Akt Protein Kinase inhibits GTP Rac1 binding through phosphorylation on the 71-serine residue of Rac1

다국어 초록 (Multilingual Abstract)

We noticed a putative Akt kinase phosphorylation site(^(64)ydRiRp1SYp^(73)) in Rac1/CDC42 and Rho family proteins(RhoA, B, C and G). To determine whether these proteins are phosphorylated by Akt kinase, we assayed Akt kinase with the recombinant Rac1 protein and the fluorescein Rac1 peptide(^(64)ydRiRp1SYp^(73)). We observed that the activated recombinant Akt kinase protein and SK-MEL 28 cell lysate(a human melanoma cell line) phosphorylates the fluorescein Rac1 peptide or the recombinant Rac1 protein. We also observed that Rac1 phosphorylation by Akt kinase inhibited its GTP binding(without GTPase activity change), whereas both GTP binding and GTPase activity of Rac1S71A(a mutant which serine residue, to be phosphorylated by Akt kinase, was replaced with alanine) were dramatically reduced, regardless of the treatment of Akt kinase. Furthermore, with the treatment of Wortmannin or LY294002(PI3 kinase inhibitor) to SK-MEL 28 cell, we noticed the down-regulation of both Akt kinase activity and Rac1 peptide phosphorylation, but the up-regulation of JNK/SAPK kinase activity. Thus, our observation suggested that Akt kinase of PI3 kinase signal transduction pathway phosphorylates on the 71-serine residue of Rac1 as one of its authentic substrate proteins, and modulates Rac1 signal transduction pathway through phosphorylation.