The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells

The differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk-cell formation in the lower eukaryote Dictyostelium discoideu m. This molecule has been shown to inhibit cell growth and induce erythroid differentiation in human leukemia K562 cells. In the present study, to cla rify the mechanism of the actions of DIF-1, we examined the effect of DIF-1 on Akt/protein kinase B (PKB) in K562 cells. Akt/PKB is a serine/threonine kinase that plays a pivotal role in the regulation of cell survival and di fferentiation in a variety of cells. A nonphosphorylated (inactive) form of Akt/PKB was ordinarily expressed in K562 cells. However, Akt/PKB was phosp horylated and potently activated within several hours of incubation with 5- 30 mu M DIF-1, and this activation was inhibited by wortmannin, an inhibito r of phosphatidylinositol 3-kinase (PI3-kinase). Calcium-increasing agents thapsigargin and A23187 also activated Akt/PKB slightly, which was inhibite d by wortmannin. By contrast, calcium-reducing agents TMB-8 and EGTA togeth er with A23187 inhibited the DIF-1-induced activation of Akt/PKB. PMA (PKC activator) also activated Akt/PKB but this activation was not inhibited by wortmannin. DIF-1 exhibited no marked effect on the activation of PKC alpha , beta, and gamma, which were activated by PMA These results indicate that DIF-1 activates Akt/PKB possibly via cytosolic calcium and subsequent activ ation of PI3-kinase and also that PMA activates Akt/PKB in a PI3-kinase-ind ependent manner. (C) 1999 Academic Press.