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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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Ras L61-induced F-actin stress fiber disassembly is antagonized by the specific PKC inhibitor GF109203X at concentrations sensitive for atypical PKC-isotypes. Representative fluorescence images of green fluorescence protein expressing (top  panels) versus TRITC-phalloidin–stained fibroblasts (bottom  panels), transiently expressing Ha-Ras L61 (B–D). (A) Vector  control; (B) Ras L61; (C) Ras L61 in the presence of 200 nM  GF109203X; (D) Ras L61 in the presence of 6 μM GF109203X.  48 h posttransfection, cells were fixed and expression of GFP was  visualized by fluorescence microscopy. F-actin filaments were  stained by using TRITC-conjugated phalloidin. 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 in Materials and Methods. Bar, 10 μm.
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Figure 1: Ras L61-induced F-actin stress fiber disassembly is antagonized by the specific PKC inhibitor GF109203X at concentrations sensitive for atypical PKC-isotypes. Representative fluorescence images of green fluorescence protein expressing (top panels) versus TRITC-phalloidin–stained fibroblasts (bottom panels), transiently expressing Ha-Ras L61 (B–D). (A) Vector control; (B) Ras L61; (C) Ras L61 in the presence of 200 nM GF109203X; (D) Ras L61 in the presence of 6 μM GF109203X. 48 h posttransfection, cells were fixed and expression of GFP was visualized by fluorescence microscopy. F-actin filaments were stained by using TRITC-conjugated phalloidin. 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 in Materials and Methods. Bar, 10 μm.

Mentions: NIH3T3 cells transiently transfected with the transforming Ha-Ras L61 oncogene exhibit dramatic morphological differences compared with their nontransfected counterparts. Normal NIH3T3 fibroblasts are spreading on the extracellular matrix, appear flat, and exhibit bundles of actin stress fibers traversing the cell (Fig. 1 A). Cells expressing the Ras oncogene are more spindle-shaped, exhibit frequently long protrusions, and are characteristically devoid of actin stress fibers (Fig. 1 B). Similar morphological effects of transforming Ras have been described by other authors (Bar-Sagi and Feramisco, 1986; Ridley and Hall, 1992; Prendergast and Gibbs, 1993; Dartsch et al., 1994; Rodriguez-Viciana et al., 1994).


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)

Ras L61-induced F-actin stress fiber disassembly is antagonized by the specific PKC inhibitor GF109203X at concentrations sensitive for atypical PKC-isotypes. Representative fluorescence images of green fluorescence protein expressing (top  panels) versus TRITC-phalloidin–stained fibroblasts (bottom  panels), transiently expressing Ha-Ras L61 (B–D). (A) Vector  control; (B) Ras L61; (C) Ras L61 in the presence of 200 nM  GF109203X; (D) Ras L61 in the presence of 6 μM GF109203X.  48 h posttransfection, cells were fixed and expression of GFP was  visualized by fluorescence microscopy. F-actin filaments were  stained by using TRITC-conjugated phalloidin. 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 in Materials and Methods. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Ras L61-induced F-actin stress fiber disassembly is antagonized by the specific PKC inhibitor GF109203X at concentrations sensitive for atypical PKC-isotypes. Representative fluorescence images of green fluorescence protein expressing (top panels) versus TRITC-phalloidin–stained fibroblasts (bottom panels), transiently expressing Ha-Ras L61 (B–D). (A) Vector control; (B) Ras L61; (C) Ras L61 in the presence of 200 nM GF109203X; (D) Ras L61 in the presence of 6 μM GF109203X. 48 h posttransfection, cells were fixed and expression of GFP was visualized by fluorescence microscopy. F-actin filaments were stained by using TRITC-conjugated phalloidin. 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 in Materials and Methods. Bar, 10 μm.
Mentions: NIH3T3 cells transiently transfected with the transforming Ha-Ras L61 oncogene exhibit dramatic morphological differences compared with their nontransfected counterparts. Normal NIH3T3 fibroblasts are spreading on the extracellular matrix, appear flat, and exhibit bundles of actin stress fibers traversing the cell (Fig. 1 A). Cells expressing the Ras oncogene are more spindle-shaped, exhibit frequently long protrusions, and are characteristically devoid of actin stress fibers (Fig. 1 B). Similar morphological effects of transforming Ras have been described by other authors (Bar-Sagi and Feramisco, 1986; Ridley and Hall, 1992; Prendergast and Gibbs, 1993; Dartsch et al., 1994; Rodriguez-Viciana et al., 1994).

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