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

Protein kinase B (PKB; also known as Akt) is pivotal for beta-cell growth and survival. The main activator of PKB is the lipid kinase phosphinositide 3-kinase (PI3K), which is active as a heterodimer of regulatory and catalytic subunits and may be inhibited by an excess of free regulatory subunits. Alternative splicing of the Pik3r1 gene yields the three PI3K regulatory isoforms p85a, p55a and p50a. Since little is known how PI3K is regulated in pancreatic beta-cells, we examined signalling by p85a, p55a and p50a in insulinoma cells (INS-1E) stimulated by IGF-1. PI3K regulatory isoforms p85a, p55a and p50a were knocked down by siRNA transfection or overexpressed by adenoviral gene delivery in INS-1E cells stimulated with glucose and IGF-1. PI3K assays were performed using phosphoinositide as a substrate. Activation of PKB, GSK-3, p70S6K was determined by immunoblotting with specific antibodies. Cell cycle regulation and rate of apoptosis were determined by FACS. First, we demonstrated expression of PI3K regulatory isoforms p85a, p55a and p50a together with p110 catalytic units in INS-1E cells and rat islets. Serial immunoprecipitations determined an excess of free regulatory isoform p85a compared with catalytic isoform p110. Si-RNA mediated knockdown of p85a by 50% elevated PKB phosphorylation. Conversely, a two-fold elevation of p85a levels by adenoviral gene transfer inhibited PKB phosphorylation indicating a negative regulatory role of free p85a on PI3K. Overexpression of p55a also reduced PKB phosphorylation demonstrating that free p55a also inhibits PI3K activity. However, this was not the case for p50a since overexpression of p50a first elevated PKB phosphorylation and then decreased it in a dose dependent manner. In addition, excess p50a but not p85a or p55a activated stress kinase p38. These results indicate that free p50a acts as an activator or inhibitor of PI3K in a dose-dependent manner. The selective activation of p38 by free p50a provides a link between proliferation and apoptosis of pancreatic beta-cells