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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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Treatment of Ras-expressing cells with the  PI3K inhibitors wortmannin  or LY294002 counteracts the  effects of Ras on the actin cytoskeleton. Shown are representative fluorescence images of fibroblasts transiently  expressing Ha-Ras L61. (A)  Ha-Ras L61 alone, or (B) in  the presence of 25 nM wortmannin, or (C) in the presence of 25 μM LY294002. 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 as described.
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Figure 8: Treatment of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61. (A) Ha-Ras L61 alone, or (B) in the presence of 25 nM wortmannin, or (C) in the presence of 25 μM LY294002. 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 as described.

Mentions: PI3K has been shown to be implicated in the Ras-induced reorganization of actin cytoskeleton (Rodriguez-Viciana et al., 1994). In agreement with these findings, treatment of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton (Fig. 8). Both inhibitors also antagonize the disassembly of actin stress fibers induced by constitutively active aPKC-λ A119E, whereas the cytoskeletal reorganization mediated by the aPKC-ζ A119E mutant was not affected (Fig. 9). These data suggest that aPKC-λ acts upstream of PI3K whereas aPKC-ζ functions either downstream or independent of PI3K. A quantitative evaluation of the effects of the PI3K inhibitors is presented in Fig. 4 B and Fig. 10.


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)

Treatment of Ras-expressing cells with the  PI3K inhibitors wortmannin  or LY294002 counteracts the  effects of Ras on the actin cytoskeleton. Shown are representative fluorescence images of fibroblasts transiently  expressing Ha-Ras L61. (A)  Ha-Ras L61 alone, or (B) in  the presence of 25 nM wortmannin, or (C) in the presence of 25 μM LY294002. 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 as described.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Treatment of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton. Shown are representative fluorescence images of fibroblasts transiently expressing Ha-Ras L61. (A) Ha-Ras L61 alone, or (B) in the presence of 25 nM wortmannin, or (C) in the presence of 25 μM LY294002. 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 as described.
Mentions: PI3K has been shown to be implicated in the Ras-induced reorganization of actin cytoskeleton (Rodriguez-Viciana et al., 1994). In agreement with these findings, treatment of Ras-expressing cells with the PI3K inhibitors wortmannin or LY294002 counteracts the effects of Ras on the actin cytoskeleton (Fig. 8). Both inhibitors also antagonize the disassembly of actin stress fibers induced by constitutively active aPKC-λ A119E, whereas the cytoskeletal reorganization mediated by the aPKC-ζ A119E mutant was not affected (Fig. 9). These data suggest that aPKC-λ acts upstream of PI3K whereas aPKC-ζ functions either downstream or independent of PI3K. A quantitative evaluation of the effects of the PI3K inhibitors is presented in Fig. 4 B and Fig. 10.

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