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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 of PI3K and Rac-1,  whereas aPKC-ζ functions  downstream of PI3K and  Rac-1. Shown are representative fluorescence images of  fibroblasts transiently expressing (CA) atypical  aPKC-λ (A–C). (A) (CA)  aPKC-λ alone, or (B) in the  presence of 25 nM wortmannin, or (C) 25 μM LY294002;  (D) (CA) aPKC-ζ A119E  alone, or (E) in the presence  of 25 nM wortmannin, or (F)  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 above.
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Figure 9: Evidence that atypical aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1. Shown are representative fluorescence images of fibroblasts transiently expressing (CA) atypical aPKC-λ (A–C). (A) (CA) aPKC-λ alone, or (B) in the presence of 25 nM wortmannin, or (C) 25 μM LY294002; (D) (CA) aPKC-ζ A119E alone, or (E) in the presence of 25 nM wortmannin, or (F) 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 above.

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)

Evidence that  atypical aPKC-λ acts upstream of PI3K and Rac-1,  whereas aPKC-ζ functions  downstream of PI3K and  Rac-1. Shown are representative fluorescence images of  fibroblasts transiently expressing (CA) atypical  aPKC-λ (A–C). (A) (CA)  aPKC-λ alone, or (B) in the  presence of 25 nM wortmannin, or (C) 25 μM LY294002;  (D) (CA) aPKC-ζ A119E  alone, or (E) in the presence  of 25 nM wortmannin, or (F)  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 above.
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Related In: Results  -  Collection

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Figure 9: Evidence that atypical aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1. Shown are representative fluorescence images of fibroblasts transiently expressing (CA) atypical aPKC-λ (A–C). (A) (CA) aPKC-λ alone, or (B) in the presence of 25 nM wortmannin, or (C) 25 μM LY294002; (D) (CA) aPKC-ζ A119E alone, or (E) in the presence of 25 nM wortmannin, or (F) 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 above.
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.

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Related in: MedlinePlus