Two New Substrates in Insulin Signaling, IRS5/DOK4 and IRS6/DOK5
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m212430200
ISSN1083-351X
AutoresDongsheng Cai, Sirano Dhe‐Paganon, Peter A. Meléndez, Jongsoon Lee, Steven E. Shoelson,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoWe have identified two new human genes that encode proteins with tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at their amino termini. Because the other known PH-PTB proteins (insulin receptor substrates: IRS-1, IRS-2, IRS-3, and IRS-4, and the downstream of kinases: DOK-1, DOK-2, and DOK-3) are substrates of insulin and insulin-like growth factor (IGF)-1 receptors, we asked whether these new proteins, termed IRS5/DOK4 and IRS6/DOK5, might also have roles in insulin and IGF-1 signaling. Northern analyses indicate that IRS5/DOK4 is ubiquitously expressed but most abundant in kidney and liver. IRS6/DOK5 expression is highest in skeletal muscle. Both proteins are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics differ. Insulin receptor-phosphorylated IRS5/DOK4 associates with RasGAP, Crk, Src, and Fyn, but not phosphatidylinositol 3-kinase p85, Grb2, SHP-2, Nck, or phospholipase Cγ Src homology 2 domains, and activates MAPK in cells. IRS6/DOK5 neither associates with these Src homology 2 domains nor activates MAPK. IRS5/DOK4 and IRS6/DOK5 represent two new signaling proteins with potential roles in insulin and IGF-1 action. We have identified two new human genes that encode proteins with tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at their amino termini. Because the other known PH-PTB proteins (insulin receptor substrates: IRS-1, IRS-2, IRS-3, and IRS-4, and the downstream of kinases: DOK-1, DOK-2, and DOK-3) are substrates of insulin and insulin-like growth factor (IGF)-1 receptors, we asked whether these new proteins, termed IRS5/DOK4 and IRS6/DOK5, might also have roles in insulin and IGF-1 signaling. Northern analyses indicate that IRS5/DOK4 is ubiquitously expressed but most abundant in kidney and liver. IRS6/DOK5 expression is highest in skeletal muscle. Both proteins are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics differ. Insulin receptor-phosphorylated IRS5/DOK4 associates with RasGAP, Crk, Src, and Fyn, but not phosphatidylinositol 3-kinase p85, Grb2, SHP-2, Nck, or phospholipase Cγ Src homology 2 domains, and activates MAPK in cells. IRS6/DOK5 neither associates with these Src homology 2 domains nor activates MAPK. IRS5/DOK4 and IRS6/DOK5 represent two new signaling proteins with potential roles in insulin and IGF-1 action. To accomplish its pivotal role in maintaining in vivo metabolic homeostasis, insulin binds and activates insulin receptors present on insulin-responsive cells. Early cellular events initiated by insulin binding include receptor tyrosine kinase activation and phosphorylation of the insulin receptor substrates (IRSs). 1The abbreviations used are: IRS, insulin receptor substrates; MAP, mitogen-activated protein; MAPK, MAP kinase; IGF, insulin-like growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; SH, Src homology; PH, pleckstrin homology; PI 3-kinase, phosphatidylinositol 3-kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; DOK, downstream of kinase; CHO, Chinese hamster ovary; PTB, phosphotyrosine binding.1The abbreviations used are: IRS, insulin receptor substrates; MAP, mitogen-activated protein; MAPK, MAP kinase; IGF, insulin-like growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; SH, Src homology; PH, pleckstrin homology; PI 3-kinase, phosphatidylinositol 3-kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; DOK, downstream of kinase; CHO, Chinese hamster ovary; PTB, phosphotyrosine binding. Phosphorylated IRSs bind and activate SH2 domain enzymes to couple the activated receptors to such downstream metabolic effects as glucose uptake and glycogen and triglyceride synthesis and storage. IRS-1 is the prototype member of the IRS family. Based on shared domain architecture and phosphorylation by insulin and related IGF-1 receptors, the immediate family has been expanded to include three additional IRS proteins (IRS-2, IRS-3, and IRS-4) and three proteins referred to as downstream of kinase (DOK-1, DOK-2, and DOK-3). These seven proteins have similar amino-terminal targeting domains comprising tandem PH and PTB domains and carboxyl-terminal phosphorylation or “activation” domains which, when tyrosine-phosphorylated, dock SH2 domain proteins. The IRS and DOK proteins are expressed differentially in varying tissues and appear to have distinct but potentially overlapping cellular functions. IRS-1 and IRS-2 are widely expressed, including in tissues thought to be most important for glucose and lipid homeostasis (1Sun 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 (1284) Google Scholar, 2Sun X.J. Wang L.-M. Zhang Y. Yenush L. Myers M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (764) Google Scholar). Both proteins are expressed in muscle, liver, fat, and pancreatic islets, although IRS-1 appears to be more important in muscle metabolism, whereas IRS-2 may play greater roles in liver and islet β cells. Phosphorylated IRS-1 and IRS-2 both bind and activate the SH2 domain proteins PI 3-kinase, SHP2, and Grb2, although PI 3-kinase activation appears to be most important in insulin-mediated glucose homeostasis. Irs1–/– mice are small and insulin-resistant (predominantly in muscle) but in general do not develop diabetes (3Araki E. Lipes M.A. Patti M.-E. Bruning J.C. Haag B.I. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1093) Google Scholar, 4Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (904) Google Scholar). Irs2–/– mice develop diabetes due to combined insulin resistance (predominantly in liver) and a diminished insulin secretory capacity (5Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1334) Google Scholar); the females are infertile (6Burks D.J. de Mora J.F. Schubert M. Withers D.J. Myers M.G. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Nature. 2000; 407: 377-382Crossref PubMed Scopus (396) Google Scholar). IRS-3 expression in rodents is restricted primarily to fat, where it binds and activates PI 3-kinase and SHP2 (7Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Irs3–/– mice appear normal. IRS-3 has not been identified in the human genome. IRS-4 is expressed predominantly in brain and thymus, where it may bind PI 3-kinase and Grb2. Irs4–/– mice appear normal with the exception of reduced fertility (8Fantin V.R. Wang Q. Lienhard G.E. Keller S.R. Am. J. Physiol. 2000; 278: E127-E133Crossref PubMed Google Scholar). Although the DOK proteins have similar domain architectures, they can be distinguished from the IRS family based on sequence homology (see below) and functional interactions. DOK-1 is phosphorylated prominently in v-Src, v-Abl, and v-Fps transformed cells and in response to receptor tyrosine kinase activation (9Ellis C. Moran M. McCormick F. Pawson T. Nature. 1990; 343: 377-381Crossref PubMed Scopus (525) Google Scholar, 10Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; : 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 11Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-212Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 12Songyang Z. Yamanashi Y. Liu D. Baltimore D. J. Biol. Chem. 2001; 276: 2459-2465Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These kinases are different from those typically associated with the IRS activation. DOK-1 was discovered through its association with RasGAP, an SH2 domain-containing protein that does not associate significantly with the IRSs and appears to interfere with MAP kinase activation downstream from B cell and FcγRIIb receptor activation (13Yamanashi Y. Tamura T. Kanamori T. Yamane H. Nariuchi H. Yamamoto T. Baltimore D. Genes Dev. 2000; 14: 11-16PubMed Google Scholar). Less is known about DOK-2 and DOK-3, although these too appear to associate with RasGAP and Nck (14Di Cristofano A. Carpino N. Dunant N. Friedland G. Kobayashi R. Strife A. Wisniewski D. Clarkson B. Pandolfi P.P. Resh M.D. J. Biol. Chem. 1998; 273: 4827-4830Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 15Jones N. Dumont D.J. Curr. Biol. 1999; 9: 1057-1060Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Jones N. Dumont D.J. Oncogene. 1998; 17: 1097-1108Crossref PubMed Scopus (133) Google Scholar, 17Cong F. Yuan B. Goff S.P. Mol. Cell. Biol. 1999; 19: 8314-8325Crossref PubMed Scopus (95) Google Scholar, 18Lemay S. Davidson D. Latour S. Veillette A. Mol. Cell. Biol. 2000; 20: 2743-2754Crossref PubMed Scopus (155) Google Scholar). All three DOK proteins appear to have functions in lymphocytes and myeloid cells. Targeted deletion of DOK-1 in mice has no overt phenotype, possibly due to a compensatory effect of DOK-2 or DOK-3 (13Yamanashi Y. Tamura T. Kanamori T. Yamane H. Nariuchi H. Yamamoto T. Baltimore D. Genes Dev. 2000; 14: 11-16PubMed Google Scholar). Knockouts of DOK-2 or DOK-3 have not been reported. We have identified two additional members of the IRS/DOK family in the human genome data base, based on their having amino-terminal PH and PTB domains, and we characterized these proteins in terms of potential functions in insulin and IGF-1 signaling. The recently reported DOK-4 and DOK-5 proteins may be the mouse orthologs (19Grimm J. Sachs M. Britsch S. Di Cesare S. Schwarz-Romond T. Alitalo K. Birchmeier W. J. Cell Biol. 2001; 154: 345-354Crossref PubMed Scopus (138) Google Scholar), although expression patterns and potential biological functions appear to be distinct. cDNA Isolation and Plasmid Construction—Full-length cDNAs encoding the human proteins were amplified from a skeletal muscle cDNA library (Clontech) by PCR methods using primers 5′-CGGAATTCATGGCGACCAATTTCAGTGAC-3′ and 5′-CCGCTCGAGTCACTG GGATGGGGTCTTG-3′ (for IRS5/DOK4) and 5′-CGGAATTCATGGCTTCCAATTTTAATGACATAG-3′ and 5′-CCGCTCGAGTCAGTGCTCAGATCTGTAGG-3′ (for IRS6/DOK5). EcoRI and XhoI restriction sites were incorporated at the 5′ ends. PCR products were purified by agarose gel electrophoresis and sequenced. Vectors for expression of FLAG-tagged proteins in eukaryotic cells were generated by inserting the IRS5/DOK4 and IRS6/DOK5 cDNAs into pCMV-Tag2 (Stratagene). Northern Blot Analyses—IRS5/DOK4 and IRS6/DOK5 cDNAs were used as probes for Northern blot analyses. cDNAs were excised from the cloning vector, labeled with [α-32P]dCTP (PerkinElmer Life Sciences) by the random hexamer method (Invitrogen), and purified by PCR purification (Qiagen). Human multiple tissue Northern blots were purchased from Clontech. Filters were serially hybridized with a human IRS5/DOK4 and IRS6/DOK5 probes using ExpressHyb hybridization solution (Clontech) at 68 °C for 2 h. Membranes were washed twice with 2× SSC containing 0.1% SDS at room temperature for 20 min and twice with 0.1× SSC containing 0.1% SDS at 55 °C for 20 min, and exposed to x-ray film overnight at –80 °C. Cell Culture and Transfections—CHO-IR (20Yamada K. Goncalves E. Kahn C.R. Shoelson S.E. J. Biol. Chem. 1992; 267: 12452-12461Abstract Full Text PDF PubMed Google Scholar) and CHO-IGF1R (21Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) cells were maintained in F-12 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma) in the presence of 0.4 mg/ml G-418 and 2 mm glutamine under 7.5% CO2 and at 37 °C. HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in the presence of 2 mm glutamine and penicillin/streptomycin under 5% CO2. Cells at 50–60% confluence were transfected with pCMV(IRS5/DOK4), pCMV(IRS6/DOK5), pCMV(IRS-1), or salmon sperm DNA as a control, using FuGENE 6 (Roche Applied Science). Cells were incubated for 24 h, serum-starved overnight, and stimulated with different doses of insulin and sodium pervanadate (prepared by mixing 10 mg of sodium vanadate (Sigma) with 5.8 μl of 30% H2O2 in 535 μl of H2O). Cells were washed (PBS containing 1.0 mm phenylmethylsulfonyl fluoride, 3.0 μm aprotinin, 10 μm leupeptin, 5.0 μm pepstatin A, 25 mm benzamidine, 25 mm sodium vanadate, 5.0 mm glycerol phosphate, 100 mm NaF, 1.0 mm ammonium molybdate, 30 mm tetrasodium pyrophosphate, 5 mm EGTA) and lysed for immunoprecipitation and Western blotting in lysis buffer (30 mm HEPES, 150 mm NaCl, 1.0 mm phenylmethylsulfonyl fluoride, 3.0 μm aprotinin, 10 μm leupeptin, 5.0 μm pepstatin A, 25 mm benzamidine, 25 mm sodium vanadate, 5.0 mm glycerol phosphate, 100 mm NaF, 1.0 mm ammonium molybdate, 30 mm tetrasodium pyrophosphate, 5.0 mm EGTA, 10% glycerol, and 1% Triton X-100, pH 7.4). Immunoprecipitation and Western Blotting—Proteins were immunoprecipitated by incubating cell lysates for 4 h at 4 °C with immobilized anti-FLAG (Stratagene) or anti-IRS-1 and anti-IR antibodies coupled to protein A-Sepharose beads (Amersham Biosciences). The beads were washed extensively, and proteins eluted with SDS sample buffer were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Blots were probed with the indicated antibodies, and proteins were detected by chemiluminescence (Pierce). Protein bands were scanned using a densitometer (Amersham Biosciences), and relative amounts were quantified (ImageQuant 5.1). Antibodies were from Upstate Biotechnology, Inc., anti-phosphotyrosine (4G10); Stratagene, anti-FLAG; Santa Cruz Biotechnology, anti-IRα, anti-Src, anti-Fyn, anti-GAP, anti-CrkII, and anti-Erk1; and Cell Signaling Technology, anti-pMAPK. Horseradish peroxidase- or alkaline phosphatase-conjugated sheep anti-rabbit or sheep anti-mouse IgG (Amersham Biosciences) were used as second antibodies for Western blotting. SH2 Domain Binding—Escherichia coli BL21 cells (Invitrogen) transformed with GST-SH2 vectors were grown to optical densities (600 nm) of 0.6–0.8. Isopropyl-1-thio-d-galactopyranoside (0.1 mm) was added, and the bacteria were incubated for 4 h at 37 °C, harvested, resuspended in lysis buffer (PBS-CMF, 0.5 m NaCl, 2 mm EDTA, 10 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 25 mm benzamidine, pH 7.4), and sonicated on ice. Debris was removed by centrifugation, and the lysate was incubated 60 min at 4 °C with glutathione-agarose (Molecular Probes). After washing with PBS-CMF containing 10 mm dithiothreitol and 0.5 m NaCl, GST fusion proteins were analyzed on a SDS-PAGE by Coomassie Blue staining. Equivalent amounts of the immobilized GST-SH2 fusion proteins were incubated for 4 h at 4 °C with cell lysates prepared from transfected, insulin-stimulated (10–7m, 15 min) CHO-IR cells. Proteins were eluted from the washed beads with Laemmli sample buffer, separated by SDS-PAGE, and detected by immunoblotting. Domain Architecture and Structural Homology—The human genome data base was searched for genes encoding new PH-PTB domain proteins with potential roles in receptor tyrosine kinase signaling. Two genes, located on chromosomes 16q13/21 and 20, were identified. The encoded proteins contain 326 and 306 residues, respectively, and have predicted molecular masses of 37.1 and 35.5 kDa (Fig. 1A). Both of the proteins have amino-terminal PH and PTB domains and short carboxyl-terminal tails containing a few tyrosine motifs. Because all known proteins with tandem PH and PTB domains have been categorized either as IRS or DOK proteins, we wondered whether these new members of the family functioned downstream from insulin receptors and whether they were more related to IRS or DOK proteins. The PH domain of the larger protein, referred to as IRS5/DOK4, shares 22% identity with IRS-1 and IRS-2, 15–19% identity with IRS-3 and IRS-4, and 16–21% identity with DOK-1, DOK-2, and DOK-3 (Fig. 1B). The PH domain of the smaller protein, IRS6/DOK5, shares 20–25% identity with IRSs and 14–23% identity with DOKs. These levels of PH domain identity are lower than those shared by the IRS (33–58%) or DOK (40–45%) proteins within their own families (Fig. 1B). Because the PH domains of IRS5/DOK4 and IRS6/DOK5 are 60% identical, we conclude that on the basis of PH domain identity, these two newly identified proteins are more closely related to one another than they are to either the IRS or DOK families. Similar comparisons were made between PTB domain sequences. The IRS5/DOK4 PTB domain shares 20–22% identity with the IRS domains and 30–34% identity with those of the DOKs. The IRS6/DOK5 PTB domain is 21–23% identical with the IRS domains and 31–37% identical the DOK domains. By contrast, IRS PTB domain sequences are 41–76% identical to each other, and the DOK PTB domain sequences are 43–53% identical. Therefore, based as well on PTB domain sequence comparisons, the IRS5/DOK4 and IRS6/DOK5 proteins are more related to each other (74% identity) than to the IRS or DOK proteins. Another short region of homology is shared between IRS5/DOK4 and IRS6/DOK5. The high identity of the PRSAYWHHIT (where Y is Tyr269) and PRSAYWQHIT (where Y is Tyr267) motifs of these proteins suggests a conserved function, possibly binding by Tyr269 or Tyr267, respectively, to SH2 domain proteins. Each protein contains additional tyrosines outside of this shared motif. IRS5/DOK4 has a total of five tyrosines outside of its PH-PTB targeting region within its putative carboxyl-terminal phosphorylation domain: Tyr255, Tyr257, Tyr269, Tyr286, and Tyr291. The EHYSYPCTP sequence encompassing Tyr255 and Tyr257 resembles the HEYIYVDPV sequence of the platelet-derived growth factor receptor that binds the SH2 domains of Src and closely related kinases (22Mori S. Ronnstrand L. Yokote K. Engstrom A. Courtneidge S.A. Claesson-Welsh L. Heldin C.H. EMBO J. 1993; 12: 2257-2264Crossref PubMed Scopus (295) Google Scholar). IRS6/DOK5 contains a total of three tyrosines in its carboxyl-terminal phosphorylation domain, Tyr282 and Tyr302 in addition to Tyr267. The 286YAGE and 291YGAA sequences of IRS5/DOK4 and the 282YRLQ and 302YRSE sequences of IRS6/DOK5 do not appear to conform to ideal motifs for SH2 recognition (23Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2381) Google Scholar, 24Songyang Z. Shoelson S.E. McGlade J. Olivier J.P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (833) Google Scholar). Northern Analyses of IRS5/DOK4 and IRS6/DOK5 Expression—Patterns of gene expression were explored using the full-length cDNAs to probe multiple human tissue mRNA blots (Fig. 2). The IRS5/DOK4 and IRS6/DOK5 probes hybridized transcripts of ∼3.0 and 2.2 kb, respectively (Fig. 2). IRS5/DOK4 expression was greatest in kidney and liver, with low levels of expression in essentially all other tissues. IRS6/DOK5 expression was very high in muscle, lower in brain, heart, and kidney, and virtually undetectable in other tissues. These are interesting expression patterns in terms of insulin action as muscle and liver are two of the most important systemic targets of insulin. Insulin-stimulated Tyrosine Phosphorylation of IRS5/DOK4 and IRS6/DOK5—To be categorized as genuine “IRSs, ” the proteins would need to be tyrosine-phosphorylated by IR in addition to having the appropriate PH-PTB domain architecture. Because we have not yet been successful in generating useful anti-IRS5/DOK4 or anti-IRS6/DOK5, we expressed FLAG-tagged versions of IRS5/DOK4 and IRS6/DOK5 in HEK293 cells. Cells transfected with pCMV(IRS5/DOK4) or pCMV(IRS6/DOK5) were exposed for 20 min to 10 nm insulin or 10 nm IGF-1, lysed, and FLAG-tagged IRS5/DOK4 or IRS6/DOK5 were immunoprecipitated with anti-FLAG antibodies. Western blots developed with anti-phosphotyrosine antibodies revealed significant increases in tyrosine phosphorylation of both proteins, in response to either insulin (Fig. 3A) or IGF-1 (Fig. 3B). Endogenous insulin and IGF-1 receptors in the 293 cells apparently phosphorylate IRS5/DOK4 or IRS6/DOK5. Additional studies conducted in CHO-IR cells determined the concentration dependence for insulin phosphorylation. Cells transfected with pCMV(IRS5/DOK4) or pCMV(IRS6/DOK5) were exposed for 15 min to 0 to 10–6m insulin and lysed, and FLAG-tagged IRS5/DOK4 and IRS6/DOK5 were immunoprecipitated with anti-FLAG antibodies. Peak phosphorylation of both proteins was observed at 10–7m insulin (Fig. 4A), the same concentration of insulin that maximally stimulated IR phosphorylation (Fig. 4B). The anti-FLAG antibodies failed to precipitate phosphoproteins from control cell lysates (data not shown). The time course for IRS5/DOK4 and IRS6/DOK5 phosphorylation was similarly studied using CHO-IR cells transfected either with pCMV(IRS5/DOK4) or pCMV(IRS6/DOK5). Amounts of the tyrosine-phosphorylated proteins were determined in cells treated for 0–40 min with 10–7m insulin. Phosphorylation of IRS5/DOK4 increased quickly, reaching half-maximum within 2–5 min (Fig. 5A, left panel). By contrast, IRS6/DOK5 phosphorylation increased linearly over the entire 40-min period (Fig. 5, A, right panel, and C). IRS5/DOK4 phosphorylation more closely matched IR phosphorylation, which was near maximal in these cells within 2 min (Fig. 5B), whereas IRS6/DOK5 phosphorylation proceeded more slowly (Fig. 5C). The expression of IRS5/DOK4 or IRS6/DOK5 did not affect IR phosphorylation. Effects of Phosphatase Inhibition on IRS5/DOK4 and IRS6/DOK5 Phosphorylation—Because net levels of phosphorylation are balanced by phosphatases, which counter the actions of kinases, we wondered whether the dissimilar kinetics for IRS5/DOK4 and IRS6/DOK5 phosphorylation might be affected differentially by protein-tyrosine phosphatase inhibition. To test this cells were treated with 1 μm pervanadate along with the insulin. In the case of IRS5/DOK4, the kinetics of insulin-stimulated phosphorylation changed significantly, becoming more linear over the 30-min time course studied (Fig. 6). By contrast, there were no discernible effects on IRS6/DOK5 phosphorylation (data not shown). The low levels of pervanadate by itself, in the absence of insulin, did not significantly stimulate the phosphorylation of IRS5/DOK4 (Fig. 6), although higher concentrations (50–100 μm) of pervanadate do increase IRS5/DOK4 and IRS-1 phosphorylation in the absence of insulin (data not shown). These findings suggested that IRS5/DOK4 might be particularly susceptible to dephosphorylation by a pervanadate-inhibitable phosphatase. Additional studies further evaluated the effects of pervanadate on IRS5/DOK4 and IRS6/DOK5 phosphorylation in comparison with related effects on IRS-1 phosphorylation. CHO-IR cells expressing IRS5/DOK4, IRS6/DOK5, or IRS-1 were treated with insulin (10–7m, 15 min) and/or pervanadate (1.0 μm, 15 min). Phosphorylation of IRS5/DOK4 and IRS-1 was significantly augmented (Fig. 7A), whereas phosphorylation of IRS6/DOK5 was unaffected (Fig. 7B). Therefore, insulin-stimulated tyrosine-phosphorylation of IRS5/DOK4 is countered by a pervanadate-inhibitable phosphatase, analogous to the situation for IRS-1 (Fig. 7, A and B). By contrast, insulin-stimulated phosphorylation of IRS6/DOK5 accumulates more slowly (Fig. 5) and is much less prone to dephosphorylation by a pervanadate-inhibitable phosphatase (Fig. 7B). IGF-1-Stimulated Tyrosine Phosphorylation of IRS5/DOK4 and IRS6/DOK5—Another characteristic of the IRS proteins is their capacity to be phosphorylated by the IGF-1 receptor (IGF-1R), which is closely related to IR in terms of primary sequence, three-dimensional structure, and mechanism. We already knew that IGF-1 stimulated the phosphorylation of IRS5/DOK4 or IRS6/DOK5 in HEK293 cells (Fig. 3). To establish further similarities and differences between the IRSs and IRS5/DOK4 or IRS6/DOK5, IGF-1 receptor-expressing CHO cells (CHO-IGF-1R) were transfected with pCMV(IRS5/DOK4) or pCMV(IRS6/DOK5). Amounts of tyrosine-phosphorylated IRS5/DOK4 and IRS6/DOK5 were determined in cells treated for 0–40 min with 10–7m IGF-1. Phosphorylation of IRS5/DOK4 increased quickly, within 2 min, reached a maximum within 5–10 min, and dropped again following longer stimulation times (Fig. 8). IRS6/DOK5 phosphorylation occurred rapidly as well and decreased correspondingly at longer stimulation times. SH2 Domain Binding to Insulin-stimulated IRS5/DOK4 and IRS6/DOK5—SH2 domain binding experiments were used to begin delineating potential signaling pathways downstream from IRS5/DOK4 and IRS6/DOK5. CHO-IR cells transfected with pCMV(IRS5/DOK4) or pCMV(IRS6/DOK5) were exposed for 15 min to 10–7m insulin in the presence of 1 μm sodium pervanadate; cell lysates were prepared, and phosphorylated proteins were precipitated with GST-SH2 fusion proteins bound to glutathione-agarose beads. Tyrosine-phosphorylated IRS5/DOK4s were precipitated by isolated SH2 domains from Src, Fyn, and Crk and by the SH2/SH3/SH2 region of RasGAP (Table I). In each case IRS5/DOK4 binding was insulin-dependent, as no protein was precipitated from lysates of unstimulated cells. By contrast, tyrosine-phosphorylated IRS5/DOK4 was not precipitated by Grb2 or Nck SH2 domains or by tandem SH2 domains from PI 3-kinase p85, SHP2, or phospholipase Cγ (Table I). Identical experiments conducted with pCMV(IRS6/DOK5)-transfected CHO-IR cells demonstrated that under these conditions, IRS6/DOK5 does not associate with any of these SH2 domain proteins (Table I).Table ISH2 domain bindingProteinDomainIRS5/DOK4IRS6/DOK5PI 3-kinase p85SH2-SH2--SHP2SH2-SH2--RasGAPSH2-SH3-SH2++-PLCγSH2-SH2--Grb2SH2--CrkSH2++-NckSH2--SrcSH2++-FynSH2++- Open table in a new tab Co-immunoprecipitation of IRS5/DOK4 with Src, Fyn, CrkII, and RasGAP—Subsequent experiments asked whether the SH2 domain pull-down results translated into corresponding interactions in cells. CHO-IR cells were transfected with pCMV(IRS5/DOK4) and stimulated for 20 min with to 10–7m insulin in the presence of 1 μm sodium pervanadate. Cell lysates were prepared, and proteins precipitated with anti-Src, anti-Fyn, anti-CrkII, and anti-RasGAP antibodies were separated by SDS-PAGE. Western blotting with anti-FLAG antibodies identified IRS5/DOK4 in all four immunoprecipitates. Insulin and pervanadate stimulated the association in all four cases (Fig. 9A), although lesser amounts of IRS5/DOK4 were also present in the anti-Fyn and anti-RasGAP immunoprecipitates from unstimulated cells. The SH3 domains of Fyn or RasGAP, rather than their SH2 domains, could mediate such constitutive association, although this was not formally tested. Western blotting with anti-phosphotyrosine antibodies further demonstrated that the immunoprecipitated IRS5/DOK4 was tyrosine-phosphorylated (Fig. 9B), as expected for SH2 domain-mediated interactions. Participation of IRS5/DOK4 in Insulin-mediated Activation of MAPK—Findings from the GST-SH2 domain pull-down and co-immunoprecipitation experiments prompted a further investigation of potential cellular consequences of SH2 domain protein activation. Pathways leading through RasGAP, Crk, Src, or Fyn could potentially feed into the MAP kinase (MAPK) cascade. We therefore looked at MAPK activation in insulin-stimulated CHO-IR cells that either were or were not transfected with pCMV(IRS5/DOK4). There was a left shift in the insulin dose response of MAPK activation, as well as an increase in its magnitude, in cells transfected with IRS5/DOK4 compared with cells transfected with control DNA (Fig. 10). These consistent findings in multiple experiments further suggested that IRS5/DOK4 may play a relevant role in insulin signaling. Now that all proteins in the human genome with tandem PH-PTB domain architectures are known, we can attempt to subcategorize the two new ones, IRS5/DOK4 and IRS6/DOK5, as either insulin receptor substrates (IRS) or downstream of kinase (DOK) proteins. The identification of IRS5/DOK4 and IRS6/DOK5 brings the total to nine: four IRS proteins (IRS-1–4), three DOK proteins (DOK-1–3), IRS5/DOK4, and IRS6/DOK5. We seriously doubt that additional genes encoding PHPTB domain proteins exist in the human or mouse genomes. Our main reason for being interested in IRS5/DOK4 and IRS6/DOK5 was to determine whether they function, like the IRS and DOK proteins, in insulin action. Although the lack of useful antibodies left us unable to look at the endogenous proteins, Northern analyses showed that the corresponding mRNAs are expressed in relevant and interesting tissues. IRS5/DOK4 message is expressed in highest abundance in kidney and liver. As major sites of in vivo glycogen storage and glucose production, these tissues are responsible for maintaining normal glucose levels during periods of fasting. A diminished capacity of insulin to suppress hepatic glucose production in type 2 diabetes contributes to hyperglycemia. IRS6/DOK5 message is strongly expressed in muscle, with much less expressed in the other human tissues tested. Muscle is the primary site of in vivo glucose disposal. Suppression of insulin-stimulated glucose disposal occurs in insulin resistance and may contribute to hyperglycemia in type 2 diabetes. Liver and muscle and possib
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