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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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Effect of GF109203X on cPKC-α, aPKC-ζ, and aPKC-λ enzyme activities.  COS-1 fibroblasts were transiently transfected with 15 μg of PKC-α, PKC-λ, and PKC-ζ  constructs encoding for wild-type PKC using the lipofectin technique as described by  the manufacturer. Enzyme activities are expressed as cofactor-dependent phosphorylation of the synthetic PKC peptide [A25S], in the absence or presence of various concentrations of GF109203X. The concentrations of synthetic substrates and cofactors  used are: 50 μg/ml [A25S] (synthetic peptide RFARKGSLRQKNVY representing the  pseudosubstrate sequence of PKC-α with a alanine-to-serine substitution), 280 μg/ml  PtdSer, 10 μM TPA, and 1 mM CaCl2. Expression of the fusion tag-peptide COOH-terminal of the recombinant PKC-isotypes was found not to affect the kinase activity  in vitro (Überall et al., 1997). Shown are kinase activities of Ni2+-batched recombinant  cPKC-α, atypical aPKC-ζ, and atypical aPKC-λ. (A–C) GF109203X, at the concentrations described above, was present for 15 min during the whole PKC assay running  time. Data are expressed as percent of stimulated controls (± SEM) of at least three  independent experiments done in triplicate.
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Figure 2: Effect of GF109203X on cPKC-α, aPKC-ζ, and aPKC-λ enzyme activities. COS-1 fibroblasts were transiently transfected with 15 μg of PKC-α, PKC-λ, and PKC-ζ constructs encoding for wild-type PKC using the lipofectin technique as described by the manufacturer. Enzyme activities are expressed as cofactor-dependent phosphorylation of the synthetic PKC peptide [A25S], in the absence or presence of various concentrations of GF109203X. The concentrations of synthetic substrates and cofactors used are: 50 μg/ml [A25S] (synthetic peptide RFARKGSLRQKNVY representing the pseudosubstrate sequence of PKC-α with a alanine-to-serine substitution), 280 μg/ml PtdSer, 10 μM TPA, and 1 mM CaCl2. Expression of the fusion tag-peptide COOH-terminal of the recombinant PKC-isotypes was found not to affect the kinase activity in vitro (Überall et al., 1997). Shown are kinase activities of Ni2+-batched recombinant cPKC-α, atypical aPKC-ζ, and atypical aPKC-λ. (A–C) GF109203X, at the concentrations described above, was present for 15 min during the whole PKC assay running time. Data are expressed as percent of stimulated controls (± SEM) of at least three independent experiments done in triplicate.

Mentions: As shown in Fig. 1 D, the PKC-specific inhibitor GF109203X (Toullec et al., 1991) is capable of reversing the dissolution of actin stress fibers by Ras. This effect is observed in the presence of 6 μM of the inhibitor (Fig. 1 D). 200 nM of the compound which had been shown to be sufficient for blocking c- and n-type PKC isotype kinase activity in cell-free extracts (Fig. 2 A) (Überall et al., 1997), is, however, without an effect on the Ras-mediated stress fiber dissolution (Fig. 1 C). 6 μM GF109203X corresponds roughly to the IC50 of the compound against the atypical PKC-ζ (Fig. 2 B, see also Martiny-Baron et al., 1993), and as shown in Fig. 2 C, exerts a similar effect on PKC-λ, suggesting that if a PKC is involved in the Ras-induced reorganization of the actin cytoskeleton, it should be an atypical (a-), rather than a conventional (c-) or novel (n-) type PKC isoform.


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)

Effect of GF109203X on cPKC-α, aPKC-ζ, and aPKC-λ enzyme activities.  COS-1 fibroblasts were transiently transfected with 15 μg of PKC-α, PKC-λ, and PKC-ζ  constructs encoding for wild-type PKC using the lipofectin technique as described by  the manufacturer. Enzyme activities are expressed as cofactor-dependent phosphorylation of the synthetic PKC peptide [A25S], in the absence or presence of various concentrations of GF109203X. The concentrations of synthetic substrates and cofactors  used are: 50 μg/ml [A25S] (synthetic peptide RFARKGSLRQKNVY representing the  pseudosubstrate sequence of PKC-α with a alanine-to-serine substitution), 280 μg/ml  PtdSer, 10 μM TPA, and 1 mM CaCl2. Expression of the fusion tag-peptide COOH-terminal of the recombinant PKC-isotypes was found not to affect the kinase activity  in vitro (Überall et al., 1997). Shown are kinase activities of Ni2+-batched recombinant  cPKC-α, atypical aPKC-ζ, and atypical aPKC-λ. (A–C) GF109203X, at the concentrations described above, was present for 15 min during the whole PKC assay running  time. Data are expressed as percent of stimulated controls (± SEM) of at least three  independent experiments done in triplicate.
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Figure 2: Effect of GF109203X on cPKC-α, aPKC-ζ, and aPKC-λ enzyme activities. COS-1 fibroblasts were transiently transfected with 15 μg of PKC-α, PKC-λ, and PKC-ζ constructs encoding for wild-type PKC using the lipofectin technique as described by the manufacturer. Enzyme activities are expressed as cofactor-dependent phosphorylation of the synthetic PKC peptide [A25S], in the absence or presence of various concentrations of GF109203X. The concentrations of synthetic substrates and cofactors used are: 50 μg/ml [A25S] (synthetic peptide RFARKGSLRQKNVY representing the pseudosubstrate sequence of PKC-α with a alanine-to-serine substitution), 280 μg/ml PtdSer, 10 μM TPA, and 1 mM CaCl2. Expression of the fusion tag-peptide COOH-terminal of the recombinant PKC-isotypes was found not to affect the kinase activity in vitro (Überall et al., 1997). Shown are kinase activities of Ni2+-batched recombinant cPKC-α, atypical aPKC-ζ, and atypical aPKC-λ. (A–C) GF109203X, at the concentrations described above, was present for 15 min during the whole PKC assay running time. Data are expressed as percent of stimulated controls (± SEM) of at least three independent experiments done in triplicate.
Mentions: As shown in Fig. 1 D, the PKC-specific inhibitor GF109203X (Toullec et al., 1991) is capable of reversing the dissolution of actin stress fibers by Ras. This effect is observed in the presence of 6 μM of the inhibitor (Fig. 1 D). 200 nM of the compound which had been shown to be sufficient for blocking c- and n-type PKC isotype kinase activity in cell-free extracts (Fig. 2 A) (Überall et al., 1997), is, however, without an effect on the Ras-mediated stress fiber dissolution (Fig. 1 C). 6 μM GF109203X corresponds roughly to the IC50 of the compound against the atypical PKC-ζ (Fig. 2 B, see also Martiny-Baron et al., 1993), and as shown in Fig. 2 C, exerts a similar effect on PKC-λ, suggesting that if a PKC is involved in the Ras-induced reorganization of the actin cytoskeleton, it should be an atypical (a-), rather than a conventional (c-) or novel (n-) type PKC isoform.

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