Insulin Stimulates PKCζ-mediated Phosphorylation of Insulin Receptor Substrate-1 (IRS-1)
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m007281200
ISSN1083-351X
AutoresYanfang Liu, Keren Paz, Avia Herschkovitz, Addy Alt, Tamar Tennenbaum, Sanford R. Sampson, Motoi Ohba, Toshio Kuroki, Derek LeRoith, Yehiel Zick,
Tópico(s)Pancreatic function and diabetes
ResumoIncubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCζ fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCζ in Fao cells, by infection of the cells with adenovirus-based PKCζ construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR·IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCζ was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC α, δ, or η. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCζ, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCζ inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCζ itself. These findings implicate PKCζ as a key element in a multistep negative feedback control mechanism of IRS-1 functions. Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCζ fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCζ in Fao cells, by infection of the cells with adenovirus-based PKCζ construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR·IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCζ was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC α, δ, or η. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCζ, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCζ inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCζ itself. These findings implicate PKCζ as a key element in a multistep negative feedback control mechanism of IRS-1 functions. The insulin receptor mediates insulin action through the phosphorylation of substrate proteins on Tyr residues (1Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 2Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 3Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (718) Google Scholar). The major substrates of the insulin receptor kinase are Shc (4Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar) and the IRS1 family of proteins, IRS-1 (5Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1276) Google Scholar), IRS-2 (6Sun X.-J. Wang L.-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (763) Google Scholar), IRS-3 (7Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), and IRS-4 (8Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). IRS proteins contain a conserved PH (pleckstrin homology) domain (9Haslam R.J. Kolde H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (386) Google Scholar,10Mayer B.J. Ren R. Clark K.L. Baltimore D. Cell. 1993; 73: 629-630Abstract Full Text PDF PubMed Scopus (379) Google Scholar) located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain. The PTB domain is present in a number of signaling molecules (11Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2219) Google Scholar) and shares 75% sequence identity between IRS-1 and IRS-2 (12Sawka V.D. Tartare D.S. White M.F. Van O.E. J. Biol. Chem. 1996; 271: 5980-5983Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). This domain interacts with the NPXY motif of the juxtamembrane region of IR and promotes IR-IRS-1 interaction (13Wolf G. Trüb T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 14Eck M.J. Dhe P.S. Trub T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 15Paz K. Voliovitch H. Hadari Y.R. Roberts C.T. LeRoith D. Zick Y. J. Biol. Chem. 1996; 271: 6998-7003Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The carboxyl-terminal region of IRS proteins is poorly conserved. It contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2 (Src homology-2) domain-containing proteins like the p85α regulatory subunit of PI3K, Grb2, Nck, Crk, Fyn, and SHP-2, which mediate the metabolic and growth-promoting functions of insulin (1Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 2Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 3Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (718) Google Scholar).IRS proteins contain more than 70 potential Ser/Thr phosphorylation sites for kinases like PKA (cAMP-dependent protein kinase), PKC, and MAPK (5Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1276) Google Scholar, 6Sun X.-J. Wang L.-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (763) Google Scholar, 16Mothe I. Van Obberghen E. J. Biol. Chem. 1996; 271: 11222-11227Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). The phosphorylation of Ser/Thr residues of IRS proteins has a dual function, serving either for a positive or negative modulation of insulin signal transduction. Phosphorylation of Ser residues within the PTB domain of IRS-1 by insulin-stimulated PKB protects IRS proteins from the rapid action of protein tyrosine phosphatases and enables the Ser-phosphorylated IRS proteins to maintain their Tyr-phosphorylated active conformation, implicating PKB as a positive regulator of IRS-1 functions (17Paz K. Yan-Fang L. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In contrast, a wortmannin-sensitive Ser/Thr kinase, different from PKB, phosphorylates IRS proteins and acts as a negative feedback control regulator that turns off insulin signals by inducing the dissociation of IRS proteins from IR (17Paz K. Yan-Fang L. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). These observations raise the question as to which kinases act as negative modulators of IRS proteins function.Several Ser/Thr kinases located downstream of PI3K are potential candidates to fulfill this role. These include the mammalian target of rapamycin (mTOR) (18Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (408) Google Scholar) and p70S6 kinase (19Ming X.-F. Burgering B.M.T. Wennstrom S. Clawsson-Welsh L. Heldin C.-H Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar), which are activated by phosphoinositide-dependent kinase-1 (PDK-1) (20Alessi 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). Other candidates are members of the PKC family. Indeed, 12-O-tetradecanoylphorbol 13-acetate, a potent activator of various PKC isoforms, effectively inhibits both IRS-1 interactions with the juxtamembrane region of the insulin receptor and insulin's ability to phosphorylate IRS proteins, thus implicating diacylglycerol-activated PKCs as potential regulators of IR-IRS interactions (21Paz K. Hemi R. LeRoith R. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 22De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 23Li J. DeFea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar).Atypical PKCs, exemplified by PKCζ, are downstream effectors of PI3K (24Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar) and PDKs (25Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) and act as mediators of insulin action. These proteins do not contain PH domains, and the exact mechanism of their activation is unknown. Atypical PKCs bind phosphatidylinositol 1,4,5-trisphosphate with reasonably high affinities, which results in their activation (26Palmer R.H. Dekker L.V. Woscholski R. LeGood J.A. Gigg R. Parker P.J. J. Biol. Chem. 1995; 270: 22412-22416Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Hence, phosphatidylinositol 1,4,5-trisphosphate may promote PKC signaling by colocalizing the enzyme in close proximity to its substrates. PKCζ can be activated by insulin, and this activation is blocked by inhibitors of PI3K and the expression of dominant negative p85 (27Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 28Bandyopadhyay G. Standaert M.L. Zhao L., Yu, B. Avignon A. Galloway L. Karnam P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 2551-2558Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Phosphorylation of PKCζ is required for its full activation; two of the regulatory phosphorylation sites on PKCζ are targets for phosphorylation by PDK-1 and PDK-2 (25Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar), thus representing another mechanism by which activation of PI3K affects the activity of PKCζ.In the present study we provide evidence that overexpression of PKCζ, but not other PKC isoforms potentiates the insulin-stimulated dissociation of IRS-1 from the insulin receptor, accelerates the rate of Tyr dephosphorylation of IRS-1, and as a result, reduces complex formation between IRS-1 and PI3K. These findings implicate PKCζ as an insulin-stimulated and a PI3K-dependent kinase that down-regulates IRS-1 functions by a tightly regulated process. Agents that induce insulin resistance, such as TNF, a known activator of PKCζ (29Muller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (469) Google Scholar, 30Berra E. Diaz M.M. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar) could take advantage of this mechanism. Activation of PKCζ by TNF in an insulin-independent manner (29Muller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (469) Google Scholar, 30Berra E. Diaz M.M. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar) could account for the enhanced Ser phosphorylation of IRS proteins and their dissociation from IR, which takes place when cells are exposed to TNF (21Paz K. Hemi R. LeRoith R. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), thus providing us with a possible molecular mechanism for the induction of an insulin-resistant state.DISCUSSIONAtypical PKC isotypes PKCζ and PKCλ are important elements in insulin signal transduction. They are activated by insulin through a PI3K-dependent mechanism (27Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 38Kotani K. Ogawa W. Matsumoto M. Ktamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar), and insulin-stimulated glucose transport depends upon the activation of one or both of these enzymes (27Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 28Bandyopadhyay G. Standaert M.L. Zhao L., Yu, B. Avignon A. Galloway L. Karnam P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 2551-2558Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 38Kotani K. Ogawa W. Matsumoto M. Ktamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar, 39Etgen G.J. Valasek K.M. Broderick C.L. Miller A.R. J. Biol. Chem. 1999; 274: 22139-22142Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In the present study we provide evidence that PKCζ also plays a major regulatory role in a negative feedback control mechanism induced by insulin to terminate its own signaling pathways. This mechanism involves PKCζ-mediated Ser/Thr phosphorylation of IRS-1 that leads to its dissociation from the insulin receptor. The dissociated IRS protein fails to undergo further Tyr phosphorylation by the insulin receptor kinase, while being subjected to the action of protein Tyr phosphatases that reduce its P-Tyr content. The resulting Tyr-dephosphorylated IRS-1 is unable to recruit downstream effectors like PI3K, and the insulin signal is therefore terminated.Several lines of evidence support such a mechanism. First, we could demonstrate that of several inhibitors tested, wortmannin, a PI3K inhibitor, effectively inhibits the dissociation of IRS-1 from the insulin receptor and the subsequent reduction in its P-Tyr content observed following a 60-min insulin treatment. Hence, a wortmannin-sensitive Ser/Thr kinase presumably acts as the feedback control regulator that turns off insulin signals. PKCζ, an insulin-stimulated Ser/Thr kinase downstream of PI3K, could fulfill this role, and indeed Go6983, an inhibitor of several PKC isoforms including PKCζ, partially prevented the reduction in P-Tyr content of IRS-1 following a 60-min insulin treatment. Second, overexpression of PKCζ by infection of Fao cells with an adenoviral-based expression vector markedly potentiated the dissociation of IR·IRS-1 complexes and the resulting reduction in P-Tyr content of IRS-1. The overexpressed PKCζ, like its endogenous counterpart, remained under the control of the insulin signaling pathway, since its effects were inhibited in the presence of wortmannin. Furthermore, overexpression of PKCζ did not impair the acute (1 min) effects of insulin on Tyr phosphorylation of IRS-1, indicating that the basal activity of the overexpressed PKCζ is rather low.The effects of PKCζ on Ser phosphorylation of IRS-1 seems to be quite unique in the sense that other PKC isoforms such as PKCα, δ, and η, when overexpressed in Fao cells, fail to mimic the effects of PKCζ following insulin stimulation. Similarly, a kinase-inactive form of PKCζ fails to mimic the inhibitory effects of its wild-type counterpart when transiently overexpressed in Fao or H-35 cells. Still, these observations do not exclude the possibility that other PKC isoforms could phosphorylate the IRS proteins in an insulin-independent manner. In fact, we have shown (21Paz K. Hemi R. LeRoith R. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) that 12-O-tetradecanoylphorbol 13-acetate, a potent activator of conventional and novel PKC isoforms, effectively inhibits both IRS-1 interactions with the juxtamembrane region of the insulin receptor and insulin's ability to phosphorylate IRS proteins (21Paz K. Hemi R. LeRoith R. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Similarly, the mutation of Ser612 of IRS-1, a potential MAPK phosphorylation site, eliminates the ability of 12-O-tetradecanoylphorbol 13-acetate to induce IR-IRS dissociation, indicating that diacylglycerol-activated PKCs (40Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar) could act as potential regulators of IR-IRS interactions (22De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 23Li J. DeFea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). It should be noted, however, that although overexpression of PKCζ potentiates insulin-mediated activation of MAPK, the latter does not seem to mediate PKCζ effects on IRS-1 phosphorylation because PD98059, which effectively inhibits activation of MAPK does not inhibit Ser/Thr phosphorylation of IRS-1 in response to insulin treatment.Although IRS proteins contain several PKC phosphorylation sites, it is presently unclear whether PKCζ phosphorylates IRS-1 directly or whether its effects are mediated by a downstream effector of PKCζ. Recent studies suggest that IRS-1 serves as an in vitrosubstrate for PKCζ (41Ravichandran L.V. Esposito D.L. Chen J. Quon M.J. J. Biol. Chem. 2000; 276: 3543-3549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Furthermore, endogenous IRS-1 coprecipitates with endogenous PKCζ, and this association is increased 2-fold upon insulin stimulation (41Ravichandran L.V. Esposito D.L. Chen J. Quon M.J. J. Biol. Chem. 2000; 276: 3543-3549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). These findings suggest that PKCζ could function as a direct IRS-1 kinase. However, several Ser/Thr kinases that are downstream effectors of PKCζ could also fulfill this role. A potential candidate is the IkB kinase β (IKKβ), which binds PKCα as well as the atypical PKCζ both in vitro and in vivo, and serves as an in vitro substrate for PKCζ (42Lallena M.-J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). Overexpression of PKCζ positively modulates IKKβ activity, whereas the transfection of a dominant negative mutant of PKCζ severely impairs the activation of IKKβ in TNF-stimulated cells (42Lallena M.-J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar).The p70 S6 kinase (19Ming X.-F. Burgering B.M.T. Wennstrom S. Clawsson-Welsh L. Heldin C.-H Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar) is also a potential candidate in view of the fact that rapamycin, which inhibits the insulin-stimulated activation of p70S6K (43Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar), partially prevents the reduction in P-Tyr content of IRS-1 following 60 min of insulin treatment. Indeed, mTOR-mediated phosphorylation on Ser632, Ser662, and Ser731 of IRS-1 was shown to inhibit its insulin-stimulated Tyr phosphorylation and its ability to bind PI3K (23Li J. DeFea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Furthermore, p70 S6 kinase is activated by PKCζ and participates in a PI3K-regulated signaling complex in some (44Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar) but not all cellular models (30Berra E. Diaz M.M. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar). Recently, the c-Jun NH2-terminal kinase (JNK) was shown to promote insulin resistance during association with IRS-1 and phosphorylation of Ser307. However it still needs to be determined whether JNK is a downstream effector of PI3K. Although earlier studies suggest that it is (45Desbois-Mouthon C. Eggelpoel M.J. Auclair M. Cherqui G. Capeau J. Caron M. Biochem. Biophys. Res. Commun. 1998; 243: 765-770Crossref PubMed Scopus (29) Google Scholar), recent findings indicate that phosphatidylinositol 3-kinase is not part of the insulin signaling pathway that leads to JNK activation (46Fukunaga K. Noguchi T. Takeda H. Matozaki T. Hayashi Y. Itoh H. Kasuga M. J. Biol. Chem. 2000; 275: 5208-5213Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Our current work and previous studies (17Paz K. Yan-Fang L. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 21Paz K. Hemi R. LeRoith R. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) indicate that Ser/Thr phosphorylation of IRS protein following insulin stimulation has a dual role, either to enhance or to terminate the insulin signal. Ser residues of the PTB domain of IRS-1, located within consensus PKB phosphorylation sites, presumably function as positive effectors of insulin signaling (17Paz K. Yan-Fang L. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Once phosphorylated by PKBα, they serve to protect IRS proteins from the rapid action of protein Tyr phosphatases. In such a way, PKBα acts to propagate and accelerate insulin signaling by phosphorylating downstream effectors and phosphorylating IRS proteins, thus generating a positive feedback loop for insulin action. Insulin also activates PKCζ, which mediates phosphorylation of yet unidentified Ser/Thr residues within the IRS protein. Phosphorylation of these sites is part of the negative feedback control mechanism induced by insulin, which leads to the dissociation of the IR·IRS complexes and results in the termination of insulin signal. Agents that induce insulin resistance, such as TNF, take advantage of this mechanism by stimulating Ser phosphorylation of IRS proteins and the dissociation of IR·IRS complexes (21Paz K. Hemi R. LeRoith R. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Interestingly, TNF induces the activation of PKCζ in an insulin-independent manner (29Muller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (469) Google Scholar, 30Berra E. Diaz M.M. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar), implicating PKCζ itself or its downstream Ser/Thr kinases (i.e. IKKβ) as potential TNF-stimulated IRS-1 kinases. Both Ser/Thr kinases, which phosphorylate IRS-1, PKB the positive regulator, and PKCζ the negative regulator, are downstream effectors of PI3K. This finding suggests that their action should be orchestrated in a way that enables sustained activation of IRS-1, as a result of its phosphorylation by PKB, prior to the activation of PKCζ, the action of which is expected to terminate insulin signal transduction. Our studies further indicate that the negative feedback control mechanism induced by PKCζ has a built-in self-attenuation signal. Accordingly, insulin-stimulated and PI3K-mediated activation of PKCζ inhibits IRS-1 functions and reduces complex formation between IRS-1 and PI3K, which then inhibits further activation of PKCζ. It is presently unclear how PKCζ promotes insulin action and glucose transport (27Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 28Bandyopadhyay G. Standaert M.L. Zhao L., Yu, B. Avignon A. Galloway L. Karnam P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 2551-2558Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 38Kotani K. Ogawa W. Matsumoto M. Ktamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar, 39Etgen G.J. Valasek K.M. Broderick C.L. Miller A.R. J. Biol. Chem. 1999; 274: 22139-22142Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) while acting as a negative feedback regulator of insulin signal transduction. Most likely, the positive and negative regulatory roles of PKCζ are subjected to a tight spatio-temporal control along the insulin signal transduction pathway, such that the positive effects are turned on and terminated before the negative regulatory functions are being activated. Further studies are required to unravel the mechanisms that govern this intricate regulatory process. The insulin receptor mediates insulin action through the phosphorylation of substrate proteins on Tyr residues (1Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 2Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 3Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (718) Google Scholar). The major substrates of the insulin receptor kinase are Shc (4Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar) and the IRS1 family of proteins, IRS-1 (5Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1276) Google Scholar), IRS-2 (6Sun X.-J. Wang L.-M. Zhang Y. Yenush L. Myers Jr., M.G. 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