Positive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene products of the p85 alpha regulatory subunit

Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metab olic actions, including stimulation of glucose transport and glycogen synth esis. The gene for the p85 alpha regulatory subunit yields three splicing v ariants, p85 alpha, AS53/p55 alpha, and p50 alpha. All three have (i) a C-t erminal structure consisting of two Src homology 2 domains flanking the p11 0 catalytic subunit-binding domain and (ii) a unique N-terminal region of 3 04, 34, and 6 amino acids, respectively. To determine if these regulatory s ubunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditio ns, we expressed each regulatory subunit in fully differentiated L6 myotube s using adenovirus-mediated gene transfer with or without coexpression of t he p110 alpha catalytic subunit. PI 3-kinase activity associated with p50 a lpha was greater than that associated with p85 alpha or AS53, Increasing th e level of p85 alpha or AS53, but not p50 alpha, inhibited both phosphotyro sine-associated and p110-associated PI 3-kinase activities. Expression of a p85 alpha mutant lacking the p110-binding site (Delta p85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated act ivity. Insulin stimulation of two kinases downstream from PI-3 kinase, AM a nd p70 S6 kinase (p70(S6K)), was decreased in cells expressing p85 alpha or AS53 but not in cells expressing p50a. Similar inhibition of PI 3-kinase, Akt, and p70(S6K) was observed, even when p110 alpha was coexpressed with p 85 alpha or AS53, Expression of p110 alpha alone dramatically increased glu cose transport but decreased glycogen synthase activity. This effect was re duced when p110 alpha was coexpressed with any of the three regulatory subu nits. Thus, the three different isoforms of regulatory subunit can relay th e signal from IRS proteins to the p110 catalytic subunit with different eff iciencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform, These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the c atalytic subunit or interaction with additional signaling molecules.