Limits...
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.

Show MeSH

Related in: MedlinePlus

Quantitative evaluation of total fiber length. Effect of  CA aPKC-λ and aPKC-ζ alone, together with (DN) N17Rac, or  after treatment with the PI3K inhibitors wortmannin and  LY294002. Total F-actin fiber length for all images were calculated as described in Fig. 4. Shown is a quantitative evaluation of  total F-actin fiber length from fibroblasts expressing CA aPKC-λ  and aPKC-ζ mutants under the influence of (DN) N17 Rac-1, and  the PI3K inhibitors wortmannin and LY294002. Final inhibitor concentrations are described above. Bars indicate means  (± SEM) of at least three independent experiments with ∼70–75  GFP-positive cells analyzed per coverslip.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132909&req=5

Figure 10: Quantitative evaluation of total fiber length. Effect of CA aPKC-λ and aPKC-ζ alone, together with (DN) N17Rac, or after treatment with the PI3K inhibitors wortmannin and LY294002. Total F-actin fiber length for all images were calculated as described in Fig. 4. Shown is a quantitative evaluation of total F-actin fiber length from fibroblasts expressing CA aPKC-λ and aPKC-ζ mutants under the influence of (DN) N17 Rac-1, and the PI3K inhibitors wortmannin and LY294002. Final inhibitor concentrations are described above. Bars indicate means (± SEM) of at least three independent experiments with ∼70–75 GFP-positive cells analyzed per coverslip.

Mentions: As shown in Fig. 6 C, N17 Rac-1 is able to overcome stress fiber disassembly induced by CA aPKC-λ A119E (compare with Fig. 5 for the effect of aPKC-λ A119E in the absence of N17 Rac-1), indicating that aPKC-λ acts upstream of Rac-1. Stress fiber disassembly by aPKC-ζ A119E, however, is not affected by N17 Rac-1 (Fig. 6 D), suggesting that aPKC-ζ acts either downstream or independent of Rac-1. A quantitative evaluation of the stress fiber alterations shown in Fig. 6 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)

Quantitative evaluation of total fiber length. Effect of  CA aPKC-λ and aPKC-ζ alone, together with (DN) N17Rac, or  after treatment with the PI3K inhibitors wortmannin and  LY294002. Total F-actin fiber length for all images were calculated as described in Fig. 4. Shown is a quantitative evaluation of  total F-actin fiber length from fibroblasts expressing CA aPKC-λ  and aPKC-ζ mutants under the influence of (DN) N17 Rac-1, and  the PI3K inhibitors wortmannin and LY294002. Final inhibitor concentrations are described above. Bars indicate means  (± SEM) of at least three independent experiments with ∼70–75  GFP-positive cells analyzed per coverslip.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Quantitative evaluation of total fiber length. Effect of CA aPKC-λ and aPKC-ζ alone, together with (DN) N17Rac, or after treatment with the PI3K inhibitors wortmannin and LY294002. Total F-actin fiber length for all images were calculated as described in Fig. 4. Shown is a quantitative evaluation of total F-actin fiber length from fibroblasts expressing CA aPKC-λ and aPKC-ζ mutants under the influence of (DN) N17 Rac-1, and the PI3K inhibitors wortmannin and LY294002. Final inhibitor concentrations are described above. Bars indicate means (± SEM) of at least three independent experiments with ∼70–75 GFP-positive cells analyzed per coverslip.
Mentions: As shown in Fig. 6 C, N17 Rac-1 is able to overcome stress fiber disassembly induced by CA aPKC-λ A119E (compare with Fig. 5 for the effect of aPKC-λ A119E in the absence of N17 Rac-1), indicating that aPKC-λ acts upstream of Rac-1. Stress fiber disassembly by aPKC-ζ A119E, however, is not affected by N17 Rac-1 (Fig. 6 D), suggesting that aPKC-ζ acts either downstream or independent of Rac-1. A quantitative evaluation of the stress fiber alterations shown in Fig. 6 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