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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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CA PKC mutants mimic the Ras-mediated disassembly of F-actin. Potency of the PKC-specific inhibitor GF109203X  to revert the disassembly of actin fibers induced by CA mutants.  Shown are representative fluorescence images of fibroblasts  transiently expressing constitutively active (CA) PKC mutants  alone (B and D), or in the presence of 6 μM GF109203X (C and  E). (A) Control, (B) (CA) atypical aPKC-λ A119E; (C) plus  GF109203X; (D) (CA) aPKC-ζ A119E; (E) plus GF109203X.  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 5: CA PKC mutants mimic the Ras-mediated disassembly of F-actin. Potency of the PKC-specific inhibitor GF109203X to revert the disassembly of actin fibers induced by CA mutants. Shown are representative fluorescence images of fibroblasts transiently expressing constitutively active (CA) PKC mutants alone (B and D), or in the presence of 6 μM GF109203X (C and E). (A) Control, (B) (CA) atypical aPKC-λ A119E; (C) plus GF109203X; (D) (CA) aPKC-ζ A119E; (E) plus GF109203X. 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: If Ha-Ras uses atypical PKC isotypes for the reorganization of the actin cytoskeleton as suggested by the data shown in Figs. 3 and 4, expression of CA aPKC-λ and (CA) aPKC-ζ mutants should affect actin stress fibers like transforming Ha-Ras. As shown in Fig. 5, B and D, this is indeed the case. A CA mutant of PKC-α A25E did not show any significant effect on stress fiber rearrangements (data not shown).


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)

CA PKC mutants mimic the Ras-mediated disassembly of F-actin. Potency of the PKC-specific inhibitor GF109203X  to revert the disassembly of actin fibers induced by CA mutants.  Shown are representative fluorescence images of fibroblasts  transiently expressing constitutively active (CA) PKC mutants  alone (B and D), or in the presence of 6 μM GF109203X (C and  E). (A) Control, (B) (CA) atypical aPKC-λ A119E; (C) plus  GF109203X; (D) (CA) aPKC-ζ A119E; (E) plus GF109203X.  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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132909&req=5

Figure 5: CA PKC mutants mimic the Ras-mediated disassembly of F-actin. Potency of the PKC-specific inhibitor GF109203X to revert the disassembly of actin fibers induced by CA mutants. Shown are representative fluorescence images of fibroblasts transiently expressing constitutively active (CA) PKC mutants alone (B and D), or in the presence of 6 μM GF109203X (C and E). (A) Control, (B) (CA) atypical aPKC-λ A119E; (C) plus GF109203X; (D) (CA) aPKC-ζ A119E; (E) plus GF109203X. 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: If Ha-Ras uses atypical PKC isotypes for the reorganization of the actin cytoskeleton as suggested by the data shown in Figs. 3 and 4, expression of CA aPKC-λ and (CA) aPKC-ζ mutants should affect actin stress fibers like transforming Ha-Ras. As shown in Fig. 5, B and D, this is indeed the case. A CA mutant of PKC-α A25E did not show any significant effect on stress fiber rearrangements (data not shown).

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