81

  • Role of hepatic STAT3 in brain-insulin action on hepatic glucose production.

    Inoue H, Ogawa W, Asakawa A, Okamoto Y, Nishizawa A, Matsumoto M, Teshigawara K, Matsuki Y, Watanabe E, Hiramatsu R, Notohara K, Katayose K, Okamura H, Kahn CR, Noda T, Takeda K, Akira S, Inui A and Kasuga M

    Department of Clinical Molecular Medicine, Division of Diabetes and Digestive and Kidney Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

    STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.

    Cell metabolism 2006;3;4;267-75

  • 3-phosphoinositide-dependent protein kinase-1 activates the peroxisome proliferator-activated receptor-gamma and promotes adipocyte differentiation.

    Yin Y, Yuan H, Wang C, Pattabiraman N, Rao M, Pestell RG and Glazer RI

    Georgetown University School of Medicine, Washington, D.C. 20007, USA.

    Adipocyte differentiation is regulated largely through the actions of the peroxisome proliferator-activated receptor (PPAR) gamma nuclear receptor and the insulin signaling pathway. 3-phosphoinositide-dependent protein kinase-1 (PDK1) serves as a critical regulatory point in insulin signaling through its ability to phosphorylate the activation loop of several protein kinase families. The present study was undertaken to determine the interrelationships between the PDK1 and PPARgamma signaling pathways, and their association with adipocyte differentiation. Coexpression of PDK1 and PPARgamma1 in 293T cells stimulated PPARgamma response element-dependent reporter gene activity in either the presence or absence of ligand. PDK1-mediated stimulation of PPARgamma1 activity was comparable in magnitude to the coactivator activated in breast cancer-1, and was blocked by either the corepressor silencing mediator of retinoid and thyroid hormone receptor or dominant-negative PAX8-PPARgamma1. Heterologous Gal4-PPARgamma1 assays indicated that PDK1 interacted with the ligand binding domain, and physically associated with PPARgamma1; however, PDK1-mediated stimulation was not dependent on phosphorylation of PPARgamma1 by PDK1. PDK1 stimulatory activity was eliminated by mutation of the alpha-helical hydrophobic motifs in PDK1, L(268)XII, and V(313)XXLL, and expression of the alpha-helical region encompassing these motifs stimulated PPARgamma response element-dependent transcription. PDK1-PPARgamma interaction was confirmed by chromatin immunoprecipitation analysis of the lipoprotein lipase and adipocyte fatty acid-binding protein promoters. In cells expressing PDK1 and PPARgamma, binding to PPARgamma response elements occurred, which was enhanced by treatment with a PPARgamma agonist. Expression of PDK1 in 3T3-L1 or COMMA-1D mammary epithelial cells promoted adipocyte differentiation in the presence of a PPARgamma agonist that was comparable to the response of PPARgamma1-transfected cells in the presence of agonist; expression of PDK1 and PPARgamma resulted in a synergistic effect. Adipocyte differentiation in the presence of a PPARgamma agonist was markedly attenuated in PDK1 null cells. These results suggest that PDK1 can function as a PPARgamma1 coactivator independently of its catalytic activity and establishes an important mechanistic link between adipocyte differentiation and the insulin signaling pathway.

    Funded by: NCI NIH HHS: N01CN15017, R01CA70896, R01CA75503, R01CA81565, R01CA86071, R01CA86072

    Molecular endocrinology (Baltimore, Md.) 2006;20;2;268-78

  • 3-phosphoinositide-dependent protein kinase-1 (PDK1) promotes invasion and activation of matrix metalloproteinases.

    Xie Z, Yuan H, Yin Y, Zeng X, Bai R and Glazer RI

    Department of Oncology, Georgetown University School of Medicine, and Lombardi Comprehensive Cancer Center, Washington, DC, USA. xiez@niaid.nih.gov

    Background: Metastasis is a major cause of morbidity and mortality in breast cancer with tumor cell invasion playing a crucial role in the metastatic process. PDK1 is a key molecule that couples PI3K to cell proliferation and survival signals in response to growth factor receptor activation, and is oncogenic when expressed in mouse mammary epithelial cells. We now present evidence showing that PDK1-expressing cells exhibit enhanced anchorage-dependent and -independent cell growth and are highly invasive when grown on Matrigel. These properties correlate with induction of MMP-2 activity, increased MT1-MMP expression and a unique gene expression profile.

    Methods: Invasion assays in Matrigel, MMP-2 zymogram analysis, gene microarray analysis and mammary isografts were used to characterize the invasive and proliferative function of cells expressing PDK1. Tissue microarray analysis of human breast cancers was used to measure PDK1 expression in invasive tumors by IHC.

    Results: Enhanced invasion on Matrigel in PDK1-expressing cells was accompanied by increased MMP-2 activity resulting from stabilization against proteasomal degradation. Increased MMP-2 activity was accompanied by elevated levels of MT1-MMP, which is involved in generating active MMP-2. Gene microarray analysis identified increased expression of the ECM-associated genes decorin and type I procollagen, whose gene products are substrates of MT1-MMP. Mammary fat pad isografts of PDK1-expressing cells produced invasive adenocarcinomas. Tissue microarray analysis of human invasive breast cancer indicated that PDK1pSer241 was strongly expressed in 90% of samples.

    Conclusion: These results indicate that PDK1 serves as an important effector of mammary epithelial cell growth and invasion in the transformed phenotype. PDK1 mediates its effect in part by MT1-MMP induction, which in turn activates MMP-2 and modulates the ECM proteins decorin and collagen. The presence of increased PDK1 expression in the majority of invasive breast cancers suggests its importance in the metastatic process.

    Funded by: NCI NIH HHS: R01CA81565

    BMC cancer 2006;6;77

  • Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling.

    Fiory F, Alberobello AT, Miele C, Oriente F, Esposito I, Corbo V, Ruvo M, Tizzano B, Rasmussen TE, Gammeltoft S, Formisano P and Beguinot F

    Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Italy.

    In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.

    Molecular and cellular biology 2005;25;24;10803-14

  • In vivo role of the phosphate groove of PDK1 defined by knockin mutation.

    Collins BJ, Deak M, Murray-Tait V, Storey KG and Alessi DR

    MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dundee, DD1 5EH, UK. b.j.collins@dundee.ac.uk

    AGC kinases are mediators of signalling responses stimulated by agonists and are activated following phosphorylation at their T-loop residue by the 3-phosphoinositide-dependent protein kinase-1 (PDK1). Agonists stimulate the activation of the AGC kinases p70 ribosomal S6 kinase (S6K), p90 ribosomal S6 kinase (RSK) and serum and glucocorticoid-induced protein kinase (SGK), by inducing the phosphorylation of these enzymes at a non-catalytic regulatory site termed the hydrophobic motif. This creates a high-affinity docking site enabling PDK1 to bind and phosphorylate the T-loop of these enzymes. The site that interacts with these substrates is located on the small lobe of the catalytic domain of PDK1 and is composed of a hydrophobic groove next to a basic phosphate groove. The disruption of the hydrophobic groove ablates activation of S6K, RSK and SGK, but the role of the phosphate groove in regulating the function of PDK1 has not been explored in vivo. We generated knockin ES cells, in which both copies of the gene encoding PDK1 were altered to express a form of PDK1 that retains catalytic activity and integrity of the hydrophobic groove, but in which the phosphate groove was disrupted. The knockin ES cells were viable, mutant PDK1 was expressed at normal levels and IGF1 induced activation of protein kinase B (PKB/Akt), which is a PDK1 substrate that does not require hydrophobic motif phosphorylation to be activated. In the phosphate-groove-knockin ES cells, the activation of S6K, RSK and SGK by agonists, although markedly impaired, was not abolished. PDK1 also phosphorylates the T-loop of protein kinase C (PKC) isoforms, which stabilizes these enzymes. However, in contrast to S6K, RSK and SGK, hydrophobic motif phosphorylation of these enzymes is not thought to control their activation by PDK1. Consistent with this notion, we employed appropriate PDK1-knockin ES cells to demonstrate that the hydrophobic groove of PDK1, but not the phosphate groove, is required for the stabilization of PKC isoforms. These findings provide genetic evidence that the phosphate groove of PDK1 is required for maximal activation of isoforms of S6K, SGK and RSK, but not PKC. We also found that no live births of homozygous phosphate-groove-knockin mice are observed, indicating a key role for this regulatory motif in normal development. The knockin embryos develop to a greater extent than PDK1-knockout and hydrophobic-groove-knockin embryos, which died between E9.5-E11.5. The knockin embryos are observed until E19.5 and displayed general growth retardation and craniofacial developmental defects.

    Funded by: Wellcome Trust: WT065629

    Journal of cell science 2005;118;Pt 21;5023-34

  • Hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN(+/-) mice.

    Bayascas JR, Leslie NR, Parsons R, Fleming S and Alessi DR

    MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom. j.bayascas@dundee.ac.uk

    Many cancers possess elevated levels of PtdIns(3,4,5)P(3), the second messenger that induces activation of the protein kinases PKB/Akt and S6K and thereby stimulates cell proliferation, growth, and survival. The importance of this pathway in tumorigenesis has been highlighted by the finding that PTEN, the lipid phosphatase that breaks down PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), is frequently mutated in human cancer. Cells lacking PTEN possess elevated levels of PtdIns(3,4,5)P(3), PKB, and S6K activity and heterozygous PTEN(+/-) mice develop a variety of tumors. Knockout of PKBalpha in PTEN-deficient cells reduces aggressive growth and promotes apoptosis, whereas treatment of PTEN(+/-) mice with rapamycin, an inhibitor of the activation of S6K, reduces neoplasia. We explored the importance of PDK1, the protein kinase that activates PKB and S6K, in mediating tumorigenesis caused by the deletion of PTEN. We demonstrate that reducing the expression of PDK1 in PTEN(+/-) mice, markedly protects these animals from developing a wide range of tumors. Our findings provide genetic evidence that PDK1 is a key effector in mediating neoplasia resulting from loss of PTEN and also validate PDK1 as a promising anticancer target for the prevention of tumors that possess elevated PKB and S6K activity.

    Funded by: NCI NIH HHS: R01 CA082783, R01 CA082783-06

    Current biology : CB 2005;15;20;1839-46

  • RGS4 and RGS5 are in vivo substrates of the N-end rule pathway.

    Lee MJ, Tasaki T, Moroi K, An JY, Kimura S, Davydov IV and Kwon YT

    Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, 3501 Terrace Street, Pittsburgh, PA 15261, USA.

    The ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1-/- embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and/or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1-/- embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and beta-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O2-ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;42;15030-5

  • Role of the PDK1-PKB-GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart.

    Mora A, Sakamoto K, McManus EJ and Alessi DR

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.

    In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.

    FEBS letters 2005;579;17;3632-8

  • Epithelium-mesenchyme interactions control the activity of peroxisome proliferator-activated receptor beta/delta during hair follicle development.

    Di-Poï N, Ng CY, Tan NS, Yang Z, Hemmings BA, Desvergne B, Michalik L and Wahli W

    Center for Integrative Genomics, Biology Building, University of Lausanne, CH-1015 Lausanne, Switzerland.

    Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

    Molecular and cellular biology 2005;25;5;1696-712

  • Deficiency of PDK1 in liver results in glucose intolerance, impairment of insulin-regulated gene expression and liver failure.

    Mora A, Lipina C, Tronche F, Sutherland C and Alessi DR

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. a.m.corral@dundee.ac.uk

    The liver plays an important role in insulin-regulated glucose homoeostasis. To study the function of the PDK1 (3-phosphoinositide-dependent protein kinase-1) signalling pathway in mediating insulin's actions in the liver, we employed CRE recombinase/loxP technology to generate L(liver)-PDK1-/- mice, which lack expression of PDK1 in hepatocytes and in which insulin failed to induce activation of PKB in liver. The L-PDK1-/- mice were not insulin-intolerant, possessed normal levels of blood glucose and insulin under normal feeding conditions, but were markedly glucose-intolerant when injected with glucose. The L-PDK1-/- mice also possessed 10-fold lower levels of hepatic glycogen compared with control littermates, and were unable to normalize their blood glucose levels within 2 h after injection of insulin. The glucose intolerance of the L-PDK1-/- mice may be due to an inability of glucose to suppress hepatic glucose output through the gluconeogenic pathway, since the mRNA encoding hepatic PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase) and SREBP1 (sterol-regulatory-element-binding protein 1), which regulate gluconeogenesis, are no longer controlled by feeding. Furthermore, three other insulin-controlled genes, namely IGFBP1 (insulin-like-growth-factor-binding protein-1), IRS2 (insulin receptor substrate 2) and glucokinase, were regulated abnormally by feeding in the liver of PDK1-deficient mice. Finally, the L-PDK1-/- mice died between 4-16 weeks of age due to liver failure. These results establish that the PDK1 signalling pathway plays an important role in regulating glucose homoeostasis and controlling expression of insulin-regulated genes. They suggest that a deficiency of the PDK1 pathway in the liver could contribute to development of diabetes, as well as to liver failure.

    The Biochemical journal 2005;385;Pt 3;639-48

  • Roles of PDK-1 and PKN in regulating cell migration and cortical actin formation of PTEN-knockout cells.

    Lim MA, Yang L, Zheng Y, Wu H, Dong LQ and Liu F

    Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.

    Mutations in the tumor suppressor protein PTEN (phosphatase and tensin homologue deleted on chromosome 10) enhance cell migration, yet the underlying molecular mechanisms remain largely uncharacterized. Loss of PTEN in mouse embryonic fibroblasts (MEFs) correlates with striking cortical actin accumulation. However, how loss of PTEN leads to cortical actin formation and whether the presence of cortical actin contributes to the increased cell migration are unclear. Here we show that overexpression of dominant-negative forms of (DN) PTEN, RhoA or its kinase-dead (KD) effector, PKN, inhibited cortical actin formation, indicating that cortical actin of Pten(-/-) MEFs is mediated by the PTEN/Rho/PKN pathway. However, neither DN RhoA nor KD PKN inhibited the enhanced migration of Pten(-/-) cells, in contrast to the inhibitory effect of DN Rac. In agreement with the previous observation that DN Akt inhibits migration of Pten(-/-) cells, we demonstrate here that overexpression of KD PDK-1, the Akt kinase, reduces Pten(-/-) cell migration. Furthermore, overexpression of DN forms of Akt, Rac, or PDK-1, all of which inhibit migration of Pten(-/-) cells, had no effect on cortical actin accumulation. Our findings suggest that PDK-1/Akt signaling pathway plays a major role in regulating cell migration induced by PTEN deficiency.

    Funded by: NIDDK NIH HHS: DK56166

    Oncogene 2004;23;58;9348-58

  • Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

    Watahiki A, Waki K, Hayatsu N, Shiraki T, Kondo S, Nakamura M, Sasaki D, Arakawa T, Kawai J, Harbers M, Hayashizaki Y and Carninci P

    Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.

    It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

    Nature methods 2004;1;3;233-9

  • Activation of Akt/PDK signaling in macrophages upon binding of receptor-recognized forms of alpha2-macroglobulin to its cellular receptor: effect of silencing the CREB gene.

    Misra UK and Pizzo SV

    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Macrophage binding of receptor-recognized forms of alpha2-macrogobulin (alpha2M*) significantly increases cAMP, CREB, and activated CREB. We have now examined the participation of the PI 3-kinase/PDK/Akt/p70s6k signaling cascade in alpha2M*-induced cellular proliferation and also studied the role of CREB in these events. Exposure of cells to alpha2M* caused an approximately 2-fold increase in CREB and its phosphorylation at Ser133, phosphorylation of the regulatory subunit of PI 3-kinase, Akt phosphorylation at Ser473 or Thr308, and phosphorylated 70s6k. Silencing of the CREB gene with dsRNA homologous in sequence to the target gene, markedly reduced the levels of CREB mRNA activation of CREB, PI 3-kinase, Akt, and p70s6k in alpha2M*-stimulated macrophages. We conclude that in murine peritoneal macrophages, alpha2M*-induced increase of cAMP is involved in cellular proliferation and this process is mediated by the PI 3-kinase signaling cascade.

    Funded by: NHLBI NIH HHS: HL-24066

    Journal of cellular biochemistry 2004;93;5;1020-32

  • Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis.

    Yamazaki T, Akada T, Niizeki O, Suzuki T, Miyashita H and Sato Y

    Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aobaku, Sendai 980-8575, Japan.

    Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) plays an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs). Here we characterize the mechanism by which PILSAP regulates the vascular endothelial growth factor (VEGF)-stimulated proliferation of ECs. The specific elimination of PILSAP expression or its enzymatic activity inhibited VEGF-stimulated G1/S transition in ECs. This G1 arrest correlated with reduced cyclin dependent kinase 4/6 (CDK4/6) activity and retinoblastoma (Rb) protein phosphorylation. Analyses of signaling molecules upstream of CDK4/6 revealed that S6 kinase (S6K) activation was affected by PILSAP, whereas that of phosphatidylinositol-3 kinase (PI3K), Akt, and extracellular signal-related kinase 1/2 (ERK1/2) was not. We further demonstrated that PILSAP bound phosphatidylinositol-dependent kinase 1 (PDK1) and removed 9 amino acids from its N-terminus, which allowed S6K to associate with PDK1 and PILSAP upon VEGF stimulation. We constructed mutant PILSAP, which lacked the aminopeptidase activity but bound PDK1. Mutant PILSAP abrogated S6K activation upon VEGF stimulation in a dominant-negative manner. An N-terminal truncated form of PDK1 abolished the dominant-negative effect of mutant PILSAP. Finally, the introduction of a mutated PILSAP gene in ECs inhibited angiogenesis and retarded tumor growth in vivo. These results indicate that PILSAP plays a crucial role in the cell cycle progression of ECs and angiogenesis via the binding and modification of PDK1.

    Blood 2004;104;8;2345-52

  • Nonobese diabetic CD4 lymphocytosis maps outside the MHC locus on chromosome 17.

    Koarada S, Wu Y, Yim YS, Wakeland EW and Ridgway WM

    Division of Rheumatology and Immunology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

    Genetic control of homeostasis of peripheral CD4+ lymphocyte levels is incompletely understood. Recent genome scans have linked mouse peripheral CD4 levels to chromosome 17, with strongest linkage to the Ea region. Nonobese diabetic (NOD) mice demonstrate peripheral T-cell lymphocytosis, and previous studies also suggested that the MHC region might control this phenotype. Here we confirm that loci on Chr 17 control NOD peripheral CD4 lymphocytosis. An elevated NOD CD4:CD8 ratio maps to the same region, and we show it is due to increased numbers of CD4+ cells. However, using NOD MHC congenic mice, we demonstrate that the MHC region is excluded, and that NOD peripheral lymphocytosis is controlled by genetic intervals adjacent to the MHC region on Chr 17.

    Funded by: NIAID NIH HHS: U19 AI056374, U19 AI056374-010002; NIDDK NIH HHS: R01 DK060714, R01 DK060714-04, R01 DK6071; PHS HHS: RFA A102

    Immunogenetics 2004;56;5;333-7

  • The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation.

    McManus EJ, Collins BJ, Ashby PR, Prescott AR, Murray-Tait V, Armit LJ, Arthur JS and Alessi DR

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK. e.j.mcmanus@dundee.ac.uk

    We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-tris-phosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.

    Funded by: Wellcome Trust: 065629

    The EMBO journal 2004;23;10;2071-82

  • The serine kinase phosphoinositide-dependent kinase 1 (PDK1) regulates T cell development.

    Hinton HJ, Alessi DR and Cantrell DA

    Lymphocyte Activation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

    T lymphocyte activation is associated with activation of diverse AGC serine kinases (named after family members protein kinase A, protein kinase G and protein kinase C). It has been difficult to assess the function of these molecules in T cell development with simple gene-deletion strategies because different isoforms of AGC kinases are coexpressed in the thymus and have overlapping, redundant functions. To circumvent these problems, we explored the consequences of genetic manipulation of phosphoinositide-dependent kinase 1 (PDK1), a rate-limiting 'upstream' activator of AGC kinases. Here we analyzed the effect of PDK1 deletion on T lineage development. We also assessed the consequences of reducing PDK1 levels to 10% of normal. Complete PDK1 loss blocked T cell differentiation in the thymus, whereas reduced PDK1 expression allowed T cell differentiation but blocked proliferative expansion. These studies show that AGC family kinases are essential for T cell development.

    Nature immunology 2004;5;5;539-45

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Nuclear translocation of 3'-phosphoinositide-dependent protein kinase 1 (PDK-1): a potential regulatory mechanism for PDK-1 function.

    Lim MA, Kikani CK, Wick MJ and Dong LQ

    Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78229, USA.

    3'-Phosphoinositide-dependent protein kinase 1 (PDK-1) phosphorylates and activates members of the AGC protein kinase family and plays an important role in the regulation of cell survival, differentiation, and proliferation. However, how PDK-1 is regulated in cells remains elusive. In this study, we demonstrated that PDK-1 can shuttle between the cytoplasm and nucleus. Treatment of cells with leptomycin B, a nuclear export inhibitor, results in a nuclear accumulation of PDK-1. PDK-1 nuclear localization is increased by insulin, and this process is inhibited by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Consistent with the idea that PDK-1 nuclear translocation is regulated by the PI3-kinase signaling pathway, PDK-1 nuclear localization is increased in cells deficient of PTEN (phosphatase and tensin homologue deleted on chromosome 10). Deletion mapping and mutagenesis studies unveiled that presence of a functional nuclear export signal (NES) in mouse PDK-1 located at amino acid residues 382 to 391. Overexpression of constitutively nuclear PDK-1, which retained autophosphorylation at Ser-244 in the activation loop in cells and its kinase activity in vitro, led to increased phosphorylation of the predominantly nuclear PDK-1 substrate p70 S6KbetaI. However, the ability of constitutively nuclear PDK-1 to induce anchorage-independent growth and to protect against UV-induced apoptosis is greatly diminished compared with the wild-type enzyme. Taken together, these findings suggest that nuclear translocation may be a mechanism to sequestrate PDK-1 from activation of the cytosolic signaling pathways and that this process may play an important role in regulating PDK-1-mediated cell signaling and function.

    Funded by: NIDDK NIH HHS: DK56166, R01 DK056166

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14006-11

  • Mouse 3-phosphoinositide-dependent protein kinase-1 undergoes dimerization and trans-phosphorylation in the activation loop.

    Wick MJ, Ramos FJ, Chen H, Quon MJ, Dong LQ and Liu F

    Departments of Pharmacology, Biochemistry, and Cellular & Structural Biology, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.

    Activation of mouse 3-phosphoinositide-dependent protein kinase-1 (mPDK1) requires phosphorylation at a conserved serine residue, Ser244, in the activation loop. However, the mechanism by which mPDK1 is phosphorylated at this site remains unclear. We have found that kinase-defective mPDK1 (mPDK1KD), but not a kinase-defective mPDK1 in which Ser244 was replaced with alanine (mPDK1KD/S244A), is significantly phosphorylated in intact cells and is a direct substrate of wild-type mPDK1 fused to the yellow fluorescence protein. Phosphoamino acid analysis and phosphopeptide mapping studies revealed that mPDK1 trans-autophosphorylation occurred mainly on Ser244. On the other hand, Ser399 and Thr516, two recently identified autophosphorylation sites of mPDK1, are phosphorylated primarily through a cis mechanism. In vivo labeling studies revealed that insulin stimulated both mPDK1KD and mPDK1KD/S244A phosphorylation in Chinese hamster ovary cells overexpressing the insulin receptor. However, Western blot analysis using a phosphospecific antibody revealed no increase in insulin-stimulated phosphorylation of Ser244 in these cells overexpressing mPDK1. mPDK1 undergoes dimerization in cells and this self-association is enhanced by kinase inactivation. Deletion of the extreme C terminus disrupts mPDK1 dimerization and Ser244 trans-phosphorylation, suggesting that dimerization is important for mPDK1 trans-phosphorylation. Taken together, our results show that mPDK1 autophosphorylation occurs at multiple sites through both cis and trans mechanisms and suggest that dimerization and trans-phosphorylation may serve as mechanisms to regulate PDK1 activity in cells.

    Funded by: NIDDK NIH HHS: DK56166

    The Journal of biological chemistry 2003;278;44;42913-9

  • Requirement for 3-phosphoinositide-kependent dinase-1 (PDK-1) in insulin-induced glucose uptake in immortalized brown adipocytes.

    Sakaue H, Nishizawa A, Ogawa W, Teshigawara K, Mori T, Takashima Y, Noda T and Kasuga M

    Division of Diabetes and Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

    To provide insight into the physiological importance of 3-phosphoinositide-dependent kinase-1 (PDK-1) in the metabolic actions of insulin, we have generated mice that harbor a PDK-1 gene containing LoxP sites (PDK-1(lox/lox) mice) and established immortalized brown preadipocyte cell lines both from these animals and from wild-type mice. Exposure to appropriate hormonal inducers resulted in the differentiation of >80% of the immortalized brown preadipocytes derived from both types of mice into mature adipocytes. Introduction of the Cre recombinase with the use of adenovirus-mediated gene transfer induced a dose-dependent decrease in the abundance of PDK-1 in PDK-1(lox/lox) adipocytes but not in the wild-type cells. In Cre-expressing PDK-1(lox/lox) adipocytes in which the abundance of PDK-1 was reduced by approximately 85%, the insulin-induced phosphorylation both of Akt on threonine 308 and of p70 S6 kinase on threonine-389 was markedly inhibited. The phosphorylation both of Akt on serine 473 and of p42 and p44 isoforms of mitogen-activated protein kinase induced by insulin was not affected by Cre expression, indicating that the latter specifically inhibits PDK-1-dependent signaling. Both glucose uptake and the translocation of glucose transporter 4 to the plasma membrane induced by insulin as well as glucose uptake induced by a constitutively active form of phosphoinositide 3-kinase were also greatly inhibited by Cre expression in PDK-1(lox/lox) adipocytes. Phosphorylation of AMP-activated protein kinase and glucose uptake induced by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) were not affected by Cre expression in PDK-1(lox/lox) adipocytes. These results indicate that PDK-1 is essential for insulin-induced glucose uptake in adipocytes.

    The Journal of biological chemistry 2003;278;40;38870-4

  • Deficiency of PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia.

    Mora A, Davies AM, Bertrand L, Sharif I, Budas GR, Jovanović S, Mouton V, Kahn CR, Lucocq JM, Gray GA, Jovanović A and Alessi DR

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

    We employed Cre/loxP technology to generate mPDK1(-/-) mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6-phosphofructo-2-kinase and production of fructose 2,6-bisphosphate, in the hearts of mPDK1(-/-) mice, consistent with PDK1 mediating these processes. All mPDK1(-/-) mice died suddenly between 5 and 11 weeks of age. The mPDK1(-/-) animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1(-/-) muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1(-/-) mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single-cell assay we found that cardiomyocytes from mPDK1(-/-) mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a deficiency of the PDK1 pathway might contribute to development of cardiac disease in humans.

    Funded by: Biotechnology and Biological Sciences Research Council: C15048; British Heart Foundation: PG/02/117/14488; Wellcome Trust: 059528/Z/99/Z/JMW/CP/JF

    The EMBO journal 2003;22;18;4666-76

  • Transformation of mammary epithelial cells by 3-phosphoinositide- dependent protein kinase-1 activates beta-catenin and c-Myc, and down-regulates caveolin-1.

    Xie Z, Zeng X, Waldman T and Glazer RI

    Department of Oncology, Georgetown University School of Medicine, Washington, DC 20057, USA.

    3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a pivotal role in coupling growth factor receptor signaling to tumor cell proliferation, survival, and invasion. Protein kinase C (PKC) alpha, but not Akt1, was found previously to be downstream of PDK1-mediated transformation of mammary epithelial cells. To determine the basis for its oncogenic activity, signal transduction pathways mediated by PDK1 in mammary epithelial cells were investigated. beta-Catenin/T-cell factor-dependent promoter activity was markedly activated in PDK1- and PKCalpha-expressing cells, but not in Akt1-expressing cells, which resulted in increased levels of the beta-catenin/T-cell factor target genes c-myc and cyclin D1. In contrast, caveolin-1, of which the transcription is suppressed by c-myc, was down-regulated in PDK1- and PKCalpha-expressing, but not in Akt1-expressing cells. Analysis of 16 breast cancer cell lines established that caveolin-1 expression was either absent or reduced compared with breast epithelial cells, and that PDK1 was elevated in all of the cell lines. Interestingly, all of the cell lines known to be invasive expressed caveolin-1 to some degree, whereas, 5 of 6 cell lines that are not invasive did not express caveolin-1. Therefore, it appears that a concomitant gain of c-myc function and a loss or reduction of caveolin-1 are major determinants of PDK1- and PKCalpha-mediated mammary oncogenesis.

    Funded by: NCI NIH HHS: CA81565

    Cancer research 2003;63;17;5370-5

  • Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1.

    Torbett NE, Casamassima A and Parker PJ

    Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

    Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1). We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress. Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation.

    The Journal of biological chemistry 2003;278;34;32344-51

  • A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome.

    Hansen J, Floss T, Van Sloun P, Füchtbauer EM, Vauti F, Arnold HH, Schnütgen F, Wurst W, von Melchner H and Ruiz P

    Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, D-85764 Neuherberg, Germany.

    A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration "hot spots." Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;17;9918-22

  • In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation.

    Collins BJ, Deak M, Arthur JS, Armit LJ and Alessi DR

    MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. b.j.collins@dundee.ac.uk

    PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.

    Funded by: Wellcome Trust: 065629

    The EMBO journal 2003;22;16;4202-11

  • Expression pattern of the Rsk2, Rsk4 and Pdk1 genes during murine embryogenesis.

    Kohn M, Hameister H, Vogel M and Kehrer-Sawatzki H

    Department of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.

    The ribosomal S6 kinase family members RSK2 (RPS6KA3) and RSK4 (RPS6KA6) belong to the group of X chromosomal genes, in which defects cause unspecific mental retardation (MRX) in humans. In this study, we investigated the spatiotemporal expression pattern of these genes during mouse development with emphasis to midgestation stages. Additionally, we analyzed the expression of the phosphoinositide-dependent protein kinase-1 gene, Pdk1 (Pspk1), which is essential for the activation of Rsk family members and thus regulates their function. During midgestation we observed specifically enhanced expression of Rsk2 first in somites, later restricted to the dermatomyotome of the somites, then in the sensory ganglia of cranial nerves and in the dorsal root ganglia of the spinal nerves. High Rsk2 expression in the cranial nerve ganglia persists throughout development and is correlated with Pdk1 expression. In the brain of 2-day-old mice, Pdk1 is expressed in the cortical plate of the cerebral cortex and in the stratum pyramidale of the hippocampus, whereas Rsk2 expression is lower in these structures. For Rsk4 ubiquitous expression at lower levels was observed throughout development.

    Gene expression patterns : GEP 2003;3;2;173-7

  • BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

    Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, Harper CA, Meng EC, Lee RE, Yee A, L'Italien L, Chuang PT, Young SG, Skarnes WC, Babbitt PC and Ferrin TE

    Department of Pharmaceutical Chemistry, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

    The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

    Funded by: NCRR NIH HHS: P41 RR001081, P41 RR01081; NHLBI NIH HHS: U01 HL066621, U01 HL66621

    Nucleic acids research 2003;31;1;278-81

  • Phosphorylation of the catalytic subunit of protein kinase A. Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1.

    Moore MJ, Kanter JR, Jones KC and Taylor SS

    Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654, USA.

    The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA.

    Funded by: NIDDK NIH HHS: DK07233; NIGMS NIH HHS: GM19301

    The Journal of biological chemistry 2002;277;49;47878-84

  • The Rb-family protein p107 inhibits translation by a PDK1-dependent mechanism.

    Makris C, Voisin L, Giasson E, Tudan C, Kaplan DR and Meloche S

    Institut de recherches cliniques de Montréal and Department of Pharmacology, University of Montreal, 110 Pine Avenue West, Montreal, Quebec H2W 1R7, Canada.

    The Rb family of proteins, which consists of Rb, p107 and p130, are critical regulators of cell proliferation. In addition to their inhibitory effects on cell cycle progression, Rb-family proteins repress transcription by RNA polymerases I and III, and may therefore restrain cell growth. However, it is not known if Rb, p107 or p130 have direct effects on protein synthesis. Here we report that ectopic expression of p107 in rat fibroblasts markedly attenuates the stimulation of mRNA translation and global protein synthesis by serum growth factors. This effect is associated with a reduction in the phosphorylation and activation of the serine-threonine kinases Akt1 and p70 S6 kinase (S6K1), two downstream targets of phosphoinositide-dependent kinase 1 (PDK1). We show that overexpression of p107 interferes with the recruitment of PDK1 to the plasma membrane in response to growth factors. Overexpression of PDK1 restores the defect in translation elicited by p107. These results suggest that p107 restricts cell growth by interfering with the phosphoinositide 3-kinase (PI3K) signaling pathway.

    Oncogene 2002;21;51;7891-6

  • 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

  • Essential role of PDK1 in regulating cell size and development in mice.

    Lawlor MA, Mora A, Ashby PR, Williams MR, Murray-Tait V, Malone L, Prescott AR, Lucocq JM and Alessi DR

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. m.a.lawlor@dundee.ac.uk

    PDK1 functions as a master kinase, phosphorylating and activating PKB/Akt, S6K and RSK. To learn more about the roles of PDK1, we generated mice that either lack PDK1 or possess PDK1 hypomorphic alleles, expressing only approximately 10% of the normal level of PDK1. PDK1(-/-) embryos die at embryonic day 9.5, displaying multiple abnormalities including lack of somites, forebrain and neural crest derived tissues; however, development of hind- and midbrain proceed relatively normally. In contrast, hypomorphic PDK1 mice are viable and fertile, and insulin injection induces the normal activation of PKB, S6K and RSK. Nevertheless, these mice are 40-50% smaller than control animals. The organ volumes from the PDK1 hypomorphic mice are reduced proportionately. We also establish that the volume of a number of PDK1-deficient cells is reduced by 35-60%, and show that PDK1 deficiency does not affect cell number, nuclear size or proliferation. We provide genetic evidence that PDK1 is essential for mouse embryonic development, and regulates cell size independently of cell number or proliferation, as well as insulin's ability to activate PKB, S6K and RSK.

    The EMBO journal 2002;21;14;3728-38

  • Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes.

    Yamada T, Katagiri H, Asano T, Tsuru M, Inukai K, Ono H, Kodama T, Kikuchi M and Oka Y

    Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Japan.

    To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.

    American journal of physiology. Endocrinology and metabolism 2002;282;6;E1385-94

  • Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta.

    Dong LQ, Ramos FJ, Wick MJ, Lim MA, Guo Z, Strong R, Richardson A and Liu F

    Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX 78229, USA. dongq@uthscsa.edu

    3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.

    Funded by: NIA NIH HHS: P03 AG13319; NIDDK NIH HHS: DK52543, DK56166

    Biochemical and biophysical research communications 2002;294;1;136-44

  • Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells.

    Wick MJ, Wick KR, Chen H, He H, Dong LQ, Quon MJ and Liu F

    Department of Pharmacology, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.

    3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.

    Funded by: NIA NIH HHS: 2T32AG00205-11; NIDDK NIH HHS: DK56166

    The Journal of biological chemistry 2002;277;19;16632-8

  • Regulation of novel protein kinase C epsilon by phosphorylation.

    Cenni V, Döppler H, Sonnenburg ED, Maraldi N, Newton AC and Toker A

    Boston Biomedical Research Institute, Watertown, MA 02472, USA.

    The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKCepsilon isoform, these are Thr(566) in the activation loop, Thr(710) in the turn motif and Ser(729) in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKCepsilon kinase activity in controlling the phosphorylation of Thr(566) and Ser(729). In NIH 3T3 fibroblasts, PKCepsilon migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr(566) and Ser(729). Cells transfected with an active PDK-1 allele also resulted in increased PKCepsilon Thr(566) and Ser(729) phosphorylation, whereas an active myristoylated PKCepsilon mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKCepsilon were not phosphorylated at Ser(729) in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr(566), an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKCepsilon in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P(3), concomitant with an increase in Ser(729) phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

    Funded by: NCI NIH HHS: CA75134; NIGMS NIH HHS: GM43154

    The Biochemical journal 2002;363;Pt 3;537-45

  • Expression cloning of protein targets for 3-phosphorylated phosphoinositides.

    Rao VR, Corradetti MN, Chen J, Peng J, Yuan J, Prestwich GD and Brugge JS

    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The phosphatidylinositol 3-kinase (PI 3'-K) family of lipid kinases play a critical role in cell proliferation, survival, vesicle trafficking, motility, cytoskeletal rearrangements, and oncogenesis. To identify downstream effectors of PI 3'-K, we developed a novel screen to isolate proteins that bind to the major products of PI 3'-K: phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P(2)) and PtdIns-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)). This screen uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitro transcription/translation reactions. The feasibility of the screen was initially demonstrated using avidin-coated beads prebound to biotinylated PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3) to specifically isolate the pleckstrin homology domain of the serine/threonine kinase Akt. We then demonstrated the utility of this technique in isolating novel 3'-phosphorylated phosphatidylinositol (3'-PPI)-binding proteins through the preliminary screening of in vitro transcribed/translated cDNAs from a small pool expression library derived from mouse spleen. Three proteins were isolated that bound specifically to 3'PPIs. Two of these proteins have been previously characterized as PIP3BP/p42(IP4) and the PtdIns-3,4,5-P(3)-dependent serine/threonine kinase phosphoinositide-dependent kinase 1. The third protein is a novel protein that contains only a Src homology 2 domain and a pleckstrin homology domain; this protein has a higher specificity for both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) than for PtdIns-4, 5-bisphosphate. Transcripts of this novel gene are present in every tissue analyzed but are most prominently expressed in spleen. We have renamed this new protein PHISH for 3'-phosphoinositide-interacting Src homology-containing protein. This report demonstrates the utility of this technique for isolating and characterizing 3'-PPI-binding proteins and has broad applicability for the isolation of binding domains for other lipid products.

    Funded by: NCI NIH HHS: CA27951, CA78773; NINDS NIH HHS: NS29632; ...

    The Journal of biological chemistry 1999;274;53;37893-900

  • Primary structure, tissue distribution, and expression of mouse phosphoinositide-dependent protein kinase-1, a protein kinase that phosphorylates and activates protein kinase Czeta.

    Dong LQ, Zhang RB, Langlais P, He H, Clark M, Zhu L and Liu F

    Department of Pharmacology, The University of Health Science Center, San Antonio, Texas 78284, USA.

    Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified serine/threonine kinase that phosphorylates and activates Akt and p70(S6K), two downstream kinases of phosphatidylinositol 3-kinase. To further study the potential role of PDK1, we have screened a mouse liver cDNA library and identified a cDNA encoding the enzyme. The predicted mouse PDK1 (mPDK1) protein contained 559 amino acids and a COOH-terminal pleckstrin homology domain. A 7-kilobase mPDK1 mRNA was broadly expressed in mouse tissues and in embryonic cells. In the testis, a high level expression of a tissue-specific 2-kilobase transcript was also detected. Anti-mPDK1 antibody recognized multiple proteins in mouse tissues with molecular masses ranging from 60 to 180 kDa. mPDK1 phosphorylated the conserved threonine residue (Thr402) in the activation loop of protein kinase C-zeta and activated the enzyme in vitro and in cells. Our findings suggest that there may be different isoforms of mPDK1 and that the protein is an upstream kinase that activates divergent pathways downstream of phosphatidylinositol 3-kinase.

    Funded by: NIDDK NIH HHS: DK52933

    The Journal of biological chemistry 1999;274;12;8117-22

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000022 G2C Mus musculus Pocklington M4 Cluster 4 (mouse) from Pocklington et al (2006) 8
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 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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