Title: The Insulin Receptor Substrate (IRS)-1 Pleckstrin Homology Domain Functions in Downstream Signaling
Abstract: The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets. The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets. insulin receptor substrate pleckstrin homology phosphatidylinositol hemagglutinin phosphotyrosine binding polymerase chain reaction green fluorescent protein Src homology triple mutant glutathione S-transferase After insulin binds to its receptor, it activates an intrinsic tyrosine kinase activity, which mediates the tyrosine phosphorylation of a variety of endogenous substrates including insulin receptor substrates (IRS)1 1–4 (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar,2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Binding of these tyrosine phosphorylated substrates to the Src homology (SH) domain-2 of the regulatory subunit of the heterodimeric p85/p110 phosphatidylinositol (PI) 3-kinase leads to a 3–5-fold stimulation in its enzymatic activity and an increase in the PI 3,4-bisphosphate and 3,4,5-trisphosphate in the cell (3Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 4Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (391) Google Scholar). As a result of the increase in these lipids, there is a pronounced stimulation in the enzymatic activity of the Ser/Thr kinases called Akt, and a more modest increase in the enzymatic activity of particular isoforms of protein kinase C, which ultimately lead to numerous biological responses (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar).The IRS 1–4 all contain an amino-terminal pleckstrin homology (PH) domain next to a phosphotyrosine binding (PTB) domain (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar). The PH domain is homologous to a region in pleckstrin and has been found in over 120 proteins including serine/threonine kinases, tyrosine kinases, phospholipases, GTPase-activating proteins, GTPases, and cytoskeletal proteins (7Shaw G. Bioessays. 1996; 18: 35-46Crossref PubMed Scopus (252) Google Scholar). PH domains are often involved in the attachment of proteins to membranes, either by directly binding to phospholipids and/or by protein-protein interactions with, for example, the βγ subunits of the heterotrimeric G proteins (8Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). In the case of the PH domain of IRS-1, both phospholipid binding as well as protein binding have been reported (9Takeuchi H. Matsuda M. Yamamoto T. Kanematsu T. Kikkawa U. Yagisawa H. Watanabe Y. Hirata M. Biochem. J. 1998; 334: 211-218Crossref PubMed Scopus (33) Google Scholar, 10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar, 11Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).In contrast, the PTB domain of IRS-1 has been documented to bind to phosphotyrosines in the motif found in the juxtamembrane region of the insulin receptor (i.e. NPXpY) (12Margolis B. J. Lab. Clin. Med. 1996; 128: 235-241Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 13Wolf G. Trub 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). Although the binding specificities of these two regions is quite distinct, they share a similar overall structure, with each containing a β-sandwich formed by two nearly orthogonal antiparallel β-sheets of 4 and 3 strands, respectively. In recent studies, the crystal structure of the region of IRS-1 containing both the PH and PTB domains has been determined (14Dhe-Paganon S. Ottinger E.A. Nolte R.T. Eck M.J. Shoelson S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8378-8383Crossref PubMed Scopus (85) Google Scholar). These data indicate that the two binding domains may act cooperatively to localize IRS-1 at the membrane in association with the receptor and thereby allow multiple tyrosine phosphorylations of IRS-1.A variety of experimental approaches have also been utilized to test the role of the PH and PTB domains of the IRS proteins in their interactions with the IR and the subsequent tyrosine phosphorylations. Mutant IRS molecules in which either the PH or PTB domain have been deleted or replaced with homologous structures of other proteins have been expressed in mammalian cells (15Paz K. Voliovitch H. Hadari Y.R. Roberts Jr., C.T. LeRoith D. Zick Y. J. Biol. Chem. 1996; 271: 6998-7003Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 16Burks D.J. Pons S. Towery H. Smith-Hall J. Myers Jr., M.G. Yenush L. White M.F. J. Biol. Chem. 1997; 272: 27716-27721Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17Yenush L. Makati K.J. Smith H.J. Ishibashi O. Myers M.J. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18Voliovitch H. Schindler D.G. Hadari Y.R. Taylor S.I. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 18083-18087Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). These studies have documented a primary role for the PH domain in the subsequent ability of IRS-1 to be tyrosine phosphorylated in intact cells after insulin stimulation. In either in vitro studies or yeast two-hybrid systems, a primary role of the IRS-1 PTB domain has been documented in receptor interactions (15Paz K. Voliovitch H. Hadari Y.R. Roberts Jr., C.T. LeRoith D. Zick Y. J. Biol. Chem. 1996; 271: 6998-7003Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (321) Google Scholar). In the case of IRS-2, a more central region of the molecule has also been identified as playing a role in receptor interactions (20Sawka-Verhelle D. Tartare-Deckert S. White M.F. Van Obberghen E. J. Biol. Chem. 1996; 271: 5980-5983Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21He W. Craparo A. Zhu Y. O'Neill T.J. Wang L.M. Pierce J.H. Gustafson T.A. J. Biol. Chem. 1996; 271: 11641-11645Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar).Although numerous studies have documented the roles of the PH and PTB domains of the IRS proteins in the interactions with the IR and the subsequent tyrosine phosphorylation of the IRS proteins, no studies have tested the roles of these two domains on subsequent signals induced by the IRS·PI 3-kinase complex. Because mutations in the PH and PTB domains interfere with the interaction of the IRS with the IR and its subsequent tyrosine phosphorylation, it is impossible to determine the effect of these mutations on the downstream signals emanating from the IRS·PI 3-kinase complex. To overcome this obstacle, we now describe a constitutively active form of IRS-1. In this chimeric molecule, the inter-SH2 domain of the p85 regulatory subunit of PI 3-kinase is attached to various portions of the IRS-1 molecule. The inter-SH2 domain of p85 has previously been documented to bind to the 110-kDa catalytic unit of the PI 3-kinase (22Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar). Thus the resultant chimeric IRS-1 molecules now constitutively bind to the PI 3-kinase. By using these chimeric molecules, we have tested the role of different regions of the IRS-1 molecule in eliciting a subsequent biological response, the stimulation of the Ser/Thr kinase Akt. We demonstrate that the PH domain alone is sufficient to target the constitutively active PI 3-kinase to induce the activation of Akt and that this requires the lipid binding properties of the PH domain.DISCUSSIONThe principal mechanism whereby the insulin receptor activates PI 3-kinase appears to be via its ability to stimulate the tyrosine phosphorylation of various endogenous proteins including the different IRS molecules, IRS-1 to 4 (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar, 2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Activation of PI 3-kinase plays a critical role in subsequent biological responses, such as stimulation of glucose uptake and regulation of gene transcription (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar). A number of downstream targets of the PI 3-kinase and the lipids it generates have been identified. These include several Ser/Thr kinases such as the PDK-1, the family of Akt/PKB kinases, and several atypical protein kinase C including PKC ζ and λ. The exact role of these different kinases in eliciting subsequent biological responses has been debated (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar).Extensive studies have investigated the mechanism whereby the IRS proteins are localized to the insulin receptor. All four IRS molecules contain both a PH domain and a PTB domain (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar, 2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). The PH domain binds various phospholipids, including PI 4,5-P2 as well PI 3,4,5-P3 (9Takeuchi H. Matsuda M. Yamamoto T. Kanematsu T. Kikkawa U. Yagisawa H. Watanabe Y. Hirata M. Biochem. J. 1998; 334: 211-218Crossref PubMed Scopus (33) Google Scholar, 10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar). In addition some studies have suggested that the PH domain may also interact with various proteins (11Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, the PTB domains bind phosphotyrosines, in particular, a phosphotyrosine in the juxtamembrane region of the insulin receptor (12Margolis B. J. Lab. Clin. Med. 1996; 128: 235-241Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 13Wolf G. Trub 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). Based on the crystal structure of the PH and PTB domains of the IRS-1 molecule, a model has been proposed of the two domains binding cooperatively to localize IRS-1 at the membrane in association with the insulin receptor (14Dhe-Paganon S. Ottinger E.A. Nolte R.T. Eck M.J. Shoelson S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8378-8383Crossref PubMed Scopus (85) Google Scholar).In the present studies, we have tested the role of these two domains in the ability of the IRS-1 molecule to elicit a subsequent biological response, the activation of the Ser/Thr kinase Akt. To accomplish this, we produced novel chimeric molecules, which contain these two domains fused to the inter-SH2 domain of the p85 subunit of the PI 3-kinase. This peptide has previously been shown to be sufficient to bind and activate the PI 3-kinase (22Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar). By adding these residues to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules, which were constitutively bound to active PI 3-kinase. This was necessary because prior studies have shown that the PH domain of the IRS-1 molecule was critical for its subsequent tyrosine phosphorylation and association with the PI 3-kinase. By adding the inter-SH2 domain to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules that were active in the absence of insulin stimulation and receptor-mediated tyrosine phosphorylation.In the present work we have shown that the PH domain is critical for the subsequent ability of these constitutively active chimeric molecules to activate Akt. The most convincing data for this conclusion comes from the studies of the point mutants of the PH-PTB domain chimeric molecule. The wild type and mutants were expressed to comparable levels and had almost identical associated PI 3-kinase activities. However, the wild type activated the endogenous Akt whereas both the single and triple PH domain mutants had a dramatically decreased ability to activate the enzymatic activities of the endogenous Akt (i.e. the introduction of either a single mutation or 3 mutations in the PH domain caused an approximate 75% decrease in the ability of the PH-PTB construct to activate Akt). Both mutations also had a similar decreased ability to bind to PI 4,5-P2 in comparison to the wild-type protein when they were expressed as PH-PTB·GST fusion proteins. The simplest interpretation of these data are that the PH domain function (i.e. binding to PI 4,5-P2 or a negatively charged protein) is critical to elicit the downstream functions. This conclusion is also supported by the finding that a chimeric molecule with only the PH domain of IRS-1 was also capable of stimulating the activation of the endogenous Akt. The somewhat weaker activity of this chimera in comparison to the PH-PTB molecule is consistent with its lower expression and somewhat decreased amount of associated PI 3-kinase activity. Because the construct, which only contained the PTB domain was expressed at lower levels and therefore had lower associated PI 3-kinase activity, it is not possible to conclude from this construct alone whether the PTB domain alone would have been capable at higher levels to induce the activation of Akt. However, the finding that the PH-PTB domain chimeric molecules whose PH domain function were impaired by either single or triple point mutations were incapable of eliciting the activation of the Akt argue that the PTB domain alone would not be sufficient to elicit this response because the PTB domain function in these chimeric molecules is still intact.A potential role of the PH domain of IRS-1 is to help localize the protein in the correct intracellular compartment. To test this hypothesis, we examined the subcellular localization of the wild-type and two mutant chimeric molecules by biochemical fractionation of the cells. The wild-type PH-PTB chimeric molecule was found to be in the particulate fraction to a much greater degree then the chimeric molecules containing the mutations in the PH domain. These results are consistent with the recent report that the IRS-1 molecule is at least partially present in a particulate fraction of the cell, possibly because of its association with the cytoskeletal complex (29Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar) as well as with a recent report that the same mutant we have studied (R28C) blocks the insulin induced translocation of a IRS·GFP construct to the membrane (10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar). Thus, the PH domain may serve to localize the chimeric molecules in the subcellular localization required for the subsequent activation of Akt.In conclusion, the present work presents the first evidence that a functional PH domain of the IRS-1 molecule is required for its ability to signal to downstream molecules such as activation of Akt. This requirement is presumably because of the ability of the PH domain of IRS-1 to direct the PI 3-kinase to the correct subcellular compartment. Compromising the function of the PH domain, for example in insulin-resistant states, could therefore both decrease the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor as well as to link to subsequent downstream targets (2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). After insulin binds to its receptor, it activates an intrinsic tyrosine kinase activity, which mediates the tyrosine phosphorylation of a variety of endogenous substrates including insulin receptor substrates (IRS)1 1–4 (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar,2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Binding of these tyrosine phosphorylated substrates to the Src homology (SH) domain-2 of the regulatory subunit of the heterodimeric p85/p110 phosphatidylinositol (PI) 3-kinase leads to a 3–5-fold stimulation in its enzymatic activity and an increase in the PI 3,4-bisphosphate and 3,4,5-trisphosphate in the cell (3Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 4Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (391) Google Scholar). As a result of the increase in these lipids, there is a pronounced stimulation in the enzymatic activity of the Ser/Thr kinases called Akt, and a more modest increase in the enzymatic activity of particular isoforms of protein kinase C, which ultimately lead to numerous biological responses (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar). The IRS 1–4 all contain an amino-terminal pleckstrin homology (PH) domain next to a phosphotyrosine binding (PTB) domain (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar). The PH domain is homologous to a region in pleckstrin and has been found in over 120 proteins including serine/threonine kinases, tyrosine kinases, phospholipases, GTPase-activating proteins, GTPases, and cytoskeletal proteins (7Shaw G. Bioessays. 1996; 18: 35-46Crossref PubMed Scopus (252) Google Scholar). PH domains are often involved in the attachment of proteins to membranes, either by directly binding to phospholipids and/or by protein-protein interactions with, for example, the βγ subunits of the heterotrimeric G proteins (8Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). In the case of the PH domain of IRS-1, both phospholipid binding as well as protein binding have been reported (9Takeuchi H. Matsuda M. Yamamoto T. Kanematsu T. Kikkawa U. Yagisawa H. Watanabe Y. Hirata M. Biochem. J. 1998; 334: 211-218Crossref PubMed Scopus (33) Google Scholar, 10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar, 11Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, the PTB domain of IRS-1 has been documented to bind to phosphotyrosines in the motif found in the juxtamembrane region of the insulin receptor (i.e. NPXpY) (12Margolis B. J. Lab. Clin. Med. 1996; 128: 235-241Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 13Wolf G. Trub 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). Although the binding specificities of these two regions is quite distinct, they share a similar overall structure, with each containing a β-sandwich formed by two nearly orthogonal antiparallel β-sheets of 4 and 3 strands, respectively. In recent studies, the crystal structure of the region of IRS-1 containing both the PH and PTB domains has been determined (14Dhe-Paganon S. Ottinger E.A. Nolte R.T. Eck M.J. Shoelson S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8378-8383Crossref PubMed Scopus (85) Google Scholar). These data indicate that the two binding domains may act cooperatively to localize IRS-1 at the membrane in association with the receptor and thereby allow multiple tyrosine phosphorylations of IRS-1. A variety of experimental approaches have also been utilized to test the role of the PH and PTB domains of the IRS proteins in their interactions with the IR and the subsequent tyrosine phosphorylations. Mutant IRS molecules in which either the PH or PTB domain have been deleted or replaced with homologous structures of other proteins have been expressed in mammalian cells (15Paz K. Voliovitch H. Hadari Y.R. Roberts Jr., C.T. LeRoith D. Zick Y. J. Biol. Chem. 1996; 271: 6998-7003Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 16Burks D.J. Pons S. Towery H. Smith-Hall J. Myers Jr., M.G. Yenush L. White M.F. J. Biol. Chem. 1997; 272: 27716-27721Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17Yenush L. Makati K.J. Smith H.J. Ishibashi O. Myers M.J. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18Voliovitch H. Schindler D.G. Hadari Y.R. Taylor S.I. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 18083-18087Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). These studies have documented a primary role for the PH domain in the subsequent ability of IRS-1 to be tyrosine phosphorylated in intact cells after insulin stimulation. In either in vitro studies or yeast two-hybrid systems, a primary role of the IRS-1 PTB domain has been documented in receptor interactions (15Paz K. Voliovitch H. Hadari Y.R. Roberts Jr., C.T. LeRoith D. Zick Y. J. Biol. Chem. 1996; 271: 6998-7003Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (321) Google Scholar). In the case of IRS-2, a more central region of the molecule has also been identified as playing a role in receptor interactions (20Sawka-Verhelle D. Tartare-Deckert S. White M.F. Van Obberghen E. J. Biol. Chem. 1996; 271: 5980-5983Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21He W. Craparo A. Zhu Y. O'Neill T.J. Wang L.M. Pierce J.H. Gustafson T.A. J. Biol. Chem. 1996; 271: 11641-11645Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Although numerous studies have documented the roles of the PH and PTB domains of the IRS proteins in the interactions with the IR and the subsequent tyrosine phosphorylation of the IRS proteins, no studies have tested the roles of these two domains on subsequent signals induced by the IRS·PI 3-kinase complex. Because mutations in the PH and PTB domains interfere with the interaction of the IRS with the IR and its subsequent tyrosine phosphorylation, it is impossible to determine the effect of these mutations on the downstream signals emanating from the IRS·PI 3-kinase complex. To overcome this obstacle, we now describe a constitutively active form of IRS-1. In this chimeric molecule, the inter-SH2 domain of the p85 regulatory subunit of PI 3-kinase is attached to various portions of the IRS-1 molecule. The inter-SH2 domain of p85 has previously been documented to bind to the 110-kDa catalytic unit of the PI 3-kinase (22Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar). Thus the resultant chimeric IRS-1 molecules now constitutively bind to the PI 3-kinase. By using these chimeric molecules, we have tested the role of different regions of the IRS-1 molecule in eliciting a subsequent biological response, the stimulation of the Ser/Thr kinase Akt. We demonstrate that the PH domain alone is sufficient to target the constitutively active PI 3-kinase to induce the activation of Akt and that this requires the lipid binding properties of the PH domain. DISCUSSIONThe principal mechanism whereby the insulin receptor activates PI 3-kinase appears to be via its ability to stimulate the tyrosine phosphorylation of various endogenous proteins including the different IRS molecules, IRS-1 to 4 (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar, 2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Activation of PI 3-kinase plays a critical role in subsequent biological responses, such as stimulation of glucose uptake and regulation of gene transcription (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar). A number of downstream targets of the PI 3-kinase and the lipids it generates have been identified. These include several Ser/Thr kinases such as the PDK-1, the family of Akt/PKB kinases, and several atypical protein kinase C including PKC ζ and λ. The exact role of these different kinases in eliciting subsequent biological responses has been debated (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar).Extensive studies have investigated the mechanism whereby the IRS proteins are localized to the insulin receptor. All four IRS molecules contain both a PH domain and a PTB domain (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar, 2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). The PH domain binds various phospholipids, including PI 4,5-P2 as well PI 3,4,5-P3 (9Takeuchi H. Matsuda M. Yamamoto T. Kanematsu T. Kikkawa U. Yagisawa H. Watanabe Y. Hirata M. Biochem. J. 1998; 334: 211-218Crossref PubMed Scopus (33) Google Scholar, 10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar). In addition some studies have suggested that the PH domain may also interact with various proteins (11Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, the PTB domains bind phosphotyrosines, in particular, a phosphotyrosine in the juxtamembrane region of the insulin receptor (12Margolis B. J. Lab. Clin. Med. 1996; 128: 235-241Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 13Wolf G. Trub 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). Based on the crystal structure of the PH and PTB domains of the IRS-1 molecule, a model has been proposed of the two domains binding cooperatively to localize IRS-1 at the membrane in association with the insulin receptor (14Dhe-Paganon S. Ottinger E.A. Nolte R.T. Eck M.J. Shoelson S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8378-8383Crossref PubMed Scopus (85) Google Scholar).In the present studies, we have tested the role of these two domains in the ability of the IRS-1 molecule to elicit a subsequent biological response, the activation of the Ser/Thr kinase Akt. To accomplish this, we produced novel chimeric molecules, which contain these two domains fused to the inter-SH2 domain of the p85 subunit of the PI 3-kinase. This peptide has previously been shown to be sufficient to bind and activate the PI 3-kinase (22Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar). By adding these residues to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules, which were constitutively bound to active PI 3-kinase. This was necessary because prior studies have shown that the PH domain of the IRS-1 molecule was critical for its subsequent tyrosine phosphorylation and association with the PI 3-kinase. By adding the inter-SH2 domain to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules that were active in the absence of insulin stimulation and receptor-mediated tyrosine phosphorylation.In the present work we have shown that the PH domain is critical for the subsequent ability of these constitutively active chimeric molecules to activate Akt. The most convincing data for this conclusion comes from the studies of the point mutants of the PH-PTB domain chimeric molecule. The wild type and mutants were expressed to comparable levels and had almost identical associated PI 3-kinase activities. However, the wild type activated the endogenous Akt whereas both the single and triple PH domain mutants had a dramatically decreased ability to activate the enzymatic activities of the endogenous Akt (i.e. the introduction of either a single mutation or 3 mutations in the PH domain caused an approximate 75% decrease in the ability of the PH-PTB construct to activate Akt). Both mutations also had a similar decreased ability to bind to PI 4,5-P2 in comparison to the wild-type protein when they were expressed as PH-PTB·GST fusion proteins. The simplest interpretation of these data are that the PH domain function (i.e. binding to PI 4,5-P2 or a negatively charged protein) is critical to elicit the downstream functions. This conclusion is also supported by the finding that a chimeric molecule with only the PH domain of IRS-1 was also capable of stimulating the activation of the endogenous Akt. The somewhat weaker activity of this chimera in comparison to the PH-PTB molecule is consistent with its lower expression and somewhat decreased amount of associated PI 3-kinase activity. Because the construct, which only contained the PTB domain was expressed at lower levels and therefore had lower associated PI 3-kinase activity, it is not possible to conclude from this construct alone whether the PTB domain alone would have been capable at higher levels to induce the activation of Akt. However, the finding that the PH-PTB domain chimeric molecules whose PH domain function were impaired by either single or triple point mutations were incapable of eliciting the activation of the Akt argue that the PTB domain alone would not be sufficient to elicit this response because the PTB domain function in these chimeric molecules is still intact.A potential role of the PH domain of IRS-1 is to help localize the protein in the correct intracellular compartment. To test this hypothesis, we examined the subcellular localization of the wild-type and two mutant chimeric molecules by biochemical fractionation of the cells. The wild-type PH-PTB chimeric molecule was found to be in the particulate fraction to a much greater degree then the chimeric molecules containing the mutations in the PH domain. These results are consistent with the recent report that the IRS-1 molecule is at least partially present in a particulate fraction of the cell, possibly because of its association with the cytoskeletal complex (29Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar) as well as with a recent report that the same mutant we have studied (R28C) blocks the insulin induced translocation of a IRS·GFP construct to the membrane (10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar). Thus, the PH domain may serve to localize the chimeric molecules in the subcellular localization required for the subsequent activation of Akt.In conclusion, the present work presents the first evidence that a functional PH domain of the IRS-1 molecule is required for its ability to signal to downstream molecules such as activation of Akt. This requirement is presumably because of the ability of the PH domain of IRS-1 to direct the PI 3-kinase to the correct subcellular compartment. Compromising the function of the PH domain, for example in insulin-resistant states, could therefore both decrease the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor as well as to link to subsequent downstream targets (2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). The principal mechanism whereby the insulin receptor activates PI 3-kinase appears to be via its ability to stimulate the tyrosine phosphorylation of various endogenous proteins including the different IRS molecules, IRS-1 to 4 (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar, 2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). Activation of PI 3-kinase plays a critical role in subsequent biological responses, such as stimulation of glucose uptake and regulation of gene transcription (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar). A number of downstream targets of the PI 3-kinase and the lipids it generates have been identified. These include several Ser/Thr kinases such as the PDK-1, the family of Akt/PKB kinases, and several atypical protein kinase C including PKC ζ and λ. The exact role of these different kinases in eliciting subsequent biological responses has been debated (5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1383) Google Scholar, 6Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (834) Google Scholar). Extensive studies have investigated the mechanism whereby the IRS proteins are localized to the insulin receptor. All four IRS molecules contain both a PH domain and a PTB domain (1White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar, 2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). The PH domain binds various phospholipids, including PI 4,5-P2 as well PI 3,4,5-P3 (9Takeuchi H. Matsuda M. Yamamoto T. Kanematsu T. Kikkawa U. Yagisawa H. Watanabe Y. Hirata M. Biochem. J. 1998; 334: 211-218Crossref PubMed Scopus (33) Google Scholar, 10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar). In addition some studies have suggested that the PH domain may also interact with various proteins (11Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, the PTB domains bind phosphotyrosines, in particular, a phosphotyrosine in the juxtamembrane region of the insulin receptor (12Margolis B. J. Lab. Clin. Med. 1996; 128: 235-241Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 13Wolf G. Trub 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). Based on the crystal structure of the PH and PTB domains of the IRS-1 molecule, a model has been proposed of the two domains binding cooperatively to localize IRS-1 at the membrane in association with the insulin receptor (14Dhe-Paganon S. Ottinger E.A. Nolte R.T. Eck M.J. Shoelson S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8378-8383Crossref PubMed Scopus (85) Google Scholar). In the present studies, we have tested the role of these two domains in the ability of the IRS-1 molecule to elicit a subsequent biological response, the activation of the Ser/Thr kinase Akt. To accomplish this, we produced novel chimeric molecules, which contain these two domains fused to the inter-SH2 domain of the p85 subunit of the PI 3-kinase. This peptide has previously been shown to be sufficient to bind and activate the PI 3-kinase (22Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar). By adding these residues to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules, which were constitutively bound to active PI 3-kinase. This was necessary because prior studies have shown that the PH domain of the IRS-1 molecule was critical for its subsequent tyrosine phosphorylation and association with the PI 3-kinase. By adding the inter-SH2 domain to the different domains of the IRS-1 molecule, we were able to produce chimeric molecules that were active in the absence of insulin stimulation and receptor-mediated tyrosine phosphorylation. In the present work we have shown that the PH domain is critical for the subsequent ability of these constitutively active chimeric molecules to activate Akt. The most convincing data for this conclusion comes from the studies of the point mutants of the PH-PTB domain chimeric molecule. The wild type and mutants were expressed to comparable levels and had almost identical associated PI 3-kinase activities. However, the wild type activated the endogenous Akt whereas both the single and triple PH domain mutants had a dramatically decreased ability to activate the enzymatic activities of the endogenous Akt (i.e. the introduction of either a single mutation or 3 mutations in the PH domain caused an approximate 75% decrease in the ability of the PH-PTB construct to activate Akt). Both mutations also had a similar decreased ability to bind to PI 4,5-P2 in comparison to the wild-type protein when they were expressed as PH-PTB·GST fusion proteins. The simplest interpretation of these data are that the PH domain function (i.e. binding to PI 4,5-P2 or a negatively charged protein) is critical to elicit the downstream functions. This conclusion is also supported by the finding that a chimeric molecule with only the PH domain of IRS-1 was also capable of stimulating the activation of the endogenous Akt. The somewhat weaker activity of this chimera in comparison to the PH-PTB molecule is consistent with its lower expression and somewhat decreased amount of associated PI 3-kinase activity. Because the construct, which only contained the PTB domain was expressed at lower levels and therefore had lower associated PI 3-kinase activity, it is not possible to conclude from this construct alone whether the PTB domain alone would have been capable at higher levels to induce the activation of Akt. However, the finding that the PH-PTB domain chimeric molecules whose PH domain function were impaired by either single or triple point mutations were incapable of eliciting the activation of the Akt argue that the PTB domain alone would not be sufficient to elicit this response because the PTB domain function in these chimeric molecules is still intact. A potential role of the PH domain of IRS-1 is to help localize the protein in the correct intracellular compartment. To test this hypothesis, we examined the subcellular localization of the wild-type and two mutant chimeric molecules by biochemical fractionation of the cells. The wild-type PH-PTB chimeric molecule was found to be in the particulate fraction to a much greater degree then the chimeric molecules containing the mutations in the PH domain. These results are consistent with the recent report that the IRS-1 molecule is at least partially present in a particulate fraction of the cell, possibly because of its association with the cytoskeletal complex (29Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar) as well as with a recent report that the same mutant we have studied (R28C) blocks the insulin induced translocation of a IRS·GFP construct to the membrane (10Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar). Thus, the PH domain may serve to localize the chimeric molecules in the subcellular localization required for the subsequent activation of Akt. In conclusion, the present work presents the first evidence that a functional PH domain of the IRS-1 molecule is required for its ability to signal to downstream molecules such as activation of Akt. This requirement is presumably because of the ability of the PH domain of IRS-1 to direct the PI 3-kinase to the correct subcellular compartment. Compromising the function of the PH domain, for example in insulin-resistant states, could therefore both decrease the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor as well as to link to subsequent downstream targets (2Pessin J.E. Saltiel A.R. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (661) Google Scholar). We thank Dr. Morris White for the IRS-1 cDNA, Dr. Garry Nolan for the Phoenix retroviral packaging cell line and the retroviral vectors, and Dr. Kozui Shii for antibodies to IRS-1 and-2.