scholarly journals Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

1993 ◽  
Vol 13 (12) ◽  
pp. 7418-7428 ◽  
Author(s):  
X J Sun ◽  
D L Crimmins ◽  
M G Myers ◽  
M Miralpeix ◽  
M F White
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Regulatory Elements ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Insulin Stimulation ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Amino Terminal

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

scholarly journals Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1

1993 ◽  
Vol 13 (12) ◽  
pp. 7418-7428
Author(s):  
X J Sun ◽  
D L Crimmins ◽  
M G Myers ◽  
M Miralpeix ◽  
M F White
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Regulatory Elements ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Insulin Stimulation ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Amino Terminal

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

scholarly journals Determination of Gab1 (Grb2-Associated Binder-1) Interaction with Insulin Receptor-Signaling Molecules

1998 ◽  
Vol 12 (7) ◽  
pp. 914-923 ◽  
Author(s):  
Stéphane Rocchi ◽  
Sophie Tartare-Deckert ◽  
Joseph Murdaca ◽  
Marina Holgado-Madruga ◽  
Albert J. Wong ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Hybrid System ◽  
Sh2 Domain ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Two Hybrid ◽  
Phosphatidylinositol 3

Abstract The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.

scholarly journals Insulin receptor substrate 1 rescues insulin action in CHO cells expressing mutant insulin receptors that lack a juxtamembrane NPXY motif.

1995 ◽  
Vol 15 (9) ◽  
pp. 4711-4717 ◽  
Author(s):  
D Chen ◽  
D J Van Horn ◽  
M F White ◽  
J M Backer
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Glycogen Synthesis ◽  
Insulin Receptors ◽  
Insulin Receptor Substrate ◽  
Insulin Stimulation ◽  
Insulin Receptor Substrate 1 ◽  
Npxy Motif ◽  
Stimulation Of

Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK.

scholarly journals pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk.

1995 ◽  
Vol 15 (5) ◽  
pp. 2635-2645 ◽  
Author(s):  
M D Schaller ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Focal Adhesions ◽  
Sh2 Domain ◽  
Binding Motif ◽  
Adapter Protein ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
High Affinity Binding Site

Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine.

scholarly journals Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

1994 ◽  
Vol 14 (2) ◽  
pp. 1308-1321 ◽  
Author(s):  
M Autero ◽  
J Saharinen ◽  
T Pessa-Morikawa ◽  
M Soula-Rothhut ◽  
C Oetken ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Lymphocyte Activation ◽  
Sh2 Domain ◽  
Protein Tyrosine Kinases ◽  
Phosphotyrosine Phosphatase ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Family Protein ◽  
Ptpase Activity

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

scholarly journals Identification of SH2B2β as an Inhibitor for SH2B1- and SH2B2α-Promoted Janus Kinase-2 Activation and Insulin Signaling

Endocrinology ◽  
2007 ◽  
Vol 148 (4) ◽  
pp. 1615-1621 ◽  
Author(s):  
Minghua Li ◽  
Zhiqin Li ◽  
David L. Morris ◽  
Liangyou Rui
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Signaling ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Pleckstrin Homology Domain ◽  
Insulin Receptor Substrate ◽  
Janus Kinase 2 ◽  
Pleckstrin Homology ◽  
Insulin Receptor Substrate 1

The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the insulin receptor, respectively. JAK2 binds to various members of the cytokine receptor family, including receptors for GH and leptin, to mediate cytokine responses. In mice, SH2B1 regulates energy and glucose homeostasis by enhancing leptin and insulin sensitivity. In this work, we identify SH2B2β as a new isoform of SH2B2 (designated as SH2B2α) derived from the SH2B2 gene by alternative mRNA splicing. SH2B2β has a DD and pleckstrin homology domain but lacks a SH2 domain. SH2B2β bound to both SH2B1 and SH2B2α, as demonstrated by both the interaction of glutathione S-transferase-SH2B2β fusion protein with SH2B1 or SH2B2α in vitro and coimmunoprecipitation of SH2B2β with SH2B1 or SH2B2α in intact cells. SH2B2β markedly attenuated the ability of SH2B1 to promote JAK2 activation and subsequent tyrosine phosphorylation of insulin receptor substrate-1 by JAK2. SH2B2β also significantly inhibited SH2B1- or SH2B2α-promoted insulin signaling, including insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. These data suggest that SH2B2β is an endogenous inhibitor of SH2B1 and/or SH2B2α, negatively regulating insulin signaling and/or JAK2-mediated cellular responses.

scholarly journals The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4218-4227 ◽  
Author(s):  
LM Wang ◽  
P Michieli ◽  
WR Lie ◽  
F Liu ◽  
CC Lee ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Mitogenic Response ◽  
Gamma Chain ◽  
Insulin Receptor Substrate 1 ◽  
Gamma Subunit ◽  
Interleukin 13 ◽  
Sh2 Domains ◽  
Stimulation Of

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3- dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin- like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL- 13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

scholarly journals ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity.

1995 ◽  
Vol 181 (1) ◽  
pp. 375-380 ◽  
Author(s):  
N Isakov ◽  
R L Wange ◽  
W H Burgess ◽  
J D Watts ◽  
R Aebersold ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Critical Event ◽  
Binding Studies ◽  
Tyrosine Residues ◽  
Sh2 Domains

Engagement of the T cell antigen receptor (TCR) results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways. TCR-mediated activation of the src-family kinases, Lck and Fyn, results in tyrosine phosphorylation of the TCR zeta and CD3 chains. The site of phosphorylation in these chains is the tyrosine-based activation motif (TAM), a 15-16 amino acid module containing two tyrosine residues. Tyrosine-phosphorylated TAMs serve as targets for binding of the zeta-associated protein (ZAP-70) tyrosine kinase via its tandem SH2 domains. This binding correlates with activation of ZAP-70, a critical event in T cell activation. To further define the structural requirements for ZAP-70 interaction with the TCR, we developed a binding assay using immobilized glutathione S-transferase fusion proteins containing the NH2- and/or COOH-terminal SH2 domains of ZAP-70, and soluble synthetic peptides with the sequence of the cytoplasmic region of the TCR zeta chain (TCR zeta cyt) or individual TCR zeta and CD3 epsilon TAM motifs. Direct binding studies demonstrated that the tandem ZAP-70 SH2 domains bind phosphorylated, but not nonphosphorylated, TCR zeta cyt. The NH2-terminal ZAP-70 SH2 domain also binds to TCR zeta cyt but with 100-fold lower affinity. No binding was observed with the COOH-terminal ZAP-70 SH2 domain. Similar studies demonstrated that the ZAP-70 tandem SH2 domain can bind a TCR zeta 3 TAM peptide in which both tyrosine residues are phosphorylated: Little or no binding was observed with peptides phosphorylated at only one tyrosine residue, or a nonphosphorylated peptide. Binding of the tandem SH2 domains to the other two TCR zeta TAM peptides and to a CD3 epsilon TAM peptide was also observed. All four doubly tyrosine phosphorylated TAM peptides cross-compete with each other for binding to the tandem SH2 domains of ZAP-70. The affinity of these peptides for the tandem SH2 construct demonstrated a hierarchy of TAM zeta 1 > or = TAM zeta 2 > TAM epsilon > or = TAM zeta 3. The results provide further evidence that the ZAP-70 interaction with the TCR requires prior phosphorylation of both tyrosine residues within a TAM motif. Binding of ZAP-70 to phospho-TAMs is notable for the high level of cooperativity between the two SH2 domains, which individually demonstrate low affinity interaction with the ligand. The cooperativity ensures higher affinity for the doubly phosphorylated ligand. Affinity differences of as much as 30-fold indicates a significant specificity of interaction of ZAP-70 SH2 domains for different phospho-TAMs.

scholarly journals Insulin stimulates the tyrosine dephosphorylation of docking protein p130cas (Crk-associated substrate), promoting the switch of the adaptor protein Crk from p130cas to newly phosphorylated insulin receptor substrate-1

1998 ◽  
Vol 334 (3) ◽  
pp. 595-600 ◽  
Author(s):  
Andrey SOROKIN ◽  
Eleanor REED
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Stable Complex ◽  
Insulin Receptor Substrate ◽  
Dependent Manner ◽  
Insulin Receptor Substrate 1 ◽  
Docking Protein

The docking protein p130cas (Crk-associated substrate) forms a stable complex with the adaptor protein CrkII in a tyrosine-phosphorylation-dependent manner. Insulin-induced tyrosine phosphorylation of insulin receptor substrates results in the redistribution of CrkII between p130cas and insulin receptor substrate-1. A decrease in the association between CrkII and p130cas in response to insulin stimulation was detected in CHO cells stably expressing insulin receptor or insulin receptor substrate-1, and in L6 rat myoblasts. Along with the decrease in the association of CrkII with p130cas, the amount of tyrosine-phosphorylated insulin receptor substrate-1 co-precipitated with CrkII increased in all cell types studied. The insulin-induced decrease in the CrkII–p130cas association was further confirmed by Far Western Blot analysis with the Src homology 2 (SH2) domain of CrkII. Insulin regulates the association of CrkII with p130cas by tyrosine dephosphorylation of p130cas and co-ordinated tyrosine phosphorylation of insulin receptor substrate-1. Tyrosine-phosphorylated insulin receptor substrate-1 serves as a docking protein for multiple adaptor proteins and competes with p130cas for CrkII.

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