IRS‐1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3′‐kinase, but p85 is not tyrosyl phosphorylated. IRS‐1 contains nine tyrosine phosphorylation sites in YXXM (Tyr‐Xxx‐Xxx‐Met) motifs. Formation of the IRS‐1‐PtdIns 3′‐kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3′‐kinase to IRS‐1 is mediated through the SH2 (src homology‐2) domains of p85. Furthermore, overexpression of IRS‐1 potentiates the activation of PtdIns 3‐kinase in insulin‐stimulated cells, and tyrosyl phosphorylated IRS‐1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3′‐kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS‐1 to the SH2 domains of p85 is the critical step that activates PtdIns 3′‐kinase during insulin stimulation.

In response to harsh environmental conditions, C. elegans larvae undergo dauer arrest at the second molt. The past decade has yielded many insights into the signaling pathways and the molecular mechanisms that govern this developmental transition. Dauer pheromone, the major physiologic signal promoting dauer arrest, has been purified, identified, and synthesized. The molecular identities of the vast majority of dauer regulatory genes isolated in initial genetic screens are now known. Physiologic ligands for DAF-12, a nuclear receptor that is the final common target of dauer regulatory pathways, have been identified. The discovery of the Hid (high temperature induction of dauer) phenotype and the results of enhancer screens have greatly expanded the repertoire of dauer regulatory genes. Genomic analysis of dauer arrest has highlighted the role of pathway crosstalk in dauer regulation. Nonetheless, critical questions remain about the mechanistic underpinnings of dauer arrest.

The C. elegans insulin/IGF-1 signaling (IIS) pathway connects nutrient levels to metabolism, growth, development, longevity, and behavior. This fundamental pathway is regulated by insulin-like peptide ligands that bind to the insulin/IGF-1 transmembrane receptor (IGFR) ortholog DAF-2. DAF-2/IGFR controls the activity of a conserved phosphoinositide 3-kinase (PI3K)/Akt kinase cascade, culminating in the regulation of a FoxO transcription factor, DAF-16, that governs most of the functions of this pathway. In light of the evolutionary conservation of the IIS pathway, its study in C. elegans is likely to shed light on its functions and regulation in higher organisms, including humans. Originally identified based on its role in the regulation of larval development and aging, IIS also controls a host of other biological processes. Here we review what is currently known about the biological functions and the molecular components of C. elegans IIS.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.

Activation of receptor-linked and cytoplasmic protein tyrosine kinases is crucial in the control of normal and abnormal cell growth and differentiation. Some substrates of protein tyrosine kinases such as phospholipase C gamma and ras GTPase-activating protein (GAP) contain sequences homologous to the src protein domains SH2 and SH3 (refs 3-9). The proto-oncogene vav is expressed in haematopoietic cells and its product Vav contains sequence motifs commonly found in transcription factors, such as helix-loop-helix, leucine-zipper and zinc-finger motifs and nuclear localization signals, as well as a single SH2 and two SH3 domains. Here we show that stimulation of T-cell antigen receptor on normal human peripheral blood lymphocytes or on human leukaemic T cells, and the crosslinking of IgE receptors on rat basophilic leukaemia cells, both promote the phosphorylation of tyrosine residues in Vav. Moreover, activation of the receptor for epidermal growth factor leads to marked tyrosine phosphorylation of Vav in cells transiently expressing vav, and Vav associates with the receptor through its SH2 domain. We propose that vav encodes a new class of substrates whose tyrosine phosphorylation may provide a mechanism for direct signal transduction linking receptors at the cell surface to transcriptional control.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a Agtll expression library, using the tyrsine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skohlik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells after growth factor treatment and the binding of GST-N-SH2 to activated growth factor receptors in vitro correlated with the amount of PI 3-kinase activity in anti-P-Tyr immunoprecipitates of lysates from HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-P-Tyr immunoprecipitates of cell lysates.However, tyrosine-phosphorylated p85 was not detectable in lysates of PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These results are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.The association of a phosphatidylinositol (PI) kinase activity with the v-src and v-ros oncoproteins (24, 43) raised the possibility that such an activity might be involved in cell transformation. This possibility was supported by studies on polyomavirus middle-T-antigen mutants that demonstrated a strong correlation between middle-T-antigen-associated PI kinase activity and transforming potential (18, 53). Demonstration that a similar PI kinase activity was induced by platelet-derived growth factor (PDGF) treatment of fibroblasts (2, 17, 52) and associated with activated PDGF receptors (9) implicated this activity as a potential participant in the normal mitogenic signaling pathway activated by PDGF. Biochemical characterization of the product of this PI kinase revealed a phospholipid which is phosphorylated at the 3' position of the inositol ring (51). This finding suggested that PI 3-kinase was part of a signal transduction pathway distinct from the inositol trisphosphate/diacylglycerol pathway mediated by phospholipase C cleavage of PI-4',5'-P2 (reviewed in reference 5). Studies with other mitogenic growth factors have shown that PI 3-kinase is also activated by colony-stimulating factor 1 (36, 39, 47), insulin (11,37), and epidermal growth factor (EGF) (4). PI 3-kinase activity has also been shown to be associated with the viral oncoproteins v-Abl, v-Crk, and v-Fps (14, 46). * Corresponding author. 981The presence of PI 3-kinase a...

Signalling proteins such as phospholipase C-y (PLC-y) or GTPase-activating expression, leading to DNA synthesis and cell growth and differentiation. Growth factor binding is followed by receptor dimerization, which is responsible for activation of the intrinsic protein-tyrosine kinase activity and receptor autophosphorylation (40). The intrinsic protein-tyrosine kinase activity was shown to be essential for the biological function of receptor tyrosine kinases (47). Several cellular proteins which directly interact with and are tyrosine phosphorylated by receptors with tyrosine kinase activity have been identified. These include phospholipase C--y (PLC-y), ras GTPase-activating protein (GAP), and pp60csn`(5, 47). Recent studies have demonstrated that these proteins bind to specific tyrosine-autophosphorylated regions in growth factor receptors via a conserved stretch of 100 amino acids called src-homology 2 (SH2) domains (20). SH2 domains were initially described as noncatalytic sequence motifs found in pp6c-sr and in other cytoplasmic tyrosine kinases (13,20,28). It has been shown that SH2 domains of signalling molecules bind to phosphorylated tyrosine residues flanked by specific short sequences (5,10,15,18,19,34,38). Different SH2 domains bind to different sequence motifs; SH2 domains of p85 bind to a YMXM motif (5), while the SH2 domains of PLC--y bind to a L/V XXXX EYL motif found in epidermal growth factor * Corresponding author.(EGF) and fibroblast growth factor (FGF) receptor (EGFR and FGFR) carboxy-terminal domains (34, 39). The binding of SH2 domains to tyrosine-phosphorylated regions of growth factor receptors is thought to provide a common mechanism by which diverse regulatory proteins can interact specifically with growth factor receptors, thereby coupling growth factor stimulation to multiple intracellular signalling pathways.SH2 domains are usually present in one or two copies per molecule and are often accompanied by a shorter conserved stretch of 50 amino acids, called the SH3 domain, whose function is still unknown (20). SH2 domains have been found in proteins with diverse enzymatic activities such as cytoplasmic tyrosine kinases (20), a tyrosine-specific phosphatase (41, 50), PLC--y (44), GAP (46,49) and PI-3 kinaseassociated protein p85 (GRB1) (11,35,42). SH2 domains were also found in protooncogene vav (4,16,17,29) and in the cytoskeletal protein tensin (9). At least three proteins, crk (30), Nck (22), and GRB2/sem-5 (7, 26), consist almost exclusively of SH2 and SH3 domains.In this report we show that Nck mRNA and proteins are widely expressed in various murine tissues and in cell lines from human, murine, and rat origins. Nck is phosphorylated on tyrosine, serine, and threonine residues in response to stimulation of EGF and platelet-derived growth factor (PDGF) in A431 and NIH 3T3 cells, respectively, and cross-linking of immunoglobulin E (IgE) receptors (FcsRI) in the rat basophilic leukemia cell line RBL-2H3. Like other SH2-containing proteins, Nck is associated with tyrosineautophosp...

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.Phosphatidylinositol 3-kinase (PI 3-kinase), an activity that phosphorylates PI at the 3'-OH of the inositol ring (63), was first identified as an activity that associates with a variety of viral oncoproteins (36,54,65). Studies with various mutants of polyomavirus middle T antigen demonstrated a correlation between association with PI 3-kinase and the ability to transform cells (15,26,58,65). The finding that PI 3-kinase also associates with activated growth factor receptor tyrosine kinases (14, 25, 61) implicated this activity as a potential participant in mitogenic signaling.PI 3-kinase phosphorylates PI, PI-4-P, and PI-4,5-P2 in vitro (8,38). Measurements of polyphosphoinositides in virally transformed cells and in cells treated with growth factors demonstrate elevations in levels of 4,9,13,20,24,34,43,44,46,52,58,60,61). Cells expressing nontransforming mutants of viral oncoproteins do not exhibit elevated levels of PI-3,4-P2 or 4,46,58,60), corroborating previous correlations between PI 3-kinase activation and the transformed phenotype. However, the observation that PI 3-kinase products are synthesized in response to nonmitogenic stimuli (31,55,56) suggests that PI 3-kinase is involved in physiological processes unrelated to mitogenesis. Since the products of PI 3-kinase are poor substrates for phospholipase C (35, 47), it has been suggested that they may constitute a second messenger pathway distinct from that involving the hydrolysis of PI-4,5-P2 to generate diacylglycerol and inositol trisphosphate (4).The association of PI 3-kinase activity with polyomavirus middle T antigen and antiphosphotyrosine immunoprecipitates from platelet-derived growth factor (PDGF)-treated fibroblasts correlated with the presence of an 85-kDa phosphoprotein (15, 25), suggesting th...