Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase - Critical roles for kinase activity and amino acids arginine211 and serine 343

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. T o become fully activated, PKB/Akt requires phosphorylation at two sites, th reonine 308 and serine 473, in a phoshpatidylinositol (PI) 3-kinase-depende nt manner. The kinase responsible for phosphorylation of threonine 308 is t he PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serin e 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has als o been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dep endent manner. Whether ILK phosphorylates this site directly or functions a s an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectro metry that biochemically purified ILK can phosphorylate PKB/Akt directly. C o-immunoprecipitation analysis of cell extracts demonstrates that ILK can c omplex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB ass ociation. The amino acid residue serine 343 of ILK within the activation lo op is required for kinase activity as well as for its interaction with PKB/ Akt. Mutational analysis of ILK further shows a crucial role for arginine 2 11 of ILK within the phosphoinositide phospholipid binding domain in the re gulation of PKB- serine 473 phosphorylation. A highly selective small molec ule inhibitor of ILK activity also inhibits the ability of ILK to phosphory late PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.