Natural variants of human p85α phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity

Abstract

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.

Regulation of Phosphoinositide 3-Kinase by Its Intrinsic Serine Kinase Activity In Vivo

“…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).…”

“…In the case of p110␣, this interaction has been shown to increase the lipid kinase activity above that caused by binding of the regulatory subunit to phosphotyrosine. With respect to the negative regulation of the PI 3-kinase activity, it has been reported that the p85 C-terminal SH2 domain binds to the enzymatic product PIP 3 . PIP 3 binding and tyrosine-phosphorylated peptide binding in the SH2 C-terminal domain have been shown to be mutually exclusive (32).…”

“…With respect to the negative regulation of the PI 3-kinase activity, it has been reported that the p85 C-terminal SH2 domain binds to the enzymatic product PIP 3 . PIP 3 binding and tyrosine-phosphorylated peptide binding in the SH2 C-terminal domain have been shown to be mutually exclusive (32). Thus, a redistribution of PI 3-kinase in the plasma membrane, caused by the displacement of phosphotyrosine by PIP 3 , was suggested as a potential feedback mechanism for the regulation of enzymatic activity.…”

In Vitro and In Vivo Studies of a Naturally Occurring Variant of the Human p85α Regulatory Subunit of the Phosphoinositide 3-Kinase

“…Swedish males after 20 years of follow-up from the age of 50 years to the age of 70 years. In vitro the Met326 and Ile326 variants of p85␣ have been shown to have similar basal binding and recruitment to phosphotyrosine-containing immunoprecipitates after insulin stimulation when coexpressed with the catalytic subunit p110␣ in HEK293 cells (14); the catalytic activities of the Met326-or Ile326-containing phosphotyrosine complexes were identical (14).…”

The Role of Single Nucleotide Polymorphisms of Untranslated Regions (Utrs) in Insulin Resistance Pathogenesis in Patients with Type 2 Diabetes

“…Described in present study results provide the first evidence for association of SNPs in UTRs of INSR and PIKR1 genes with genes expression rates. So far, all investigated in these genes SNPs were localized mainly in coding regions (Kusari et al, 1991;Baynes et al, 2000;Almind et al, 2002;Jamshidi et al, 2006;Højlund et al, 2006). Presented results stand for the first report where evaluation of SNPs in UTRs of INSR and PIK3R1 genes with gene expression levels was performed in peripheral lymphocytes and adipose tissues.…”