Cloning and Characterization of PHIP, a Novel Insulin Receptor Substrate-1 Pleckstrin Homology DomainInteracting Protein

Abstract

Insulin receptor substrate-1 (IRS-1) protein is a major substrate of the insulin receptor tyrosine kinase and is essential for transducing many of the biological effects of insulin including mitogenesis, gene expression, and glucose transport. The N terminus of IRS-1 contains a pleckstrin homology (PH) domain that is critical for recognition and subsequent phosphorylation of IRS-1 by the activated insulin receptor. Here we report the isolation of a novel protein, PHIP (PH-interacting protein), which selectively binds to the PH domain of IRS-1 in vitro and stably associates with IRS-1 in vivo. Importantly, mutants of the IRS-1 PH domain that disrupt the PH fold fail to bind to PHIP. Anti-phosphotyrosine immunoblots of PHIP revealed no discernible insulin receptor-regulated phosphorylation, suggesting that PHIP is not itself a substrate of the insulin receptor. In contrast to full-length PHIP, overexpression of the PHbinding region of PHIP has a pronounced inhibitory effect on insulin-induced IRS-1 tyrosine phosphorylation levels. Furthermore, expression of this dominantnegative PHIP mutant leads to a marked attenuation of insulin-stimulated mitogen-activated protein kinase activity. We conclude that PHIP represents a novel protein ligand of the IRS-1 PH domain that may serve to link IRS-1 to the insulin receptor. Upon ligand stimulation of insulin receptors (IR),1 IRS-1 is rapidly phosphorylated on multiple tyrosine residues, which serve as docking sites for the assembly and activation of Src homology 2-containing signaling proteins that function in eliciting many insulin-dependent biological responses (1). The N terminus of IRS-1 contains a PH domain followed by the structurally homologous phosphotyrosine-binding (PTB) domain that has been shown to cooperatively contribute in mediating productive receptor/substrate interactions (2-4). The PTB domain of IRS-1 binds directly to phosphorylated Tyr 960 within the NPEY motif in the juxtamembrane region of the activated IR (5, 6). However, the exact molecular mechanism by which the IRS-1 PH domain promotes receptor coupling is not known. Previous studies have demonstrated that deletion of the PH domain attenuates IRS-1 phosphorylation and subsequent insulin-mediated mitogenesis (2-4). Moreover, heterologous PH domains from the ␤-adrenergic receptor kinase, phospholipase C␥, or spectrin, known to promote plasma membrane targeting by binding phosphatidylinositol phospholipids, fail to restore IRS-1-specific signaling, suggesting that the IRS-1 PH domain is not simply a membrane-targeting device but may interact with specific cellular ligands (7). This ligand does not appear to be the insulin receptor itself as in vitro binding studies and yeast two hybrid analysis failed to detect a direct interaction between the IRS-1 PH domain and IR (8 -11). Although a recent report by Burks et al. (12) revealed that the IRS-1 PH domain can bind to acidic motifs in the nucleolar protein, nucleolin, physiological ligands for the IRS-1 PH domain have yet to be defined. In an attempt t...

Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program

“…Structural studies of PH domains have revealed common motifs that bind to phosphoinositides present in cellular membranes with different degrees of affinity and specificity (reviewed in Lemmon and Ferguson, 2000;Maffucci and Falasca, 2001;Lemmon et al, 2002). Although PH domains act to target proteins to phosphoinositide-rich membrane domains, some evidence supports a role for PH domains in protein-protein interactions (Burks et al, 1998;Farhang-Fallah et al, 2000). A full understanding of the physiological relevance of such interactions is still lacking and has not been addressed for Gab family members.…”

Role of IRS and PHIP on Insulin-Induced Tyrosine Phosphorylation and Distribution of IRS Proteins

“…It has been recently suggested using in vitro binding assays that phosphoinositides are the ligands for the PH domains of IRS proteins (Dhe-Paganon et al, 1999;Razzini et al, 2000), while two-hybrid analyses revealed that several proteins such as nucleolin and PHIP bind IRS PH domains (Burks et al, 1998;Farhang-Fallah et al, 2000). To test the roles of these in vitro ligands for the PH domains of IRS proteins in insulin-induced signal transduction, we firstly assessed the effects of the PH domains on tyrosine phosphorylation of IRS-1, -2, or -3, which revealed that none of the PH domains affected tyrosine phosphorylation of IRS-1~3 (data not shown).…”

“…Two-hybrid analysis recently revealed that PHIP binds the PH domains of IRS-1 and IRS-2, and that the expression of the PH-binding region of PHIP impairs tyrosine phosphorylation of IRS-1 leading to a marked attenuation of insulin actions such as mitogenesis and Glut4 translocation (Farhang-Fallah et al, 2000;Farhang-Fallah et al, 2002). To test whether the expression of the PH-binding region of PHIP affects tyrosine phosphorylation of each IRS molecule, we expressed IRS proteins, the mutant PHIP containing the N-terminal region interacting with the PH domain of IRS-1 (PHIP-N) and the human insulin receptor in Cos-1 Fig.…”

“…The cDNAs encoding the Cterminal FLAG tagged PH and PTB domain-containing region of IRS-1 (amino acid 1-270), IRS-2 (amino acid 1-293), or IRS-3 (amino acid 1-284) were also synthesized using PCR, and inserted into pcDNA3.1 (+) (Invitrogen) (pcDNA3.1-IRS1-N, pcDNA3.1-IRS2-N, and pcDNA3.1-IRS3-N, respectively). Human PHIP cDNA (Farhang-Fallah et al, 2000) was generated from human muscle cDNA (Clontech, Palo Alto, CA, USA) by PCR. Triple tandem FLAG tag sequence was inserted into the N-terminus of human PHIP cDNA containing the region interacting with the PH domain of IRS-1 (amino acid 1-209) (Kaburagi et al, 2001) and the expression vector (pcDNA3.1 (-), Invitrogen) containing the cDNA was constructed (pcDNA3.1-PHIP-N).…”

“…In their studies, the PH domain of IRS-1 preferentially binds to both PI(3,4,5)P3 and PI(4,5)P2 (Dhe-Paganon et al, 1999) or only PI(3,4,5)P 3 (Razzini et al, 2000), that of IRS-2 to PI(3,4)P3 (Razzini et al, 2000) and that of IRS-3 to PI(3)P3 (Razzini et al, 2000). Moreover, several proteins such as pleckstrin homology domain-interacting protein (PHIP) (Farhang-Fallah et al, 2000) have been reported to be the in vitro ligands for the PH domains of IRS proteins. However, the contributions of these candidate PH targets in insulininduced cell signaling mediated by each IRS protein in intact cells have not been critically evaluated.…”