Characterization of the Met326Ile variant of phosphatidylinositol 3-kinase p85α

Phosphatidylinositol 3-kinase is a key step in the metabolic actions of insulin. Two amino acid substitutions have been identified in the gene for the regulatory subunit of human p85α, Met-326Ile, and Asn-330Asp, and the former has been associated with alterations in glucose/insulin homeostasis. When the four human p85α proteins were expressed in yeast, a 27% decrease occurred in the level of protein expression of p85α(Ile/Asp) (P = 0.03) and a 43% decrease in p85α(Ile/Asn) (P = 0.08) as compared with p85α(Met/Asp). Both p85α(Ile/Asp) and p85α(Ile/Asn) also exhibited increased binding to phospho-insulin receptor substrate-1 by 41% and 83%, respectively (P < 0.001), as compared with p85α(Met/Asp). The expression of p85α(Ile) was also slightly decreased and the binding to insulin receptor substrate-1 slightly increased in brown preadipocytes derived from p85α knockout mice. Both p85α(Met) and p85α(Ile) had similar effects on AKT activity and were able to reconstitute differentiation of the preadipocytes, although the triglyceride concentration in fully differentiated adipocytes and insulin-stimulated 2-deoxyglucose uptake were slightly lower than in adipocytes expressing p85α(Met). Thus, the Met-326Ile variant of p85α is functional for intracellular signaling and adipocyte differentiation but has small alterations in protein expression and activity that could play a role in modifying insulin action

ADP-ribosylation Factor 1 Controls the Activation of the Phosphatidylinositol 3-Kinase Pathway to Regulate Epidermal Growth Factor-dependent Growth and Migration of Breast Cancer Cells*S⃞

Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110α) to the plasma membrane, and the association of the regulatory subunit (p85α) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression

Cancer-derived mutations in the regulatory subunit p85α of phosphoinositide 3-kinase function through the catalytic subunit p110α

Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85α, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85α. The mutant proteins are still able to bind to the catalytic subunits p110α and p110β. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110α, and p110α is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85α and p110α while preserving the stabilizing interaction between p85α iSH2 and the adapter-binding domain of p110α