Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding

Piccione, E.; Case, Randi D.; Domchek, Susan M.; Hu, Patrick J.; Chaudhuri, Manas; Backer, Jonathan M.; Schlessinger, Joseph; Shoelson, Steven E.

doi:10.1021/bi00064a001

Cited by 165 publications

(123 citation statements)

References 48 publications

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“…We cotransfected a variety of GFP-PIKE-L constructs into HEK293 cells with HA-p85 and Myc-p110, respectively. In the absence of the regulatory subunit of PI3-kinase, p85, no PI3-kinase activity was observed, despite which PIKE-L construct was transfected, consistent with the previous reported results (1,15). The strongest PI3-kinase activity occurs when WT PIKE-L and both subunits are cotransfected.…”

Section: Ph Domain Of Pike Mediates Its Activation Of Pi3-kinase and supporting

confidence: 91%

Phosphoinositol lipids bind to phosphatidylinositol 3 (PI3)-kinase enhancer GTPase and mediate its stimulatory effect on PI3-kinase and Akt signalings

Liu

2005

Proc. Natl. Acad. Sci. U.S.A.

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Phosphatidylinositol 3 (PI3)-kinase enhancer (PIKE) is a nuclearGTPase that enhances PI3-kinase activity in a GTP-dependent manner. Both PIKE-L and -A isoforms contain GTPase, pleckstrin homology (PH), ADP ribosylation factor-GTPase-activating protein, and two ankyrin repeats domains, and C-terminal ADP ribosylation factor-GTPase-activating protein activates its internal GTPase activity. However, whether PH domain modulates the intramolecular action and subsequently influences its downstream signalings remains elusive. Here we show that PH domain from PIKE-L robustly binds PI(3,4,5)P3 and exclusively resides in the nucleus. By contrast, the mutant (K679,687N), unable to bind phosphoinositol lipids, translocates to the cytoplasm. This mutation substantially compromises the stimulatory effects on PI3-kinase by PIKE-L. Surprisingly, PH domain from PIKE-A distributes in the cytoplasm. Similar mutation in PH domain of PIKE-A abolishes its binding to PI(3,4,5)P3 and significantly decreases its activation of Akt. Moreover, amplified PIKE-A from human cancers contains mutations and highly stimulates Akt kinase activity, correlating with its GTPase activity. Thus, phosphatidylinositols regulate PIKE GTPase activity, mediating its downstream PI3-kinase͞Akt signaling through a feedback mechanism by binding to its PH domain.pleckstrin homology domain ͉ nuclear translocation ͉ negative feedback ͉ GTPase activation P hosphatidylinositol 3 (PI3)-kinase enhancer (PIKE) is a recently identified brain-specific nuclear GTPase that binds PI3-kinase and stimulates its lipid kinase activity. PIKE activates nuclear PI3-kinase in a GTP-dependent manner (1). Nerve growth factor elicits membrane-associated 4.1N nuclear translocation and interaction with PIKE, which was initially identified as a 4.1N-binding partner in a yeast two-hybrid screening. The binding of 4.1N to PIKE prevents its interactions with nuclear PI3-kinase, leading to inactivation of nuclear PI3-kinase. To date, three forms of PIKE have been characterized, PIKE-S, PIKE-L, and PIKE-A. PIKE-S is the initially identified shorter isoform (1). PIKE-L, a longer isoform of PIKE gene, differs from PIKE-S by C-terminal extension containing ADP ribosylation factor-GTPase activating protein (Arf-GAP) and two ankyrin repeats domains. In contrast to the exclusive nuclear localization of PIKE-S, PIKE-L occurs in both the nucleus and the cytoplasm (2). In addition, we show that WT merlin, binds PIKE-L and inhibits PI3-kinase activity. This suppression of PI3-kinase activity results from merlin disrupting the binding of PIKE-L to PI3-kinase (3). Recently, a third PIKE isoform, PIKE-A, was identified in human glioblastoma multiforme brain cancers. We have reported that PIKE-A is coamplified with cyclin-dependent kinase 4 in a variety of human cancers. Unlike the brain-specific PIKE-L and -S isoforms, PIKE-A distributes in various tissues. PIKE-A contains the same domains present in PIKE-L but lacks N-terminal proline-rich domain, which binds PI3-kinase and PLC-␥1. Instead, PIKE-A specif...

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Section: Ph Domain Of Pike Mediates Its Activation Of Pi3-kinase and supporting

confidence: 91%

Phosphoinositol lipids bind to phosphatidylinositol 3 (PI3)-kinase enhancer GTPase and mediate its stimulatory effect on PI3-kinase and Akt signalings

Liu

2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The peptides used were: zeta1=NQLpYNELNLGRRGEpYDVLDKR, lnk=HLRAIDNQpYTPLSQL and Dlnk=DNQpYTP-LSQL. These were synthesized by FMOC chemistry and HPLC puri®ed before use (Piccione et al, 1993).…”

Section: Peptides and Reagentsmentioning

confidence: 99%

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

et al. 1998

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Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated z chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/ 38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/ 38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.

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“…Phosphopeptide Interactions-Phosphopeptides containing Tyr(P)-953 (LpYASSNPEYLSASDV), Tyr(P)-960 (SSNPEpYLSASD), Tyr(P)-1146 (DIpYETDYYRKG), Tyr(P)-1150 (DIYETDpYYRKG), Tyr(P)-1151 (DIYETDYpYRKG), Tyr(P)-1316 (KRSpYEEHIPY), and Tyr(P)-1322 (HIPpYTHMNGG) were immobilized on Affi-Gel 10 (Bio-Rad) (25), washed extensively with 20 mM Tris, 100 mM NaCl, pH 7.4, and incubated with 10 g of GST-SH2 for 2 h at 22°C in a total volume of 200 l. After washing the bound proteins were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining.…”

Section: Construction Of Fusion Proteins and Proteinmentioning

confidence: 99%

Interaction between the Grb10 SH2 Domain and the Insulin Receptor Carboxyl Terminus

Hansen

Svensson

Zhu

et al. 1996

Journal of Biological Chemistry

100

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Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor ␤-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.The insulin receptor plays important roles in metabolism and growth regulation of target tissues (1-3). Upon insulin stimulation the receptor becomes autophosphorylated on at least six or seven tyrosine residues located in the juxtamembrane, kinase, and carboxyl-terminal regions of its ␤-subunit (4 -7). Many signals are relayed through the insulin receptor substrate-1 (IRS-1), 1 which is phosphorylated by the receptor on various tyrosine residues (8). Phosphorylation of a tyrosine at position 960 in the juxtamembrane region of the insulin receptor is essential for signaling through IRS-1 (9, 10). Among the proteins that bind to IRS-1 are the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), Grb2, the tyrosine phosphatase Syp/SHPTP2, and Nck (2). IRS-1 is required for insulin-mediated mitogenesis (11, 12); however, studies using homozygous IRS-1 knockout mice predict important IRS-1-independent insulin signaling pathways (13,14). A promising candidate, the structurally closely related protein IRS-2, was recently identified, which has signaling functions similar to IRS-1 (15, 16). The Shc proteins represent additional receptor tyrosine kinase substrates that mediate p21 ras activation in the mitogenic pathway of insulin action (17). We employed the yeast two-hybrid system to identify signaling mediators for alternative pathways analogous to signals emerging from other receptor tyrosine kinases. Here we describe the identification of Grb10 as an IRS-1-independent interactive protein of the activated insulin receptor, and we define the sites of interaction in the receptor and Grb10. MATERIALS AND METHODSAntibodies-The antibody against the insulin re...

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Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding

Cited by 165 publications

References 48 publications

Phosphoinositol lipids bind to phosphatidylinositol 3 (PI3)-kinase enhancer GTPase and mediate its stimulatory effect on PI3-kinase and Akt signalings

Phosphoinositol lipids bind to phosphatidylinositol 3 (PI3)-kinase enhancer GTPase and mediate its stimulatory effect on PI3-kinase and Akt signalings

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

Interaction between the Grb10 SH2 Domain and the Insulin Receptor Carboxyl Terminus

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