Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase-and heterotrimeric G-proteinlinked receptors. We report herein the cloning and characterization of p110␦, a novel class I PI3K. Like p110␣ and p110␤, other class I PI3Ks, p110␦ displays a broad phosphoinositide lipid substrate specificity and interacts with SH2͞SH3 domaincontaining p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110␣ and ␤, p110␦ is exclusively found in leukocytes. In these cells, p110␣ and ␦ both associate with the p85␣ and ␤ adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110␦ suggest divergent functional͞regulatory capacities for this PI3K. Unlike p110␣, p110␦ does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110␦ catalytic domain contains unique potential proteinprotein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110␦ in white blood cells are discussed.Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3Ј OH position of the inositol ring of inositol lipids, generating phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. PI3K enzymes have been identified in plants, slime molds, yeast, fruit flies, and mammals (1) and play a role in signal transduction via tyrosine kinase-and G-protein-linked receptors (2-5). In addition, PI3Ks have a function in membrane trafficking events, either constitutive or induced upon receptor stimulation (for review, see ref. 6).

We identified SH2-Balpha as an insulin-receptor-binding protein based on interaction screening in yeast hybrid systems and co-precipitation in cells. SH2-Balpha contains pleckstrin-homology ('PH') and Src homology 2 (SH2) domains and is closely related to APS (adapter protein with a PH domain and an SH2 domain) and lnk, adapter proteins first identified in lymphocytes. SH2-Balpha is ubiquitously expressed and is present in rat epididymal adipose tissue, liver and skeletal muscle, physiological sites of insulin action. On SDS/PAGE, SH2-Balpha migrates at a molecular mass of 98 kDa, although the predicted size of SH2-Balpha is 79.6 kDa. Insulin causes an electrophoretic mobility shift. SH2-Balpha can be immunoprecipitated using anti-(insulin receptor) antibody from insulin-stimulated cells. Anti-phosphotyrosine antibody or the growth factor receptor-binding protein 2 (Grb2) SH2 domain precipitate SH2-Balpha after insulin stimulation, suggesting that SH2-Balpha is tyrosine-phosphorylated and may be a substrate for the insulin receptor. The SH2-Balpha SH2 domain did not interact with insulin-receptor substrate (IRS) proteins or epidermal-growth-factor receptor. Mutation of the juxtamembrane and C-terminus of the insulin receptor did not abolish the interaction with the SH2 domain. This was further confirmed using a panel of activation-loop single point mutants where mutation of Tyr1158, Tyr1162 and Tyr1163 abolished interaction. Thus SH2-Balpha is a likely component in the insulin-signalling pathway and may function as an alternative signalling protein by interacting with the activation loop of the insulin-receptor cytoplasmic domain.

APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction.