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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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Effects of oncogenic Ras and constitutively active Rac  on kinase activities of aPKC-ζ and aPKC-λ. Shown are representative immunocomplex kinases assays (B–D) and an in vitro kinase assay (A) of magnetically separated COS-1 cells. The data  are designed as bar graphs (top panels) and as corresponding autoradiograms of the myelin basic protein (MBP) assay (bottom  panels). Equal amounts of recombinant proteins used in the experiment were employed using a standard Western blotting  technique as described by Kampfer et al. (1998). In brief, logarithmically growing cells were transiently cotransfected with (A)  Ha-Ras L61 together with aPKC-λ/ι, (B) Ha-Ras L61 together  with aPKC-ζ, (C) CA Rac-1 V12 together with aPKC-ζ, and (D)  DN Rac-1 N17 together with aPKC-ζ. Concerning magnetic bead  separation of positively transfected cells, a truncated CD4 surface marker was cotransfected in B and C. 48 h posttransfection,  cells were separated by using magnetic beads as described by the  manufacturer and PKC assays were done as described under Materials and Methods. Enzyme activities are expressed as cofactor-independent phosphorylation of (A) a synthetic PKC-α peptide  (A25S, for details see Fig. 2 legend) or myelin basic protein  (B–D). Computer-assisted calculation of PKC-ζ or PKC-λ activities were done after scanning the corresponding PVDF membranes by using the Scanner Controller Sci System.
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Figure 7: Effects of oncogenic Ras and constitutively active Rac on kinase activities of aPKC-ζ and aPKC-λ. Shown are representative immunocomplex kinases assays (B–D) and an in vitro kinase assay (A) of magnetically separated COS-1 cells. The data are designed as bar graphs (top panels) and as corresponding autoradiograms of the myelin basic protein (MBP) assay (bottom panels). Equal amounts of recombinant proteins used in the experiment were employed using a standard Western blotting technique as described by Kampfer et al. (1998). In brief, logarithmically growing cells were transiently cotransfected with (A) Ha-Ras L61 together with aPKC-λ/ι, (B) Ha-Ras L61 together with aPKC-ζ, (C) CA Rac-1 V12 together with aPKC-ζ, and (D) DN Rac-1 N17 together with aPKC-ζ. Concerning magnetic bead separation of positively transfected cells, a truncated CD4 surface marker was cotransfected in B and C. 48 h posttransfection, cells were separated by using magnetic beads as described by the manufacturer and PKC assays were done as described under Materials and Methods. Enzyme activities are expressed as cofactor-independent phosphorylation of (A) a synthetic PKC-α peptide (A25S, for details see Fig. 2 legend) or myelin basic protein (B–D). Computer-assisted calculation of PKC-ζ or PKC-λ activities were done after scanning the corresponding PVDF membranes by using the Scanner Controller Sci System.

Mentions: The data presented so far suggest that Ras mediates the effects on the cytoskeleton via a pathway containing aPKC-λ–Rac-1 and aPKC-ζ. If this model is correct, Ras should activate aPKC-λ and aPKC-ζ whereas Rac should be able to stimulate aPKC-ζ. Unfortunately, this question could not be addressed in NIH3T3 cells due to the low transfection efficiencies in this cell type. Therefore, these studies were performed with COS cells. As shown in Fig. 7 A, cotransfection of COS cells with plasmids encoding Ras L61 and 6× His-tagged aPKC-λ leads to a significant activation of the kinase activity of aPKC-λ. Coexpression of Ras L61 and aPKC-ζ results in a marked stimulation of aPKC-ζ as demonstrated in Fig. 7 B. Furthermore, cotransfection of a plasmid encoding CA V12Rac with a construct encoding aPKC-ζ also revealed an activation of aPKC-ζ by Rac (Fig. 7 C). Surprisingly, V12Rac in addition to aPKC-ζ also activated aPKC-λ (data not shown). However, this finding is not in conflict with data or models presented so far. Possible interpretations for this effect will be presented in the Discussion. Our conclusion that Ras activates aPKC-ζ by a Rac-1–dependent mechanism is supported by the fact that expression of DN N17Rac blocks Ras-mediated stimulation of aPKC-ζ (Fig. 7 D). N17Rac does not inhibit Ras-mediated activation of PKC-λ (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)

Effects of oncogenic Ras and constitutively active Rac  on kinase activities of aPKC-ζ and aPKC-λ. Shown are representative immunocomplex kinases assays (B–D) and an in vitro kinase assay (A) of magnetically separated COS-1 cells. The data  are designed as bar graphs (top panels) and as corresponding autoradiograms of the myelin basic protein (MBP) assay (bottom  panels). Equal amounts of recombinant proteins used in the experiment were employed using a standard Western blotting  technique as described by Kampfer et al. (1998). In brief, logarithmically growing cells were transiently cotransfected with (A)  Ha-Ras L61 together with aPKC-λ/ι, (B) Ha-Ras L61 together  with aPKC-ζ, (C) CA Rac-1 V12 together with aPKC-ζ, and (D)  DN Rac-1 N17 together with aPKC-ζ. Concerning magnetic bead  separation of positively transfected cells, a truncated CD4 surface marker was cotransfected in B and C. 48 h posttransfection,  cells were separated by using magnetic beads as described by the  manufacturer and PKC assays were done as described under Materials and Methods. Enzyme activities are expressed as cofactor-independent phosphorylation of (A) a synthetic PKC-α peptide  (A25S, for details see Fig. 2 legend) or myelin basic protein  (B–D). Computer-assisted calculation of PKC-ζ or PKC-λ activities were done after scanning the corresponding PVDF membranes by using the Scanner Controller Sci System.
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Figure 7: Effects of oncogenic Ras and constitutively active Rac on kinase activities of aPKC-ζ and aPKC-λ. Shown are representative immunocomplex kinases assays (B–D) and an in vitro kinase assay (A) of magnetically separated COS-1 cells. The data are designed as bar graphs (top panels) and as corresponding autoradiograms of the myelin basic protein (MBP) assay (bottom panels). Equal amounts of recombinant proteins used in the experiment were employed using a standard Western blotting technique as described by Kampfer et al. (1998). In brief, logarithmically growing cells were transiently cotransfected with (A) Ha-Ras L61 together with aPKC-λ/ι, (B) Ha-Ras L61 together with aPKC-ζ, (C) CA Rac-1 V12 together with aPKC-ζ, and (D) DN Rac-1 N17 together with aPKC-ζ. Concerning magnetic bead separation of positively transfected cells, a truncated CD4 surface marker was cotransfected in B and C. 48 h posttransfection, cells were separated by using magnetic beads as described by the manufacturer and PKC assays were done as described under Materials and Methods. Enzyme activities are expressed as cofactor-independent phosphorylation of (A) a synthetic PKC-α peptide (A25S, for details see Fig. 2 legend) or myelin basic protein (B–D). Computer-assisted calculation of PKC-ζ or PKC-λ activities were done after scanning the corresponding PVDF membranes by using the Scanner Controller Sci System.
Mentions: The data presented so far suggest that Ras mediates the effects on the cytoskeleton via a pathway containing aPKC-λ–Rac-1 and aPKC-ζ. If this model is correct, Ras should activate aPKC-λ and aPKC-ζ whereas Rac should be able to stimulate aPKC-ζ. Unfortunately, this question could not be addressed in NIH3T3 cells due to the low transfection efficiencies in this cell type. Therefore, these studies were performed with COS cells. As shown in Fig. 7 A, cotransfection of COS cells with plasmids encoding Ras L61 and 6× His-tagged aPKC-λ leads to a significant activation of the kinase activity of aPKC-λ. Coexpression of Ras L61 and aPKC-ζ results in a marked stimulation of aPKC-ζ as demonstrated in Fig. 7 B. Furthermore, cotransfection of a plasmid encoding CA V12Rac with a construct encoding aPKC-ζ also revealed an activation of aPKC-ζ by Rac (Fig. 7 C). Surprisingly, V12Rac in addition to aPKC-ζ also activated aPKC-λ (data not shown). However, this finding is not in conflict with data or models presented so far. Possible interpretations for this effect will be presented in the Discussion. Our conclusion that Ras activates aPKC-ζ by a Rac-1–dependent mechanism is supported by the fact that expression of DN N17Rac blocks Ras-mediated stimulation of aPKC-ζ (Fig. 7 D). N17Rac does not inhibit Ras-mediated activation of PKC-λ (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