Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B ( PKB/ Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3- kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/ Akt activation is regulated by phosphoinositide phospholipid- mediated plasma membrane anchoring and by phosphorylation on Thr- 308 and Ser- 473. Whereas the Thr- 308 site is phosphorylated by PDK- 1, the identity of the Ser- 473 kinase has remained unclear and controversial. The integrin- linked kinase ( ILK) is a potential regulator of phosphorylation of PKB/ Akt on Ser- 473. Utilizing double- stranded RNA interference ( siRNA) as well as conditional knock- out of ILK using the Cre- Lox system, we now demonstrate that ILK is essential for the regulation of PKB/ Akt activity. ILK knock- out had no effect on phosphorylation of PKB/ Akt on Thr- 308 but resulted in almost complete inhibition of phosphorylation on Ser- 473 and significant inhibition of PKB/ Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/ Akt Ser- 473 phosphorylation was rescued by kinase- active ILK but not by a kinase- deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/ Akt phosphorylation. ILK knock- out also resulted in the suppression of phosphorylation of GSK- 3 beta on Ser- 9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/ Akt activation.