Regulation of Ribosomal S6 Kinase 2 by Effectors of the Phosphoinositide 3-Kinase Pathway
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m006969200
ISSN1083-351X
AutoresKathleen A. Martin, Stefanie S. Schalm, Celeste Richardson, Angela Romanelli, Kristen L. Keon, John Blenis,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoRibosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5′-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitroand is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase Cζ but that S6K2 is more sensitive to basal activation by myristoylated protein kinase Cζ than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases. Ribosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5′-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitroand is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase Cζ but that S6K2 is more sensitive to basal activation by myristoylated protein kinase Cζ than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases. ribosomal S6 kinase phosphoinositide 3-kinase mammalian target of rapamycin phosphoinositide-dependent kinase 1 mitogen-activated protein-extracellular signal-regulated kinase kinase nuclear localization signal protein kinase C myristoylated PKC glutathione S-transferase hemagglutinin The 70-kDa ribosomal S6 kinase 1 (S6K1)1 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in response to mitogen stimulation (1Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar). S6 phosphorylation up-regulates translation of mRNAs with 5′-terminal oligopyrimidine tracts, many of which encode ribosomal proteins and translation elongation factors (2Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (806) Google Scholar). S6K1 activation thus up-regulates ribosome biosynthesis and enhances the translational capacity of the cell.Deletion of S6K1 in Drosophila and mice has implicated S6K1 in regulation of cell size. The Drosophila knockout has a high incidence of embryonic lethality, but surviving flies exhibit a marked reduction in size that is cell autonomous (3Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (621) Google Scholar). Mice lacking S6K1 through targeted disruption also exhibit a small animal phenotype (4Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). Interestingly, S6 phosphorylation and 5′-terminal oligopyrimidine tract mRNA translation were found to be normal in fibroblasts derived from mice lacking S6K1, suggesting a compensatory mechanism for these S6K1 functions. Our lab and others have recently identified S6K2, a homolog of S6K1 that phosphorylates S6 in vitro (4Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 5Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar, 7Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar). Elevated S6K2 mRNA levels have been reported in the S6K1 knockout mice (4Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). Drosophila are thought to express only S6K1, which may account for the more severe S6K1 knockout phenotype in flies. S6K2 is a good candidate kinase that may supply some but not all of the functions of S6K1 in the knockout mouse, because the small animal phenotype persists despite the presence of S6K2, S6 phosphorylation, and 5′-terminal oligopyrimidine tract mRNA translation.There are two isoforms of both S6K1 and S6K2 derived from alternative splicing at the N terminus. The p70S6K1αII isoform is cytosolic, whereas p85 S6K1αI is nuclear (8Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar). In contrast, both isoforms of S6K2 (p54 S6K2βII, p60 S6K2βI) (5Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar) are primarily nuclear, because of the presence of a C-terminal putative nuclear localization signal sequence (NLS) (7Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar) not found in S6K1. Point mutation of the putative NLS (K474M) results in cytosolic immunolocalization of S6K2βII (7Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar). The S6K2 βI and βII isoforms may reside in distinct nuclear compartments based on subcellular fractionation studies (6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar).S6K1 and S6K2 are highly homologous overall, with the greatest sequence homology in the kinase domain and adjacent regulatory linker domain. A schematic diagram of S6K1 and S6K2 outlining regions of homology, features unique to S6K2, and regulatory phosphorylation sites is provided in Fig. 1. Seven of eight mitogen-stimulated regulatory phosphorylation sites identified in S6K1 are conserved in human S6K2. There are interesting differences in S6K2 primary structure that may confer differential regulation and functions to this kinase. There are regions of sequence divergence between S6K1 and S6K2 in the N- and C-terminal domains. In addition to the nuclear localization signal, the C terminus of S6K2 also contains a proline-rich domain not found in S6K1.In both S6K1 and S6K2, the conserved core catalytic and linker domains are flanked by regulatory N- and C-terminal domains. For S6K1, it is thought that interaction between N-terminal acidic residues and C-terminal basic residues inhibits the kinase by allowing a C-terminal pseudosubstrate region to occlude the kinase domain. Mitogen-stimulated phosphorylation of the C-terminal Ser/Thr-Pro motifs is believed to disrupt these interactions, relieving autoinhibition of S6K1 and exposing other regulatory sites, including the major rapamycin-sensitive site, T389, and the catalytic activation loop site, Thr229 (9Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar, 10Weng Q. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar).The PI3 kinase (PI3-K) and mTOR signaling pathways mediate multiple mitogen-stimulated phosphorylation events that lead to S6K1 activation. Consistent with the important roles of these pathways in S6K1 regulation, S6K1 activation is inhibited by pharmacological inhibitors of these pathways (11Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1018) Google Scholar, 12Chung J. Grammer T. Lemon K. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (656) Google Scholar). The immunosuppressant rapamycin, an inhibitor of mTOR, leads to rapid and complete dephosphorylation and inactivation of S6K1 (11Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1018) Google Scholar). The role of mTOR in S6K1 activation may be suppression of an S6K1 phosphatase (13Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (424) Google Scholar). There is also evidence suggesting that mTOR may directly phosphorylate S6K1 (14Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (924) Google Scholar). S6K2 activation is also sensitive to rapamycin, and the analogous rapamycin-sensitive mitogen-stimulated S6K1 phosphorylation sites are conserved in S6K2 (Thr388, Ser410, Ser417, and Ser423) (4Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 5Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar, 7Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar).Multiple PI3-K effectors provide distinct inputs to S6K1 activation. Phosphoinositide-dependent kinase 1 (PDK1) is a constitutively active kinase whose access to many substrates is regulated by PI3-K-derived phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (15Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Phosphorylation of Thr229 in the S6K1 catalytic domain activation loop by PDK1 is a critical activating input (15Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 16Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar). PDK1 may also phosphorylate Thr389 (17Balendran A. Currie R. Armstrong C.G. Avruch J. Alessi D.R. J. Biol. Chem. 1999; 274: 37400-37406Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The PI3-K- and PDK1-regulated atypical PKC isoforms ζ and λ have also been implicated in S6K1 regulation (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar, 19Akimoto K. Nakaya M. Yamanaka T. Tanaka J. Matsuda S. Weng Q.P. Avruch J. Ohno S. Biochem. J. 1998; 335: 417-424Crossref PubMed Scopus (67) Google Scholar). These atypical PKCs interact with S6K1, but it is not yet known whether they directly phosphorylate S6K1. We have reported growth factor-independent coimmunoprecipitation of PDK1, PKCζ, and S6K1, suggesting the existence of preassembled complexes of PI3-K-regulated signaling molecules (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar). Our lab has also demonstrated association of the PI3-K-regulated Rho family G proteins Cdc42 and Rac with S6K1 and has shown that these G proteins contribute to S6K1 activation (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Interestingly, Cdc42 and PKCζ or PKCλ have recently been shown to associate (21Coghlan M.P. Chou M.M. Carpenter C.L. Mol. Cell. Biol. 2000; 20: 2880-2889Crossref PubMed Scopus (83) Google Scholar). The mechanism of Cdc42/Rac activation of S6K1 is not yet known but requires isoprenylation of the G protein, suggesting that membrane targeting is important (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Preliminary studies using pharmacological inhibitors suggest that, like S6K1, S6K2 is regulated by the PI3-K and mTOR signaling pathways (4Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 5Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar, 7Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar). We have shown that a constitutively active PI3-K p110 subunit activates S6K2 and that S6K2 is activated by PDK1 (6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar). Others have found that S6K2, like S6K1, can be regulated by the PI3-K effector Akt/PKB (7Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar). However, further characterization of the signaling intermediates that regulate S6K2 has not yet been addressed. Given the differences in subcellular localization and primary sequence and the lack of complete functional redundancy between these homologs in the S6K1 knockout mice (4Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar), we aimed to examine in detail the regulation of S6K2. Here, we report that although S6K2 is regulated similarly to S6K1 by PI3-K effectors, the C terminus of S6K2 exerts a more potent inhibitory effect on the kinase.RESULTSS6K2, like S6K1, is regulated by the PI3-K pathway. S6K2 is activated by constitutively active p110 PI3-K and inhibited when cells are treated with wortmannin (6Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (119) Google Scholar). Wortmannin-sensitive phosphorylation sites found in S6K1 are conserved in S6K2 and S6K1. Multiple PI3-K pathway effectors have been implicated in S6K1 activation. Given the differences in primary structure and subcellular localization between S6K2 and S6K1, we sought to determine whether S6K2 is regulated by the same downstream PI3-K effectors known to regulate S6K1 and to investigate the roles of divergent S6K2 C-terminal sequences.Rho Family G Proteins Regulate S6K2Evidence suggests that PI3-K regulates the Rho family G proteins Cdc42 and Rac through activation of their guanine nucleotide exchange factors, such as Dbl and Vav (23Leevers S.J. Vanhaesebroeck B. Waterfield M.D. Curr Opin Cell Biol. 1999; 11: 219-225Crossref PubMed Scopus (567) Google Scholar). We have previously shown that Cdc42 and Rac regulate S6K1; cotransfection of GTPase-deficient constitutively active point mutants of Cdc42 and Rac (G12V) activates HA-S6K1, and dominant negative point mutants (T17N) with high GDP affinity (which sequester guanine nucleotide exchange factors) antagonize growth factor activation of S6K1 (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Similarly, transient cotransfection of HEK293 cells with HA-S6K2 and GST-Cdc42V12 or GST-RacV12 enhanced basal (2–5-fold) and insulin-stimulated (2–3-fold) activity as measured in immune complex kinase assays (Fig.2 A). Consistent with a role for Rho family G proteins in regulation of S6K2, cotransfection of HA-S6K2 with the dominant negative GST-Cdc42N17 mutant inhibits insulin stimulation of HA-S6K2, as well as HA-S6K1, activity (Fig.2 B). PDK1 activates S6K1 by phosphorylating Thr229 in the catalytic activation loop (15Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 16Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar). It is likely that Cdc42 contributes an S6K-activating function distinct from that of PDK1, because cotransfection of submaximally activating levels of Myc-PDK1 and GST-Cdc42V12 cooperatively activate S6K2. We demonstrate here for the first time that this is also the case with S6K1 (Fig. 3).Figure 2Rho family G proteins regulate S6K2. A, constitutively active mutants of Cdc42 or Rac activate HA-S6K2. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 and the indicated amounts of GST-Cdc42V12/pEBG, GST-RacV12/pEBG, or pEBG vector as described under "Experimental Procedures." Transfected cells were quiesced in serum-free medium for 24 h prior to 30 min of stimulation with 100 nminsulin. Cells were lysed as described, and protein expression levels were assayed by immunoblotting with anti-HA or -GST antibodies. HA-S6K2 expression levels were normalized by quantitative immunoblotting using a Bio-Rad FluorS MultiImager prior to immune complex kinase assay. Kinase activity in anti-HA immunoprecipitates was quantitated with a phosphorimager (Bio-Rad). These results are representative of at least two experiments. B, a dominant negative Cdc42 mutant inhibits HA-S6K2 activity. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 or 0.5 μg of HA-S6K1/pRK7 and the indicated amounts of GST-Cdc42N17/pEBG or pEBG vector and starved, insulin-stimulated, and lysed as above. Data from immune complex kinase assay (top panels) and anti-HA or -GST Western blots (bottom panels) are shown and are representative of at least three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Cdc42 and PDK1 cooperatively activate HA-S6K2 and HA-S6K1. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 or 0.5 μg of HA-S6K1/pRK7 and submaximally activating doses (0.5 μg) of GST-Cdc42V12/pEBG and/or Myc-PDK1/pcDNA3 or pEBG vector. Cells were starved, insulin-stimulated, and lysed as in Fig. 2. Activity of HA-S6K2 or HA-S6K1 is indicated in the top panels. Anti-HA, -GST, or -Myc Western blots are shown in the bottom panels. Data are representative of two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The mechanism by which Cdc42 activates S6K1 is not yet known, but Cdc42V12 and S6K1 coimmunoprecipitate and a mutation that prevents Cdc42 isoprenylation (C189S) fails to activate S6K1 (20Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). This suggests that membrane targeting of Cdc42 may be required. An attractive hypothesis is that association with Cdc42 may transiently target S6K1 to a cellular membrane in the course of its activation. This membrane targeting may be important for access to other membrane-associated S6K1 activators such as PDK1 and PKCζ. Because S6K2 is thought to be primarily nuclear, it is notable that the cytosolic proteins Cdc42 and Rac can regulate this kinase. We determined that isoprenylation of GST-Cdc42V12 is required for this effect, because GST-Cdc42V12,C189S fails to activate HA-S6K2 when cotransfected (Fig. 4). These data suggest that despite localization primarily to the nucleus, S6K2 is regulated by cytosolic, isoprenylated low molecular weight G proteins. We hypothesize therefore that S6K2 may shuttle in and out of the nucleus and target to a membrane during the course of its activation.Figure 4A prenylation-deficient mutant Cdc42V12 fails to activate HA-S6K2. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 with 2 μg of GST-Cdc42V12/pEBG, GST-Cdc42V12/C189S/pEBG, or pEBG vector. Cells were starved, insulin-stimulated, and lysed as in Fig. 2. Activity of HA-S6K2 is indicated in the top panel and anti-HA or -GST Western blots are presented in the bottom panel. Data are representative of two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Atypical PKCζ Regulates S6K2S6K1 associates with and is regulated by the atypical PKCζ (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar). PKCζ is activated by binding PI3-K-derived phosphatidylinositol 3,4,5-trisphosphate and by interaction with and phosphorylation by PDK1 (22Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.-S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar, 24Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (969) Google Scholar). Although PKCζ is not sufficient to activate S6K1 under basal conditions, when coexpressed with PDK1, a strong S6K1 activation is observed (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar). Coimmunoprecipitation of S6K1, PDK1, and PKCζ suggests their participation in a PI3-K-regulated signaling complex (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar). To address whether PKCζ can regulate S6K2 in vivo, we cotransfected a constitutively active FLAG-tagged, myristoylated PKCζ (myr-PKCζ) construct with HA-S6K2, which resulted in basal and insulin-stimulated HA-S6K2 activation (Fig. 5 A). There was a notable difference between S6K1 and S6K2, because HA-S6K1 was activated only modestly (up to 2-fold) by cotransfection of myr-PKCζ in quiescent cells (Fig. 5 A) and (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar), whereas HA-S6K2 was activated 5–30-fold under basal conditions (Fig.5 A), suggesting that S6K2 may be more sensitive to regulation by atypical PKCs. Further supporting a role for PKCζ in activation of HA-S6K2 was the observation that insulin-stimulated activity was inhibited by cotransfection of the dominant negative FLAG-PKCζK281W (PKCζK/W) (Fig. 5 B), as is the case with S6K1 (18Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar). In addition, HA-S6K2 is cooperatively activated by combined cotransfection of submaximally activating levels of myr-PKCζ and PDK1 (Fig. 6). Under these conditions, the constitutively active myr-PKCζ is not further activated by PDK1 (22Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.-S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar), but the modest overexpression of both activators results in synergistic activation of S6K2. Although both S6K1 and S6K2 are inhibited by dominant negative PKCζ(K/W) and cooperatively activated by PDK1 and myr-PKCζ, only S6K2 basal activity is substantially activated by myr-PKCζ alone. These data provide the first evidence that S6K1 and S6K2 can be differentially regulated.Figure 5Atypical PKC ζ regulates HA-S6K2. A, myristoylated PKCζ activates HA-S6K2. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 or 0.5 μg of HA-S6K1/pRK7 and 1.0 μg of FLAG-myr-PKCζ/pCMV6. Cells were quiesced, insulin-stimulated, and lysed as in Fig. 2. HA-S6K activity is presented in the top panel. Anti-HA and anti-PKCζ Western blots are shown in thebottom panel. Data are representative of three experiments.B, HA-S6K2 activation is inhibited by dominant negative PKCζ. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 and 4.0 μg of FLAG-PKCζK281W/pCMV6 as indicated. Cells were quiesced, insulin-stimulated, and lysed as in Fig. 2. HA-S6K2 activity is presented in the top panel. Anti-HA and anti-PKCζ, Western blots are shown in the bottom panel. Data are representative of at least two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6PKC ζ and PDK1 cooperatively activate HA-S6K2. HEK293 cells were transfected with 1.0 μg of HA-S6K2/pcDNA3 and 1.0 μg of FLAG-myr-PKCζ/pCMV6 and/or 0.5 μg of Myc-PDK1/pcDNA3 as indicated. Cells were quiesced, insulin-stimulated, and lysed as in Fig. 2. HA-S6K2 activity is presented in the top panel. Anti-HA, -PKCζ, and -Myc Western blots are shown in the bottom panel. Data are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)C-terminal Truncation Potentiates S6K2 ActivationStructure-function mutational analyses have provided important insight into regulation and activation of S6K1 (9Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar, 10Weng Q. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar). We employed this approach to further study the regulation of S6K2. Because the C-terminal domain is a region of divergence between S6K1 and S6K2, we sought to determine the effect of deletion of this domain on regulation of S6K2. Amino acids 399–482 encoding the pseudosubstrate and proline-rich regions, as well as the NLS, were deleted from HA-S6K2 (Fig. 1), and the activity of the resulting mutant (HA-S6K2-ΔCT) was assayed in transfected HEK293 cells. Surprisingly, deletion of the C terminus resulted in enhanced basal and insulin-stimulated activity (Figs. 7 and 8). By contrast, the analogous truncation mutant of S6K1 is mitogen-regulated but is less active than the full-length kinase and is not more sensitive to insulin (9Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar, 10Weng Q. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar) and (Fig. 8). The S6K2 C-terminal truncation did not significantly alter sensitivity of the kinase to wortmannin (Fig. 7 B), suggesting that essential inputs from the PI3-K pathway are primarily integrated in regions upstream of amino acid 399.Figure 7C-terminal truncation potentiates HA-S6K2 activation. A, enhanced basal and PDK1 or insulin-stimulated activity of HA-S6K2-ΔCT. HEK293 cells were transfected with 1.0 μg of HA-S6K2 wild type (wt) or 2 μg of HA-S6K2-ΔCT (ΔCT) in the pcDNA3 vector and 1.0 μg of Myc-PDK1/pcDNA3 as indicated. Cells were serum-starved, insulin-stimulated, and lysed as in Fig. 2. Lysates were normalized for HA-S6K2 expression levels after Western blotting, and kinase activity was assayed. Data are representative of at least two experiments. B, HA-S6K2 wild type and HA-S6K2-ΔCT are sensitive to wortmannin. HEK293 cells were transfected with 1.0 μg of HA-S6K2 wild type (wt) or 2 μg of HA-S6K2-ΔCT in the pcDNA3 vector. Cells were serum-starved for 24 h and then pretreated with 100 nmwortmannin or vehicle for 30 min prior to a 30-min stimulation with 100 nm insulin. Cells were lysed and subjected to immunoblotting and kinase assay as in Fig. 2. Activity and anti-HA Western blots are shown. Data are representative of two experiments.View Large Image Figure ViewerDownload Hi-res image
Referência(s)