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

Uberall F, Hellbert K, Kampfer S, Maly K, Villunger A, Spitaler M, Mwanjewe J, Baier-Bitterlich G, Baier G, Grunicke HH - J. Cell Biol. (1999)

Bottom Line: 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 lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes.Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Chemistry and Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria. florian.ueberall@uibk.ac.at

ABSTRACT
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 lambda and zeta, 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-lambda and aPKC-zeta, 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-alpha 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-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 plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

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Evidence that atypical aPKC-λ acts upstream and  aPKC-ζ acts downstream of Rac-1. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras  L61 (A and B). (A) Ha-Ras L61 alone, or (B) together with  DN Rac-1 N17; (C) (CA) aPKC-λ A119E together with (DN)  Rac-1 N17; or (D) (CA) aPKC-ζ A119E together with (DN)  Rac-1 N17. 48 h posttransfection, cells were fixed and visualized  as described in Materials and Methods. Representative cells of at  least three different experiments are shown for all panels. Stacks  of images were exported into Adobe Photoshop and printed on a  color laser copier system.
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Figure 6: Evidence that atypical aPKC-λ acts upstream and aPKC-ζ acts downstream of Rac-1. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61 (A and B). (A) Ha-Ras L61 alone, or (B) together with DN Rac-1 N17; (C) (CA) aPKC-λ A119E together with (DN) Rac-1 N17; or (D) (CA) aPKC-ζ A119E together with (DN) Rac-1 N17. 48 h posttransfection, cells were fixed and visualized as described in Materials and Methods. Representative cells of at least three different experiments are shown for all panels. Stacks of images were exported into Adobe Photoshop and printed on a color laser copier system.

Mentions: As should be expected, DN N17 Rac-1 inhibits the Ras-mediated disassembly of stress fibers (Fig. 6, A and B). To obtain some information whether PKC-λ and PKC-ζ cooperate with Rac-1 in the same pathway, cells were cotransfected with combinations of either CA aPKC-λ A119E and N17 Rac-1 or CA aPKC-ζ A119E and N17 Rac-1.


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

Uberall F, Hellbert K, Kampfer S, Maly K, Villunger A, Spitaler M, Mwanjewe J, Baier-Bitterlich G, Baier G, Grunicke HH - J. Cell Biol. (1999)

Evidence that atypical aPKC-λ acts upstream and  aPKC-ζ acts downstream of Rac-1. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras  L61 (A and B). (A) Ha-Ras L61 alone, or (B) together with  DN Rac-1 N17; (C) (CA) aPKC-λ A119E together with (DN)  Rac-1 N17; or (D) (CA) aPKC-ζ A119E together with (DN)  Rac-1 N17. 48 h posttransfection, cells were fixed and visualized  as described in Materials and Methods. Representative cells of at  least three different experiments are shown for all panels. Stacks  of images were exported into Adobe Photoshop and printed on a  color laser copier system.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132909&req=5

Figure 6: Evidence that atypical aPKC-λ acts upstream and aPKC-ζ acts downstream of Rac-1. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61 (A and B). (A) Ha-Ras L61 alone, or (B) together with DN Rac-1 N17; (C) (CA) aPKC-λ A119E together with (DN) Rac-1 N17; or (D) (CA) aPKC-ζ A119E together with (DN) Rac-1 N17. 48 h posttransfection, cells were fixed and visualized as described in Materials and Methods. Representative cells of at least three different experiments are shown for all panels. Stacks of images were exported into Adobe Photoshop and printed on a color laser copier system.
Mentions: As should be expected, DN N17 Rac-1 inhibits the Ras-mediated disassembly of stress fibers (Fig. 6, A and B). To obtain some information whether PKC-λ and PKC-ζ cooperate with Rac-1 in the same pathway, cells were cotransfected with combinations of either CA aPKC-λ A119E and N17 Rac-1 or CA aPKC-ζ A119E and N17 Rac-1.

Bottom Line: 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 lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes.Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Chemistry and Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria. florian.ueberall@uibk.ac.at

ABSTRACT
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 lambda and zeta, 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-lambda and aPKC-zeta, 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-alpha 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-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 plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

Show MeSH
Related in: MedlinePlus