One potentially important mechanism for regulating class Ia phosphoinositide 3-kinase (PI 3-kinase) activity is autophosphorylation of the p85␣ adapter subunit on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit, as this downregulates the lipid kinase activity in vitro. Here we investigate whether this phosphorylation can occur in vivo. We find that p110␣ phosphorylates p85␣ Ser608 in vivo with significant stoichiometry. However, p110␤ is far less efficient at phosphorylating p85␣ Ser608, identifying a potential difference in the mechanisms by which these two isoforms are regulated. The p85␣ Ser608 phosphorylation was increased by treatment with insulin, platelet-derived growth factor, and the phosphatase inhibitor okadaic acid. The functional effects of this phosphorylation are highlighted by mutation of Ser608, which results in reduced lipid kinase activity and reduced association of the p110␣ catalytic subunit with p85␣. The importance of this phosphorylation was further highlighted by the finding that autophosphorylation on Ser608 was impaired, while lipid kinase activity was increased, in a p85␣ mutant recently discovered in human tumors. These results provide the first evidence that phosphorylation of Ser608 plays a role as a shutoff switch in growth factor signaling and contributes to the differences in functional properties of different PI 3-kinase isoforms in vivo.Numerous studies have documented the fundamental importance of class IA phosphoinositide 3-kinases (PI 3-kinases) for a multitude of cellular functions including cell survival, growth, proliferation, intermediary metabolism, and cytoskeletal rearrangements (7,37,45).PI 3-kinases catalyze the transfer of phosphate to the 3Ј-OH position of inositol lipids to produce phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ), which in turn act as second messengers by recruiting proteins containing pleckstrin homology (PH) domains to the plasma membrane to assemble signaling complexes (44). In addition to the lipid kinase activity, in vitro experiments have demonstrated that class I PI 3-kinases possess an intrinsic protein serine kinase activity (9,15,40,42). This protein kinase activity has attracted much interest, but its functional consequences in vivo have not been defined (24).The typical form of class IA PI 3-kinase is a heterodimer with an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (37). Two isoforms of the 85-kDa regulatory subunit have been identified: p85␣ and p85␤, which are products of different genes. Also, several splice variants of p85␣ exist. Furthermore, a third gene product, termed p55␥, has been identified. Three isoforms of the 110-kDa subunit have been identified in complex with p85: p110␣, p110␤, and p110␦. The ␣ and ␤ isoforms are widely expressed, whereas the ␦ isoform is expressed predominantly in leukocytes.It is well recognized that the activity of class-Ia PI 3-kinase is regulated by a range of mechanisms acting via the various modular domains...

Severe insulin resistance is found in a heterogeneous group of uncommon disorders characterised by acanthosis nigricans, impaired glucose tolerance or diabetes mellitus and in women, features of hyperandrogenism such as oligomenorrhoea and hirsutism [1±3]. The mechanisms underlying severe insulin resistance in human disease remain poorly understood, but mutations in the insulin receptor gene or autoantibodies to the insulin receptor are responsible in only a small minority of cases [4±6]. The increasing knowledge of the complexity of intracellular insulin signalling path- Diabetologia (2000) AbstractAims/hypothesis. Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. Methods. Phosphoinositide 3-kinase p85a regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85a were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85a-GST fusion proteins were examined by surface plasmon resonance. Results. The common p85a variant, Met 326 I1e, was identified in 9 of the 20 subjects. Functional studies of the Met 326 Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85a. A novel heterozygous mutation, Arg 409 Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg 409 Gln p85a subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15 %, p < 0.05, n = 5). The recruitment of Arg 409 Gln p85a into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85a showed identical binding to phosphopeptides in surface plasmon resonance studies. Conclusion/interpretation. Mutations in p85a are uncommon in subjects with syndromes of severe insulin resistance. The Met 326 Ile p85a variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg 409 Gln mutant suggests that it could contribute to the insulin resistance seen in this family. [Diabetologia (2000) 43: 321±331] Keywords Keywords Genetics, insulin signalling, phosphatidylinositol 3-kinase.

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