Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-λ, ε and ζ
1998; Springer Nature; Volume: 17; Issue: 14 Linguagem: Inglês
10.1093/emboj/17.14.4046
ISSN1460-2075
AutoresSonja Kampfer, Karina Hellbert, Andreas Villunger, Wolfgang Doppler, Gottfried Baier, H. Grunicke, Florian Überall,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle15 July 1998free access Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-λ, ϵ and ζ Sonja Kampfer Sonja Kampfer Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Karina Hellbert Karina Hellbert Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Andreas Villunger Andreas Villunger Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Wolfgang Doppler Wolfgang Doppler Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Gottfried Baier Gottfried Baier Human Genetics, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Hans H. Grunicke Hans H. Grunicke Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Florian Überall Corresponding Author Florian Überall Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Sonja Kampfer Sonja Kampfer Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Karina Hellbert Karina Hellbert Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Andreas Villunger Andreas Villunger Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Wolfgang Doppler Wolfgang Doppler Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Gottfried Baier Gottfried Baier Human Genetics, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Hans H. Grunicke Hans H. Grunicke Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Florian Überall Corresponding Author Florian Überall Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria Search for more papers by this author Author Information Sonja Kampfer1, Karina Hellbert1, Andreas Villunger1, Wolfgang Doppler1, Gottfried Baier2, Hans H. Grunicke1 and Florian Überall 1 1Institute of Medical Chemistry and Biochemistry and the Institute of Medical Biology, University of Innsbruck, A-6020 Innsbruck, Austria 2Human Genetics, University of Innsbruck, A-6020 Innsbruck, Austria *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:4046-4055https://doi.org/10.1093/emboj/17.14.4046 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The implication of protein kinase C (PKC) isoforms cPKC-α, nPKC-ϵ, aPKC-λ and aPKC-ζ in the transcriptional activation of a c-fos promoter-driven CAT-reporter construct by transforming Ha-Ras has been investigated. This was achieved by employing antisense constructs encoding RNA directed against isoform-specific 5′ sequences of the corresponding mRNA, and expression of PKC mutants representing either kinase-defective, dominant negative, or constitutively active forms of the PKC isoforms. The data indicate that in HC11 mouse mammary epithelial cells, transforming Ha-Ras requires the activities of the three PKC isozymes: aPKC-λ, nPKC-ϵ and aPKC-ζ, not, however, of cPKC-α, for the transcriptional activation of c-fos. Co-expression of oncogenic Ha-Ras with combinations of kinase-defective, dominant negative and constitutively active mutants of the various PKC isozymes are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the c-fos promoter, aPKC-λ acts upstream whereas aPKC-ζ functions downstream of nPKC-ϵ. Introduction It is well-documented that the mitogenic activity of Ha-Ras is protein kinase C (PKC)-dependent (Lacal et al., 1987; Wolfman and Macara, 1987; Morris et al., 1989; Gauthier-Rouvière et al., 1990; Hsiao et al., 1990; Beckman, 1992; Überall et al., 1994). Evidence for an activation of PKC in Ha-Ras-transformed cells has been presented (Morris et al., 1989; Chiarugi et al., 1990; Gauthier-Rouvière et al., 1990; Überall et al., 1994). However, which PKC isoforms are employed by Ha-Ras and what their intracellular targets are, has remained insufficiently understood. PKC represents a family of structurally related serine/threonine protein kinases presently comprising 11 isotypes. The various PKC isoforms are classified into three major subgroups: the classical or conventional PKC isotypes (cPKCs) are Ca2+- and diacylglycerol (DAG)-dependent and consist of the isozymes cPKC-α, cPKC-β1, cPKC-β2 and cPKC-γ; novel PKCs (nPKCs) are Ca2+-independent but DAG-responsive and comprise the isoforms nPKC-δ, nPKC-ϵ, nPKC-η and nPKC-θ; the PKC isozymes aPKC-λ/ι and aPKC-ζ require neither Ca2+ nor DAG for activation and have been classified as atypical PKC isoforms (aPKCs) (Nishizuka, 1992, 1995; Genot et al., 1995). In contrast to c- or n-type PKCs, aPKC isoforms also do not respond to phorbol ester treatment (Stabel, 1994). The PKC isoforms cPKC-α (Dean et al., 1996), cPKC-β2 (Sauma and Friedman, 1996; Sauma et al., 1996), nPKC-ϵ (Perletti et al., 1996), aPKC-ζ (Powell et al., 1996) and aPKC-λ (Bjorkoy et al., 1997) have been correlated to Ha-Ras-mediated signaling or transformation. As not all of these PKC isoforms are expressed in all cellular systems, cell type-specific differences are to be expected. Little is known with regard to the role of PKC in Ha-Ras transformed mouse mammary epithelial cells. Results of studies performed with this system will be presented here. Furthermore, with respect to the function of the various PKC isotypes in Ha-Ras transformed cells, all studies presented so far have focussed on single PKC isozymes. Whether Ha-Ras requires the cooperative activity of several PKC isoforms has not yet been described and is addressed in this paper. As a read-out system for Ha-Ras activity, we employed the Ha-Ras-mediated transcriptional activation of a c-fos promoter driven chloramphenicol acetyltransferase (CAT) reporter. The Raf-1/mitogen-activated protein (MAP) kinase pathway is considered to be a major route for the transmission of signals from Ha-Ras to the c-fos promoter (Bos, 1995; Treisman, 1995). The central role of the Raf-1/MAP kinase pathway in Ha-Ras-mediated transformation of fibroblasts is supported by observations indicating that kinase-deficient mutants of Raf-1, MAPK/ERK kinase (MEK) and MAP kinase inhibit Ha-Ras signaling and transformation (Kölch et al., 1991; Schaap et al., 1993; Khosravi-Far et al., 1995; Okazaki and Sagata, 1995; Qiu et al., 1995). An implication of PKC isozymes in the regulation of the Ha-Ras–MAP kinase pathway has been demonstrated in a variety of systems ranging from yeast to higher eukaryotes (Marshall, 1995; Morrison et al., 1996; Liao et al., 1997). It is well-established that the association of Raf to GTP-charged Ha-Ras is insufficient for Raf-1 activation and that additional phosphorylations on tyrosine and serine/threonine residues are required (Jelinek et al., 1996; Zou et al., 1996; Cai et al., 1997). In mammalian cells, activation of PKC by phorbol ester or bryostatin 1 stimulates Raf phosphorylation and activation. In vitro, cPKC-α and nPKC-ϵ have been shown to activate Raf-1 (Cai et al., 1997). In a cell-free system the activation of MAP kinase by a PKC, Raf, and MEK-dependent mechanism has been demonstrated (Marquardt et al., 1994). Evidence for an implication of the aPKC-λ and aPKC-ζ in the regulation of the MAP kinase pathway has also been presented (Berra et al., 1995; Bjorkoy et al., 1997; Liao et al., 1997). Stimulation of the MAP kinase pathway leads to the phosphorylation of TCF/Elk-1 and SAP-1 and the induction of c-fos (Treisman, 1995). We and others had previously demonstrated that transforming Ha-Ras employs a phorbol ester-sensitive PKC for the transcriptional activation of c-fos and circumstantial evidence indicated that this phorbol ester-sensitive PKC might be nPKC-ϵ (Überall et al., 1994). An implication of nPKC-ϵ would be in accordance with the putative function of this isoform in the activation of Raf-1 obtained in in vitro studies (Cai et al., 1997). The mouse mammary epithelial cells employed in our studies express the phorbol ester-sensitive isotypes cPKC-α, nPKC-ϵ, and in traces, nPKC-δ (Marte et al., 1994; Überall et al., 1994). It was, therefore, decided to further identify the phorbol ester-sensitive PKC isozyme which is employed by Ha-Ras for the transcriptional activation of c-fos. With regard to published data supporting a role of aPKC-ζ and aPKC-λ in the regulation of the MAP kinase pathway and Ha-Ras-mediated signaling, the implication of these phorbol ester-non-responsive PKC isotypes in transcriptional activation of c-fos by transforming Ha-Ras has also been addressed. Both atypical PKC isoforms ζ and λ/ι are expressed in HC11 cells. In view of the remarkable sequence homology of aPKC-ζ and aPKC-λ (Akimoto et al., 1994; Diaz-Meco et al., 1996), it also appeared to be of interest to determine whether only one or both isoforms are required for the transmission of signals from Ha-Ras to the c-fos promoter or whether one can substitute for the other. Furthermore, if Ha-Ras requires a combination of several PKC isozymes for the transcriptional activation of c-fos, this raises the question of whether the different isotypes act in separate but cooperating pathways or whether they function in a hierarchically ordered sequence. By employing a combination of mRNA antisense constructs, kinase-defective, dominant negative (DN) and constitutively active (CA) PKC mutants, for the first time evidence is presented that in HC11 cells, transforming Ha-Ras employs nPKC-ϵ, aPKC-λ and aPKC-ζ for the transcriptional activation of c-fos. Furthermore, our data support a tentative model suggesting a hierarchical sequence Ha-Ras–atypical aPKC-λ–novel nPKC-ϵ and atypical aPKC-ζ. Results Depletion of novel nPKC-ϵ, atypical aPKC-λ and atypical aPKC-ζ by expression of isoform-specific antisense RNA constructs suppresses Ha-Ras-mediated transcriptional activation of c-fos Selective depletion of PKC isoforms was achieved by transient transfection with vectors encoding antisense RNA directed against 5′-sequences of the corresponding PKC-isotype mRNA. Efficiency and specificity of PKC depletion was determined by selectively collecting the transfected cells. This was performed by cotransfection with a vector encoding a truncated form of the CD4 surface marker. CD4-positive cells were separated by the magnetic cell separation (MAC)-select technique employing magnetic columns and CD4-specific magnetic microbeads. PKC isozyme protein levels were determined by Western blotting. Expression of the targeted antisense constructs resulted in a significant and selective depletion of the corresponding PKC isozyme protein (Figure 1A) but did not affect the expression levels of other PKC isozymes (data not shown). Figure 1.Depletion of novel nPKC-ϵ, atypical aPKC- ζ and atypical aPKC-λ by expression of isoform-specific antisense RNA constructs suppresses Ha-Ras-mediated transcriptional activation of c-fos. (A) Isoenzyme-specific depletion of cPKC-α, nPKC-ϵ, aPKC- ζ and aPKC-λ. HC11 cells were cotransfected with 4 μg pMACS4 (truncated CD4 surface marker) and 8 μg of either one of the PKC isoform-specific antisense or sense constructs per 6-well plate, respectively. Thirty-six h post-transfection, CD4-positive cells (transfected cells) were separated from the negative cells (non-transfected cells) as described in Materials and methods. Total cellular extracts (corresponding to ∼1.5 mg/ml protein) from both fractions were prepared, and Western blot analysis of cPKC-α, nPKC-ϵ and aPKC- ζ for each sample was performed as described in Materials and methods. (B) Suppression of Ha-Ras mediated c-fos induction by cPKC-α, cPKC-ϵ, aPKC- ζ and aPKC-λ antisense constructs. HC11 cells were transfected with 2 μg pEJ-Ha-Ras, the corresponding vector control pOPI3-RSV, 2 μg pcfos-DSE-FAP-tk-CAT, 2 μg pAG-Luc and 6 μg PKC isoform specific antisense or sense constructs as indicated per 6-well plate. Forty-eight h post-transfection, cells were harvested and CAT expression was determined as described in Materials and methods. Data are expressed as the means (± SE, n = 9) of at least three independent experiments done in triplicate. Download figure Download PowerPoint After the efficacy and specificity of the antisense constructs had been established, it was investigated which antisense RNA would interfere with the Ha-Ras-mediated transcriptional activation of c-fos. Expression of nPKC-ϵ as well as aPKC-ζ and aPKC-λ antisense RNA caused a dramatic repression of the Ha-Ras-mediated transcriptional activation of the c-fos–CAT reporter construct (Figure 1B). Compared with aPKC-ζ and aPKC-λ antisense, which caused a complete suppression of Ras-mediated transcriptional activation of c-fos, nPKC-ϵ antisense proved to be slightly less effective. This may be explained by the observation that in contrast to aPKC-ζ/λ antisense, the antisense directed against nPKC-ϵ achieved only an incomplete depletion of the corresponding enzyme (Figure 1A). A marginal inhibition was also observed in cells expressing the cPKC-α antisense construct. However, in cells expressing the corresponding cPKC-α sense construct the reduction in Ras-mediated c-fos induction was even more pronounced. This is in marked contrast to the behaviour of the nPKC-ϵ and aPKC-ζ/λ sense controls which did not interfere with the transcriptional activation of c-fos by Ras (Figure 1B). It appeared conceivable, therefore, that the effects observed after expression of the cPKC-α sense and antisense constructs are due to non-specific, toxic side effects. In order to confirm the conclusion that the Ras-mediated transcriptional activation of the c-fos–CAT construct requires the PKC isotypes ζ/λ and ϵ, but probably not α, it was decided to employ kinase-defective PKC mutants which had been shown to act in a transdominant fashion (Baier-Bitterlich et al., 1996; Überall et al., 1997). In order to exclude non-specific effects of the PKC antisense constructs we determined the effect of the antisense vectors on the transcriptional activation of a thymidine kinase (tk)-CAT, as well as a cytomegalovirus (CMV)-driven luciferase reporter. Neither the tk-CAT nor the corresponding CMV–luciferase reporter was found to be affected (data not shown). Expression of kinase-defective, dominant negative mutants of PKC isoform nPKC-ϵ K436R, atypical aPKC-ζ K275W and aPKC-λ K275W, but not of cPKC-α K368R depress the transcriptional activation of c-fos by Ha-Ras Replacement of the critical lysine at the ATP-binding site by an arginine or tryptophan has been shown to result in kinase-defective PKC mutants which compete with the endogenous wild-type enzymes and act as isoformselective DN inhibitors (Genot et al., 1995; Baier-Bitterlich et al., 1996; Überall et al., 1997). The biochemical and biological properties of (DN)cPKC-α K368R, (DN)nPKC-ϵ K436R and (DN)aPKC-λ K275W have been described previously (Überall et al., 1997). The expression of the DN PKC mutants in transiently transfected HC11 cells was determined by cotransfection with a truncated CD4 surface marker and separation of CD4-expressing cells by the MAC-select technique described above. Transfection by all vectors encoding DN mutants of PKC isoforms α, ϵ, ζ and λ led to a significant overexpression of the corresponding mutant isotype (Figure 2A). Figure 2.Expression of kinase-defective DN mutants of PKC isoforms nPKC-ϵ K436R, atypical aPKC-ζ K275W and aPKC-λ K275W, but not of cPKC-α K368R depress the transcriptional activation of c-fos by Ha-Ras. (A) Expression pattern of PKC isoenzymes in cells transfected with plasmids encoding kinase-defective, DN PKC mutants. HC11 cells were cotransfected with 4 μg pMACS4 and 8 μg of one of the PKC isoform-specific DN constructs per 6-well plate, respectively. Thirty-six h post-transfection, CD4-positive cells were separated from the negative cells and analyzed by Western blotting as described in Materials and methods. (B) Inhibition of Ha-Ras mediated c-fos induction by kinase-defective PKC mutants. HC11 cells were transfected with 2 μg pEJ-Ha-Ras, the corresponding vector control pOPI3-RSV, 2 μg pc-fos-DSE-FAP-tk-CAT, 2 μg pAG-Luc and 6 μg of kinase-defective, (DN)cPKC-α K368R, (DN)nPKC-ϵ K436R, (DN)aPKC-λ K275W and (DN)aPKC-ζ K275W expression vectors per 6-well plate. Forty-eight h post-transfection, cells were harvested and CAT expression was determined as described in Materials and methods. Data are expressed as the means (± SE, n = 9) of at least three independent experiments done in triplicate. Download figure Download PowerPoint In accordance with the data obtained with the PKC antisense constructs, Ras-induced transcriptional activation of c-fos was suppressed in cells expressing kinase-deficient, dominant negative mutants of nPKC-ϵ K436R, aPKC-λ K275W and aPKC-ζ K275W. Expression of the kinase-defective, dominant negative mutant of cPKC-α K368R did not interfere with Ras-mediated c-fos induction, supporting our supposition that the inhibitions observed in cells expressing cPKC-α sense or antisense RNA were due to non-specific side effects. The data presented in Figures 1 and 2 suggest that in HC11 cells transforming Ha-Ras employs PKC isotypes ϵ, λ and ζ , but not α, for the transcriptional activation of the c-fos–CAT reporter construct. This conclusion was further substantiated by using constitutively active PKC mutants. Expression of constitutively active novel nPKC-ϵ A159E, atypical aPKC-ζ A119E and aPKC-λ A119E, but not cPKC-α A25E results in a Ras-independent transcriptional activation of c-fos It has previously been demonstrated that substitution of an alanine by a glutamate within the pseudosubstrate domain of PKC generates constitutively active mutants with reduced cofactor requirements. Biochemical and biological properties of these mutants have been described in preceding publications (Baier-Bitterlich et al., 1996; Überall et al., 1997). The expression in HC11 cells of the various mutants following transient transfection with constructs encoding the different mutated PKC isozymes is illustrated in Figure 3A. Figure 3.Expression of CA novel nPKC-ϵ A159E, atypical aPKC-ζ A119E and aPKC-λ A119E, but not of conventional cPKC-α A25E results in a transcriptional activation of c-fos independent of oncogenic Ras. (A) Expression pattern of PKC isoenzymes in cells transfected with constructs encoding CA PKC mutants. HC11 cells were cotransfected with 4 μg pMACS4 and 8 μg of one of the PKC isoform-specific (CA) constructs per 6-well plate, respectively. Thirty-six h post-transfection, CD4 positive cells were separated from the negative cells and analyzed by Western blotting as described in Materials and methods. (B) Transcriptional activation of c-fos by CA PKC mutants. HC11 cells were transfected with the corresponding vector control pEF-neo (mock), 3 μg pcfos-DSE-FAP-tk-CAT, 3 μg pAG-Luc and 6 μg of PKC (CA)cPKC-α A25E, (CA)nPKC-ϵ A159E, (CA)aPKC-λ A119E and (CA)aPKC-ζ A119E expression vector per 6-well plate. Forty-eight h post-transfection, cells were harvested and CAT expression was determined as described in Materials and methods. Data are expressed as the means (± SE, n = 9) of at least three independent experiments done in triplicate. Download figure Download PowerPoint Figure 3B demonstrates that expression of the CA forms of the PKC isotypes ζ, ϵ and λ leads to a significant transcriptional activation of the cotransfected c-fos–CAT reporter construct, whereas constitutively active cPKC-α is obviously unable to induce c-fos in this system. In agreement with the data reported in the previous sections, the results obtained with the constitutively active PKC isoforms demonstrate that in HC11 cells, PKC isotypes ϵ, λ and ζ are implicated in signal transmission to the c-fos promoter. Evidence that signaling from Ha-Ras to the c-fos promoter requires atypical aPKC-λ, novel PKC-ϵ and atypical aPKC-ζ in a hierarchically ordered sequence The data presented so far indicate that Ha-Ras requires novel nPKC-ϵ, together with the two atypical isoforms λ and ζ for transcriptional activation of the c-fos promoter. In order to obtain further information on whether the different PKC isozymes act in separate, but cooperating pathways or whether they act in a sequential order, cells were cotransfected with combinations of kinase-defective, DN and CA PKC mutants. The inhibition of the Ha-Ras-mediated transcriptional activation of c-fos by the kinase-defective, (DN)nPKC-ϵ K436R mutant can be overcome by (CA)aPKC-ζ A119E mutant, but not by the (CA)aPKC-λ A119E mutant, suggesting that aPKC-λ acts upstream and aPKC-ζ downstream of nPKC-ϵ (Figure 4A). Figure 4.Transcriptional activation of c-fos mediated by oncogenic Ras in HC11 cells requires the combined activities of aPKC-λ, nPKC-ϵ, and aPKC-ζ. (A) Blockade of Ha-Ras mediated transcriptional activation of c-fos–CAT by a kinase-defective, (DN)nPKC-ϵ K436R mutant can be overcome by (CA)aPKC-ζ not, however, by (CA)aPKC-λ or (CA)cPKC-α. Shown are co-expressions of kinase-defective, (DN)nPKC-ϵ K436R with (CA)cPKC-α A25, (CA)aPKC-λ A119E or (CA)aPKC-ζ A119E in the absence (open bars) or presence (closed bars) of transforming Ha-Ras. HC11 cells growing in 6-well plates were cotransfected with the corresponding vector control pEF-neo, 1 μg pEJ-Ha-Ras, 1 μg pcfos-DSE-FAP-tk-CAT, 1 μg pAG-Luc, and 4.5 μg (DN)nPKC-ϵ K436R plus (CA)nPKC-α A25E, (CA)cPKC-λ A119E or (CA)aPKC-ζ A119E, respectively. Forty-eight h post-transfection, cells were harvested and CAT expression was determined as described elsewhere (Überall et al., 1994). (B) CA isoforms of both nPKC-ϵ and aPKC-ζ are able to overcome a blockade of Ras-mediated c-fos induction exerted by kinase-defective, (DN)aPKC-λ K275W. Demonstrated are the results obtained after co-expression of kinase-defective, (DN)aPKC-λ K275W with (CA)cPKC-α A25E, (CA)nPKC-ϵ A159E or (CA)aPKC-ζ A119E in the absence (open bars) or presence (closed bars) of transforming Ha-Ras. Transfection was performed as described for Figure 4A employing equivalent concentrations of vector DNA. (C) The transcriptional activation of c-fos by (CA)aPKC-λ or (CA)nPKC-ϵ demonstrated in Figure 3B is abrogated in the presence of (DN)aPKC-ζ K275W. If transforming Ras is expressed (closed bars), (DN)aPKC-ζ still blocks the activity of (CA)nPKC-ϵ, however, the blockade by (DN)aPKC-ζ is antagonized by (CA)aPKC-λ. Shown are co-expression studies of kinase-defective, (DN)aPKC-ζ K275W with (CA)cPKC-α A25E, (CA)nPKC-ϵ A159E or (CA)aPKC-λ A119E in the absence (open bars) or presence (closed bars) of transforming Ha-Ras. Transfection was performed as described for Figure 4A employing equivalent concentrations of vector DNA. (D) Expression of kinase-defective, (DN)cPKC-α K368R does not affect the transcriptional activation of c-fos. Shown are co-expression studies of kinase-defective, (DN)cPKC-α K368R with (CA)nPKC-ϵ A159E, (CA)aPKC-λ A119E, or (CA)aPKC-ζ A119E in the absence (open bars) or presence (closed bars) of transforming Ha-Ras. Transfection was performed as described for Figure 4A employing equivalent concentrations of vector DNA. Data in Figure 4A–D are expressed as the means (± SE, n = 9) of at least three independent experiments done in triplicate. Download figure Download PowerPoint If the conclusion that transforming Ras transmits signals to the c-fos promoter through a pathway containing the PKC isoforms λ, ϵ and ζ in this sequential order is correct, then CA isoforms of both PKC-ϵ and ζ should be able to overcome a blockade of Ras-mediated c-fos induction exerted by (DN)aPKC-λ K275W mutant. As demonstrated in Figure 4B, this is indeed the case. Furthermore, if PKC-ζ acts downstream of both aPKC-λ and nPKC-ϵ, neither (CA)nPKC-ϵ nor (CA)aPKC-λ should be capable of overcoming a blockade on the level of aPKC-ζ. In the absence of transforming Ras this is indeed to be seen (Figure 4C, open bars). The transcriptional activation of c-fos by (CA)aPKC-λ or dPKC-ϵ demonstrated in Figure 3B is abrogated in the presence of (DN)aPKC-ζ K275W. If transforming Ras is expressed (Figure 4C, closed bars), (DN)aPKC-ζ still blocks the activity of (CA)nPKC-ϵ; however, the blockade by (DN)aPKC-ζ is antagonized by (CA)aPKC-λ (Figure 4C). This could be interpreted to indicate that in cells expressing the (CA)aPKC-λ A119E mutant, Ras may be able to induce c-fos by a separate, independent mechanism. However, co-expression of the (DN)nPKC-ϵ K436R mutant completely blocks Ras-mediated c-fos induction in these cells expressing (CA)aPKC-λ A119E and (DN)aPKC-ζ K275W (Figure 5A). Thus, transforming Ras transmits signals to the c-fos promoter via nPKC-ϵ which apparently acts downstream of aPKC-λ. The conclusion previously reached that aPKC-λ operates upstream and aPKC-ζ downstream of nPKC-ϵ was independently confirmed by studies demonstrating that Ras-mediated c-fos induction in cells expressing the (CA)nPKC-ϵ A159E mutant was not affected by the kinase-defective, (DN)aPKC-λ K275W isoform, but was abrogated by the kinase-defective, (DN)aPKC-ζ K275W mutant (Figure 5B). Figure 5.(A) Kinase-defective (DN)nPKC-ϵ blocks the transcriptional activation of c-fos in cells expressing (CA)aPKC-λ and (DN)aPKC-ζ in the absence (open bars) or the presence (closed bars) of transforming Ha-Ras. HC11 cells growing in 6-well plates were cotransfected with the corresponding vector control pEF-neo, 1 μg pEJ-Ha-Ras, 1 μg pcfos-DSE-FAP-tk-CAT, 1 μg pAG-Luc, 4.5 μg (CA)aPKC-λ A119E, plus (DN)aPKC-ζ K275W, or in combination 4.5 μg (CA)aPKC-λ A119E, (DN)aPKC-ζ K275W and (DN)nPKC-ϵ K436R as described. (B) Kinase-defective (DN)aPKC-ζ blocks the transcriptional activation of c-fos in cells expressing (CA)nPKC-ϵ and (DN)aPKC-λ in the absence (open bars) or the presence (closed bars) of transforming Ha-Ras. Cotransfection with the corresponding vector control pEF-neo, 1 μg pEJ-Ras, 1 μg pcfos-DSE-FAP-tk-CAT, 1 μg pAG-Luc, 4.5 μg (CA)nPKC-ϵ A159E, plus (DN)aPKC-λ K275W or in combination 4.5 μg (CA)nPKC-ϵ A159E plus 4.5 μg (DN)aPKC-λ K275W and (DN)aPKC-ζ K275W, respectively. Forty-eight h post-transfection, cells were harvested and CAT expression was determined as described elsewhere (Überall et al., 1994). Data are expressed as the means (± SE, n = 9) of at least three independent experiments done in triplicate. Download figure Download PowerPoint However, if aPKC-ζ acts downstream of aPKC-λ and nPKC-ϵ, why does a DN mutant of aPKC-ζ not inhibit Ras-mediated c-fos induction in cells expressing the CA mutant of PKC-λ? In view of the marked homology between the two atypical PKC isoforms (Akimoto et al., 1994) it appeared conceivable that the expressed CA PKC-λ may interfere with the inhibitory activity of DN PKC ζ resulting in an incomplete inhibition of the endogenous aPKC-ζ. In order to test this hypothesis, endogenous aPKC-ζ was depleted by the antisense technique outlined before. Under these conditions, and in accordance with the model which places aPKC-λ upstream of aPKC-ζ, a significant, albeit incomplete, inhibition of Ras-mediated c-fos induction by depletion of aPKC-ζ can be registered even in presence of the (CA)aPKC-λ A119E mutant (Figure 6A). In these cells, Ras-mediated c-fos induction is completely abrogated by the additional expression of the kinase-defective, (DN)nPKC-ϵ K436R mutant, confirming the assumption that aPKC-λ acts upstream of both nPKC-ϵ and aPKC-ζ during signal transmission from Ha-Ras to the c-fos promoter. Figure 6.Antisense mediated depletion of aPKC-ζ suppresses transcriptional activation of c-fos by (CA)aPKC-λ (A) or (CA)nPKC-ϵ (B) in the absence (open bars) or presence (closed bars) of transforming Ha-Ras. (A) HC11 cells growing in 6-well plates were cotransfected with the corresponding vector control pEF-neo, 1 μg pEJ-Ras, 1 μg pcfos-DSE-FAP-tk-CAT, 1 μg pAG-Luc, 4.5 μg (CA)aPKC-λ A119E, plus 3 μg antisense/sense PKC-ζ or in combination 4.5 μg (DN)nPKC-ϵ K436R, (CA)aPKC-λ A119E, (DN)aPKC-ζ K275W and (DN)nPKC-ϵ K436R as described in Materials and methods. (B) Cotransfection with the corresponding vector control pEF-neo, 1 μg pEJ-Ha-Ras, 1 μg pcfos-DSE-FAP-tk-CAT, 1 μg pAG-Luc, 4.5 μg (CA)nPKC-ϵ A159E, plus 3 μg aPKC-ζ antisense/sense vector, respectively. Forty-eight h post-transfection, cells were harvested and CAT expression was determined as described elsewhere (Überall et al., 1994). Data are expressed as the means (± SE, n = 9) of at least three independent experiments done in triplicate.
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