Evidence that atypical protein kinase C-lambda and atypical protein kinaseC-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morph ological alterations which include a disassembly of actin stress fibers. Th e Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of th e atypical aPKC isotypes lambda and zeta, whereas lower concentrations of t he inhibitor which block conventional and novel PKC isotypes are ineffectiv e. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant- negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense co nstructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the act in cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively acti ve (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers independent of oncogenic Ha-Ras, Coexpression of (DN) Rac -1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with p lasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a st imulation of the kinase activity of both enzymes. Furthermore, the Ras-medi ated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.