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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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Quantitative evaluation of total fiber length. Effects of kinase-defective, DN  PKC-isotypes. Total F-actin  fiber length was calculated  after digitalizing TRITC-phalloidin stained F-actin by  using the MetaMorph image  processing software S/N  3542A. The edges of the cells  were detected by the aid of a  convolution kernel. This  means that the brightness of  the neighboring pixels were  compared. After thresholding and separating from the  background specimen, fiber  lengths were calculated and  expressed as the means of total fiber length compared  with the fiber length of mock-transfected fibroblasts. Shown are morphological alterations of Ras-mediated stress fiber disassembly under the influence of (A) (DN) aPKC-λ K275W, antisense aPKC-λ, (DN) aPKC-ζ, antisense aPKC-ζ, and (B), (DN) N17 Rac-1, and the  PI3K inhibitors wortmannin and LY294002. Bars indicate means (± SEM) of at least three independent experiments with ∼60–75  GFP-positive cells which were separately analyzed per coverslip.
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Figure 4: Quantitative evaluation of total fiber length. Effects of kinase-defective, DN PKC-isotypes. Total F-actin fiber length was calculated after digitalizing TRITC-phalloidin stained F-actin by using the MetaMorph image processing software S/N 3542A. The edges of the cells were detected by the aid of a convolution kernel. This means that the brightness of the neighboring pixels were compared. After thresholding and separating from the background specimen, fiber lengths were calculated and expressed as the means of total fiber length compared with the fiber length of mock-transfected fibroblasts. Shown are morphological alterations of Ras-mediated stress fiber disassembly under the influence of (A) (DN) aPKC-λ K275W, antisense aPKC-λ, (DN) aPKC-ζ, antisense aPKC-ζ, and (B), (DN) N17 Rac-1, and the PI3K inhibitors wortmannin and LY294002. Bars indicate means (± SEM) of at least three independent experiments with ∼60–75 GFP-positive cells which were separately analyzed per coverslip.

Mentions: A quantitative evaluation of Ras-mediated stress fiber depolymerization and the effects of (DN) aPKC-λ K275W, (DN) aPKC-ζ K275W, aPKC-λ, and aPKC-ζ antisense on Ras-induced actin fiber organization was performed by measuring F-actin fiber length with the aid of the MetaMorph image processing software (Fig. 4, A and B).


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)

Quantitative evaluation of total fiber length. Effects of kinase-defective, DN  PKC-isotypes. Total F-actin  fiber length was calculated  after digitalizing TRITC-phalloidin stained F-actin by  using the MetaMorph image  processing software S/N  3542A. The edges of the cells  were detected by the aid of a  convolution kernel. This  means that the brightness of  the neighboring pixels were  compared. After thresholding and separating from the  background specimen, fiber  lengths were calculated and  expressed as the means of total fiber length compared  with the fiber length of mock-transfected fibroblasts. Shown are morphological alterations of Ras-mediated stress fiber disassembly under the influence of (A) (DN) aPKC-λ K275W, antisense aPKC-λ, (DN) aPKC-ζ, antisense aPKC-ζ, and (B), (DN) N17 Rac-1, and the  PI3K inhibitors wortmannin and LY294002. Bars indicate means (± SEM) of at least three independent experiments with ∼60–75  GFP-positive cells which were separately analyzed per coverslip.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132909&req=5

Figure 4: Quantitative evaluation of total fiber length. Effects of kinase-defective, DN PKC-isotypes. Total F-actin fiber length was calculated after digitalizing TRITC-phalloidin stained F-actin by using the MetaMorph image processing software S/N 3542A. The edges of the cells were detected by the aid of a convolution kernel. This means that the brightness of the neighboring pixels were compared. After thresholding and separating from the background specimen, fiber lengths were calculated and expressed as the means of total fiber length compared with the fiber length of mock-transfected fibroblasts. Shown are morphological alterations of Ras-mediated stress fiber disassembly under the influence of (A) (DN) aPKC-λ K275W, antisense aPKC-λ, (DN) aPKC-ζ, antisense aPKC-ζ, and (B), (DN) N17 Rac-1, and the PI3K inhibitors wortmannin and LY294002. Bars indicate means (± SEM) of at least three independent experiments with ∼60–75 GFP-positive cells which were separately analyzed per coverslip.
Mentions: A quantitative evaluation of Ras-mediated stress fiber depolymerization and the effects of (DN) aPKC-λ K275W, (DN) aPKC-ζ K275W, aPKC-λ, and aPKC-ζ antisense on Ras-induced actin fiber organization was performed by measuring F-actin fiber length with the aid of the MetaMorph image processing software (Fig. 4, A and B).

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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Related in: MedlinePlus