© The Rockefeller University Press,
0021-9525/1999//413 $5.00
The Journal of Cell Biology, Volume 144, Number 3,
, 1999 413-425
Evidence That Atypical Protein Kinase C-
and Atypical Protein Kinase C-
Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton
Florian Überall*,
Karina Hellbert*,
Sonja Kampfer*,
Karl Maly*,
Andreas Villunger*,
Martin Spitaler*,
James Mwanjewe*,
Gabriele Baier-Bitterlich*,
Gottfried Baier
, and
Hans H. Grunicke*
* Institute of Medical Chemistry and Biochemistry and
Institute of Medical Biology and Human Genetics, University of Innsbruck, A-6020 Innsbruck, Austria
Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes
and
, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-
and aPKC-
, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-
was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-
and aPKC-
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-
acts upstream of PI3K and Rac-1, whereas aPKC-
functions downstream of PI3K and Rac-1.
This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-
or aPKC-
results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-
was abrogated by coexpression of DN Rac-1 N17.
Key Words: Ha-Ras L61 atypical PKC F-actin Rac-1 PI3K
Abbreviations used in this paper: a, atypical; c, conventional; CA, constitutively active; DN, dominant-negative; GFP, green fluorescence protein; n, novel; PI3K, phosphatidylinositol-3' kinase; PKC, protein kinase C; PVDF, polyvinylene difluoride.
F. Überall and K. Hellbert contributed equally to this work.

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