Recombinant Human Insulin Receptor Substrate-1 Protein

Siemeister, Gerhard; Al-Hasani, Hadi; Klein, Helmut W.; Kellner, S; Streicher, Rüdiger; Krone, Wilhelm; Müller‐Wieland, Dirk

doi:10.1074/jbc.270.9.4870

Cited by 19 publications

(15 citation statements)

References 37 publications

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“…To analyze whether the C‐terminal tyrosine phosphorylation has an influence on the catalytic properties of the IR kinase domain (IRKD), we have constructed a kinase mutant, in which the two tyrosine residues (Tyr 1316 and Tyr 1322 ) were replaced by phenylalanine (IRKD‐Y2F). In the present study, we have compared the enzymatic properties of the mutant kinase with the wild‐type enzyme IRKD, a widely used model for the IR [16–21]. Our data indicate that the phosphorylation of the C‐terminal tyrosine residues appears to be play a role in fine‐tuning of the kinase activity.…”

Section: Introductionmentioning

confidence: 91%

Autophosphorylation of the two C‐terminal tyrosine residues Tyr¹³¹⁶ and Tyr¹³²² modulates the activity of the insulin receptor kinase in vitro

et al. 2000

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Previously, several studies have demonstrated that autophosphorylation of the C-terminal tyrosine residues (Tyr 1316 and Tyr 1322 ) affects the signaling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C-terminal phosphorylation in vitro, we have constructed, purified and characterized 45 kDa soluble insulin receptor kinase domains (IRKD), either with (IRKD) or without (IRKD-Y2F) the two C-terminal tyrosine phosphorylation sites, respectively. According to HPLC phosphopeptide mapping, autophosphorylation of the three tyrosines in the activation loop of the IRKD-Y2F kinase (Tyr 1146 , Tyr 1150 , and Tyr 1151 ) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the K m values for exogenous substrates. However, the mutation in IRKD-Y2F resulted in a decrease in the maximum velocities of the phosphotransferase reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD-Y2F led to an increase in the apparent K m values for ATP, suggesting a cross-talk of the C-terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, conformational changes of the enzyme following autophosphorylation were abolished by the removal of the two C-terminal tyrosines. These data suggest a regulatory role of the two C-terminal phosphorylation sites in the phosphotransferase activity of the insulin receptor. ß

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Section: Introductionmentioning

confidence: 91%

Autophosphorylation of the two C‐terminal tyrosine residues Tyr¹³¹⁶ and Tyr¹³²² modulates the activity of the insulin receptor kinase in vitro

et al. 2000

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“…Siemester et al (58) have reported that a 262-amino acid IRS-1 region comprising five tyrosine phosphorylation sites within YXXM motifs is an excellent substrate of the insulin receptor; and after this IRS-1 domain is tyrosine phosphorylated, it binds more tightly to the insulin receptor.…”

Section: F18mentioning

confidence: 99%

Differential Modulation of the Tyrosine Phosphorylation State of the Insulin Receptor by IRS (Insulin Receptor Subunit) Proteins

Solow

Harada

Goldstein

et al. 1999

Molecular Endocrinology

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In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR؉IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR؉IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR؉IRS-1

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“…It is not clear which receptor and IRS-1 domains mediate this stable association, nor what role this stable association plays in IRS-1 phosphorylation in intact cells. However, a recent study has suggested that regions in the C-terminal half of IRS-1 may play a role in binding to the insulin receptor [16]. Studies with the two-hybrid system have identified a domain present in IRS-2 that may contribute to receptor-IRS-2 interactions [ 171.…”

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confidence: 99%

In Vitro Binding and Phosphorylation of Insulin Receptor Substrate 1 by the Insulin Receptor

Backer

Wjasow

Zhang

1997

European Journal of Biochemistry

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Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor in most cells. The N terminus of IRS-1 contains a phosphotyrosine binding (PTB) domain and a pleckstrin homology (PH) domain, both of which have been identified as important for insulin-stimulated phosphorylation in intact cells. The PTB domain binds to a phosphorylated motif, NPEY(P)960, that is present in the juxtamembrane region of the insulin receptor. A direct interaction between the PH domain of IRS-1 and the receptor has not been demonstrated. In this study, we examine the role of the IRS-1 PTB and PH domains during IRS-1-receptor binding and IRS-1 phosphorylation in intact cells and in vitro. Abrogation of binding of the PTB domain to NPXY(P) by mutation of Tyr960 of the insulin receptor did not reduce the binding of phosphorylated IRS-1 to insulin receptors in intact cells, and had no effect on binding of insulin receptors to IRS-1 or on IRS-1 phosphorylation in vitro. We examined the phosphorylation and receptor binding of a mutant recombinant IRS-I that lacks the N-terminal PH domain (APH-IRS-1). Although phosphorylation of APH-IRS-1 by wild-type or [Ala960]insulin receptors was similar to that of IRS-1, binding of insulin receptor to APH-IRS-1 was markedly reduced relative to that to IRS-1. We conclude that stable association of IRS-1 with the insulin receptor is unaffected by disruption of PTB-domainTyr960 interactions but requires the IRS-1 PH domain, and that efficient phosphorylation of IRS-1 in intact cells correlates with the formation of stable receptor . IRS-1 complexes.Keywords: insulin receptor; insulin-receptor substrate-1 ; pleckstrin-homology domain ; insulin action.The insulin receptor is a ligand-activated tyrosine-specific protein kinase that mediates cellular responses to insulin (reviewed in [ 11). Unlike tyrosine-kinase receptors that dimerize after ligand binding, the insulin receptor exists as a heterotetramer composed of two a subunits and two membrane-spanning subunits. Activation of the receptor by autophosphorylation is followed by rapid tyrosyl phosphorylation of the endogenous substrates Shc and insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) [2-41. These phosphoproteins recruit signaling molecules that contain Src-homology-2 domains, including the lipid kinase p85lpl10 phosphatidylinositol 3'-kinase, the adapter Grb-2, and the tyrosine-specific phosphatase SHPTP2 [ 11. The recruitment of these signaling components activate multiple signaling pathways, which regulate metabolic and proliferative responses.The recognition of physiological substrates by the insulin receptor may in part result from the primary structures of potential phosphorylation sites, as K,, values for the phosphorylation of peptides derived from IRS-1 are significantly lower than for poly(G1u80Tyr20) or other model peptides [5]. However, phosphorylation of endogenous substrates appears to involve the phosphotyrosine-binding domain (PTB domain), which is present In the present study, we examine the contribution of inter...

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Recombinant Human Insulin Receptor Substrate-1 Protein

Cited by 19 publications

References 37 publications

Autophosphorylation of the two C‐terminal tyrosine residues Tyr¹³¹⁶ and Tyr¹³²² modulates the activity of the insulin receptor kinase in vitro

Autophosphorylation of the two C‐terminal tyrosine residues Tyr¹³¹⁶ and Tyr¹³²² modulates the activity of the insulin receptor kinase in vitro

Differential Modulation of the Tyrosine Phosphorylation State of the Insulin Receptor by IRS (Insulin Receptor Subunit) Proteins

In Vitro Binding and Phosphorylation of Insulin Receptor Substrate 1 by the Insulin Receptor

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Recombinant Human Insulin Receptor Substrate-1 Protein

Cited by 19 publications

References 37 publications

Autophosphorylation of the two C‐terminal tyrosine residues Tyr1316 and Tyr1322 modulates the activity of the insulin receptor kinase in vitro

Autophosphorylation of the two C‐terminal tyrosine residues Tyr1316 and Tyr1322 modulates the activity of the insulin receptor kinase in vitro

Differential Modulation of the Tyrosine Phosphorylation State of the Insulin Receptor by IRS (Insulin Receptor Subunit) Proteins

In Vitro Binding and Phosphorylation of Insulin Receptor Substrate 1 by the Insulin Receptor

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Autophosphorylation of the two C‐terminal tyrosine residues Tyr¹³¹⁶ and Tyr¹³²² modulates the activity of the insulin receptor kinase in vitro

Autophosphorylation of the two C‐terminal tyrosine residues Tyr¹³¹⁶ and Tyr¹³²² modulates the activity of the insulin receptor kinase in vitro