MOLECULAR-BASIS FOR THE SUBSTRATE-SPECIFICITY OF PROTEIN-KINASE-B - COMPARISON WITH MAPKAP KINASE-1 AND P70 S6 KINASE

The substrate specificity of protein kinase-B alpha (PKB alpha, also k nown as RAC kinase or Akt) mas investigated using synthetic peptide su bstrates related to the sequence surrounding the phosphorylation site on glycogen synthase kinase-3 (GSK3), The minimum sequence motif requi red for efficient phosphorylation was Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hyd, where Xaa is any amino acid, Yaa and Zaa are small residues other tha n glycine and Hyd is a bulky hydrophobic residue (Phe, Leu), The most effective substrate, Arg-Pro-Arg-Thr-Ser-Ser-Phe, was phosphorylated w ith a K-m of 5 mu M and V-max of 260 U/mg. PKB alpha phosphorylated hi stone H2B K-m 5 mu M, V-max 68 U/mg) specifically at Ser-36 which also lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif, The peptide Arg-Pro-Arg -Ala-Ala-Thr-Phe may be a relatively specific substrate for PKB alpha because, unlike other substrates, it is not phosphorylated by p70 S6 k inase or MAP kinase activated protein (MAPKAP) kinase-1.