p21-Activated Kinase 1 Phosphorylates the Death Agonist Bad and Protects Cells from Apoptosis

Abstract

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-x_L. Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x_L and its association with 14-3-3τ. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-x_L and the increased association of Bad with 14-3-3τ. Our findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways.

ACKNOWLEDGMENTS

We acknowledge the expert technical assistance of Benjamin P. Bohl (TSRI), the support of Jon Chernoff (Fox Chase Cancer Center) and Klaus M. Hahn (TSRI), and secretarial assistance by Antonette Lestelle. Charles C. King provided much appreciated assistance with figure preparation. We thank S. J. Korsmeyer, M. E. Greenberg, and C. Thompson for providing reagents used in these studies.

This work was supported by Deutsche Forschungs Gemeinschaft fellowship Schu750/2-1 to A.S., U.S. Army grant DAMD 17-97-1-7230 to L.C.S. NIH grants CA-69381 (to J.C.R.) and AG15430 (to K. Hahn), and California Breast Cancer Research Program Award 3PB-0062 (to G.M.B.).