Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k.

Activation of the phosphatidylinositol 3-kinase (PI3K) plays an important role in the mitogenic response of many cell types. Recently, two serine/threonine kinases Akt and p70(S6k) have been identified as physiological targets of PI3K. Observations that expression of activated forms of Akt led to the activation of p70(S6k) implied Akt might mediate mitogenic signaling through activation of p70(S6k). To clarify the relationship between signaling through these two kinases, we have examined their regulation by various mitogenic stimuli. In this study we have focused on the role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts. Depletion of intracellular calcium stores by EGTA pretreatment has no effect on growth factor-induced Akt activation but completely abolishes p70(S6k) stimulation. Increase of intracellular calcium induced by ionomycin or thapsigargin results in a full activation of p70(S6k), whereas little or no activation of Akt is observed. Furthermore, although PI3K in anti-phosphotyrosine immunoprecipitates is only very weakly activated by ionomycin, the calcium-induced stimulation of p70(S6k) is completely inhibited by the specific PI3K inhibitor wortmannin. We conclude Akt and p70(S6k) lie on separate signaling pathways. Activation of signaling to Akt is insufficient for the activation of p70(S6k), which can be achieved independently of Akt. p70(S6k) requires a separate calcium-dependent and wortmannin-sensitive process that is likely to be independent of type IA PI3K family members.