PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

Abstract

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.