scholarly journals The Pleckstrin Homology and Phosphotyrosine Binding Domains of Insulin Receptor Substrate 1 Mediate Inhibition of Apoptosis by Insulin

1998 ◽  
Vol 18 (11) ◽  
pp. 6784-6794 ◽  
Author(s):  
Lynne Yenush ◽  
Christine Zanella ◽  
Tohru Uchida ◽  
Dolores Bernal ◽  
Morris F. White
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Receptors ◽  
Insulin Receptor Substrate ◽  
Phosphorylation Sites ◽  
Pleckstrin Homology ◽  
Insulin Stimulation ◽  
Kinase Cascade ◽  
Irs Proteins ◽  
Ptb Domains

ABSTRACT Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32DIR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32DIR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32DIR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH2-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32DIR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during 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 Tyrosine Phosphorylation of Insulin Receptor Substrate-1in VivoDepends upon the Presence of Its Pleckstrin Homology Region

1995 ◽  
Vol 270 (30) ◽  
pp. 18083-18087 ◽  
Author(s):  
Hedva Voliovitch ◽  
Daniel G. Schindler ◽  
Yaron R. Hadari ◽  
Simeon I. Taylor ◽  
Domenico Accili ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Pleckstrin Homology ◽  
Homology Region

scholarly journals Insulin Receptor Substrate 3 (IRS-3) and IRS-4 Impair IRS-1- and IRS-2-Mediated Signaling

2001 ◽  
Vol 21 (1) ◽  
pp. 26-38 ◽  
Author(s):  
Kaku Tsuruzoe ◽  
Renee Emkey ◽  
Kristina M. Kriauciunas ◽  
Kohjiro Ueki ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Protein Level ◽  
Fibroblast Cell ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Receptor Substrate ◽  
Akt Phosphorylation ◽  
Irs Proteins

ABSTRACT To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression (∼1.8- and ∼2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.

scholarly journals The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

1996 ◽  
Vol 16 (5) ◽  
pp. 2509-2517 ◽  
Author(s):  
L Yenush ◽  
R Fernandez ◽  
M G Myers ◽  
T C Grammer ◽  
X J Sun ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Human Insulin ◽  
Mammalian Cells ◽  
Insulin Response ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Receptor Substrate ◽  
Human Insulin Receptor ◽  
Irs Proteins ◽  
Drosophila Insulin Receptor

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.

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 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 Suppressor of Cytokine Signaling 1 (SOCS-1) and SOCS-3 Cause Insulin Resistance through Inhibition of Tyrosine Phosphorylation of Insulin Receptor Substrate Proteins by Discrete Mechanisms

2004 ◽  
Vol 24 (12) ◽  
pp. 5434-5446 ◽  
Author(s):  
Kohjiro Ueki ◽  
Tatsuya Kondo ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cultured Cells ◽  
Glycogen Synthesis ◽  
Cytokine Signaling ◽  
Insulin Receptor Substrate ◽  
Suppressor Of Cytokine Signaling ◽  
Necrosis Factor Alpha ◽  
Irs Proteins

ABSTRACT Insulin resistance is a pathophysiological component of type 2 diabetes and obesity and also occurs in states of stress, infection, and inflammation associated with an upregulation of cytokines. Here we show that in both obesity and lipopolysaccharide (LPS)-induced endotoxemia there is an increase in suppressor of cytokine signaling (SOCS) proteins, SOCS-1 and SOCS-3, in liver, muscle, and, to a lesser extent, fat. In concordance with these increases by LPS, tyrosine phosphorylation of the insulin receptor (IR) is partially impaired and phosphorylation of the insulin receptor substrate (IRS) proteins is almost completely suppressed. Direct overexpression of SOCS-3 in liver by adenoviral-mediated gene transfer markedly decreases tyrosine phosphorylation of both IRS-1 and IRS-2, while SOCS-1 overexpression preferentially inhibits IRS-2 phosphorylation. Neither affects IR phosphorylation, although both SOCS-1 and SOCS-3 bind to the insulin receptor in vivo in an insulin-dependent fashion. Experiments with cultured cells expressing mutant insulin receptors reveal that SOCS-3 binds to Tyr960 of IR, a key residue for the recognition of IRS-1 and IRS-2, whereas SOCS-1 binds to the domain in the catalytic loop essential for IRS-2 recognition in vitro. Moreover, overexpression of either SOCS-1 or SOCS-3 attenuates insulin-induced glycogen synthesis in L6 myotubes and activation of glucose uptake in 3T3L1 adipocytes. By contrast, a reduction of SOCS-1 or SOCS-3 by antisense treatment partially restores tumor necrosis factor alpha-induced downregulation of tyrosine phosphorylation of IRS proteins in 3T3L1 adipocytes. These data indicate that SOCS-1 and SOCS-3 act as negative regulators in insulin signaling and serve as one of the missing links between insulin resistance and cytokine signaling.

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