p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1 L and hTid-1 S forms of the human TID1 gene that exhibit opposing effects on apoptosis. We demonstrate that both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells.

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...

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