Phosphatidylinositol 3‐kinase (PI 3‐kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein‐serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N‐terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3‐kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter‐subunit serine phosphorylation in the regulation of the PI 3‐kinase in vivo.

Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85a) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85a and a related protein p853 are shown to form stable complexes with recombinant pllO in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the pllO protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85ct protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the pllO protein defined 88 amino acids in the N-terminal region of pllO which mediate the binding of this subunit to either the p85a or the p853 proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel c-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed.

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Insulin, IGF‐1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3‐kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3‐kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate‐1 (IRS‐1) with PI 3‐kinase in a cell‐free system and also inhibited insulin‐ or IGF‐1‐induced, but not EGF‐induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild‐type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS‐1 with PI 3‐kinase in a cell‐free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3‐kinase activity, inhibited insulin‐ or IGF‐1‐induced membrane ruffling. These results suggest that the association of IRS‐1 with PI 3‐kinase followed by the activation of PI 3‐kinase are required for insulin‐ or IGF‐1‐induced, but not for EGF‐induced, membrane ruffling.

There are three conserved phosphorylation sites in protein kinase C (PKC) isotypes that have been termed priming sites and play an important role in PKC function. The requirements and pathways involved in novel (nPKC) phosphorylation have been investigated here. The evidence presented for nPKC␦ shows that there are two independent kinase pathways that act upon the activation loop (Thr-505) and a C-terminal hydrophobic site (Ser-662) and that the phosphorylation of the Ser-662 site is protected from dephosphorylation by the Thr-505 phosphorylation. Both phosphorylations require C1 domain-dependent allosteric activation of PKC. The third site (Ser-643) appears to be an autophosphorylation site. The serum-dependent phosphorylation of the Thr-505 and Ser-662 sites increases nPKC␦ activity up to 80-fold. Phosphorylation at the Ser-662 site is independently controlled by a pathway involving mammalian TOR (mTOR) because the rapamycin-induced block of its phosphorylation is overcome by co-expression of a rapamycin-resistant mutant of mTOR. Consistent with this role of mTOR, amino acid deprivation selectively inhibits the serum-induced phosphorylation of the Ser-662 site in nPKC␦. It is established that nPKC⑀ behaves in a manner similar to nPKC␦ with respect to phosphorylation at its C-terminal hydrophobic site, Ser-729. The results define the regulatory inputs to nPKC␦ and nPKC⑀ and establish these PKC isotypes downstream of mTOR and on an amino acid sensing pathway. The multiple signals integrated in PKC are discussed.