A balanced activation of regulatory subunits of phosphoinositide 3-kinase defines proliferation and survival in pancreatic beta-cells

Objectives: Signaling by the lipid kinase phosphoinositide-3 kinase (PI3K) is pivotal for beta-cell growth. PI3K is active as a heterodimer of regulatory and catalytic subunits, but is inhibited by an excess of free regulatory subunits. Alternative splicing of the Pik3r1 gene yields the three PI3K isoforms p85a, p55a and p50a. W examined signalling by p85a, p55a and p50a in insulinoma cells (INS-1E) stimulated by IGF-1.

Methods: Immunoprecipitation, immunoblotting, lipid kinase assays and FACS analysis were performed according to standard protocolls.

Results: Serial immunoprecipitations determined an excess of free regulatory isoform p85a compared with p110 catalytic isoforms. Si-RNA mediated knockdown of p85a by 50% elevated PKB phosphorylation. Conversely, a two-fold elevation of p85a and p55a levels by adenoviral gene transfer inhibited PKB phosphorylation indicating a negative regulatory role of free p85a and p55a on PI3K. In contrast, over-expression of p50a first elevated PKB phosphorylation and then decreased it in a dose dependent manner. These results indicate that free p50a acts as an dose-dependent activator or inhibitor of PI3K. Alterations in the relations between regulatory and catalytic subunits had profound impact upon cell cycle regulation. Reduction of p85a led to decreased S-phase. Over-expression of p50a led to a massive increase in S-phase in contrast to over-expressed p55a indicating that signalling by p50a is different from p55a. When INS-1E cells underwent apoptosis, p85a was reduced and p50a was markedly up regulated demonstrating that p50a may be a mediator of apoptosis. Indeed, we found that over-expression of p50a led to marked rise of MAPK p38, a mediator of apoptosis and stress response.

Conclusion: In INS-1E cells, excess of p85a and p55a inhibits PI3K and PKB. However, free p50a acts differently by activating or repressing PI3K in a dose-dependent manner and elevating S-phase in insulinoma cells. In addition, free p50a activates stress kinase p38 linking proliferation and apoptosis.