Regulation of phospholipase D isoenzymes by transforming Ras and atypical protein kinase C-iota.

The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-iota with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-iota, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-iota, whereas PLD2 was less effective in this respect. The data suggest that PKC-iota acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-iota. The data are discussed as indicating a putative signalling cascade comprising Ras-->PLD1b-->PKC-iota-->PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.

Protein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras.

Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEK- and Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes epsilon and zeta, but not lambda are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-epsilon or aPKC-zeta inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-zeta, indicating that aPKC-zeta functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-epsilon, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. Evidence is presented that aPKC-lambda acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes epsilon and zeta act downstream of Rac and are required for the activation of ERKs.

Novel membrane-targeted ERK1 and ERK2 chimeras which act as dominant negative, isotype-specific mitogen-activated protein kinase inhibitors of Ras-Raf-mediated transcriptional activation of c-fos in NIH 3T3 cells.

Expression of constructs encoding fusion proteins of ERK1 and ERK2 containing a C-terminal farnesylation motif (CAAX) is predominantly localized at the cell membrane and was activated by coexpression of constitutively active Ha-RasL61 and epidermal growth factor. Both fusion proteins significantly inhibit the transcriptional activation of a c-fos-chloramphenicol acetyltransferase reporter induced by RasL61, constitutively active MEK1, or constitutively active RafBXB. The corresponding SAAX chimeras or overexpression of the wild-type ERKs did not interfere with the transcriptional activation of c-fos. The inhibition of the Ras-mediated c-fos induction by ERK2-CAAX can in part be rescued by coexpression of a wild-type ERK2 but not by wild-type ERK1. We find that ERK1-CAAX acts in the same fashion, indicating that mitogen-activated protein kinase (MAPK)-CAAX chimeras interact in an isotype-specific manner. It is demonstrated that both ERK1-CAAX and ERK2-CAAX associate with the corresponding endogenous ERKs, which explains the isotype-specific inhibitory effects of the ERK-CAAX chimeras. Evidence is presented that expression of ERK-CAAX fusion proteins inhibits the nuclear translocation of the corresponding endogenous ERKs. Disruption of MAPK translocation by membrane targeting provides additional, independent proof that nuclear translocation of ERKs is essential for the transcriptional activation of c-fos.

Evidence that atypical protein kinase C-lambda and atypical protein kinase C-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton.

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.

PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) P-glycoprotein by bryostatin 1.

Bryostatin 1 is a new antitumor agent which modulates the enzyme activity of protein kinase C (PKC, phospholipid-Ca2+-dependent ATP:protein transferase, EC 2.7.1.37). Several reports have suggested that the pumping activity of the multidrug resistance gene 1 (MDR1)-encoded multidrug transporter P-glycoprotein (PGP) is enhanced by a PKC-mediated phosphorylation. It was shown here that bryostatin 1 was a potent modulator of multidrug resistance in two cell lines over-expressing a mutant MDR1-encoded PGP, namely KB-C1 cells and HeLa cells transfected with an MDR1-V185 construct (HeLa-MDR1-V185) in which glycine at position 185 (G185) was substituted for valine (V185). Bryostatin 1 is not able to reverse the resistance of cells over-expressing the wild-type form (G185) of PGP, namely CCRF-ADR5000 cells and HeLa cells transfected with a MDR1-G185 construct (HeLa-MDR1-G185). Treatment of HeLa-MDR1-V185 cells with bryostatin 1 was accompanied by an increase in the intracellular accumulation of rhodamine 123, whereas no such effect could be observed in HeLa-MDR1-G185 cells. HeLa-MDR1-V185 cells expressed the PKC isoforms alpha, delta and zeta. Down-modulation of PKC alpha and delta by 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect the drug accumulation by bryostatin 1. Bryostatin 1 depleted PKC alpha completely and PKC delta partially. In HeLa-MDR1-V185 cells, short-term exposure to bryostatin 1, which led to a PKC activation, was as efficient in modulating the pumping activity of PGP as long-term exposure leading to PKC depletion. Bryostatin 1 competed with azidopine for binding to PGP in cells expressing the MDR1-V185 and MDR1-G185 forms of PGP. It is concluded that bryostatin 1: i) interacts with both the mutated MDR1-V185 and the wild-type MDR1-G185; ii) reverses multidrug resistance and inhibits drug efflux only in PGP-V185 mutants; and iii) that this effect is not due to an interference of PKC with PGP. For gene therapy, it is important to reverse the specific resistance of a mutant in the presence of a wild-type transporter and vice versa. Our results show that it is possible to reverse a specific mutant PGP.

Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-lambda, epsilon and zeta.

The implication of protein kinase C (PKC) isoforms cPKC-alpha, nPKC-epsilon, aPKC-lambda and aPKC-zeta 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-lambda, nPKC-epsilon and aPKC-zeta, not, however, of cPKC-alpha, 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-lambda acts upstream whereas aPKC-zeta functions downstream of nPKC-epsilon.

Molecular basis of targeted chemotherapy: novel concepts with special reference to the treatment of Hodgkin's disease.

Concepts for the treatment of Hodgkin's lymphomas based on novel insights of the molecular mechanisms responsible for the maintenance of the transformed phenotype of Reed-Sternberg cells, their proliferation and sensitivity to radiation and anti-tumor agents are discussed. The potentials of some recently developed new signal transduction inhibitors for the treatment of Hodgkin's lymphomas are discussed in greater detail and comprise agents directed against Janus kinase 2 (JAK 2); Signal Transducers and Activators of Transcription (STAT factors); agents directed against SH 2-domains: the fes/fps oncogene, Ras; protein kinase C (PKC) isotypes and means of inducing radiation or drug-induced apoptosis.

Azinyl and diazinyl hydrazones derived from aryl N-heteroaryl ketones: synthesis and antiproliferative activity.

A series of N-heteroaryl hydrazones derived from aryl N-heteroaryl or bis-N-heteroaryl methanones was prepared in search for potential novel antitumor agents. The stereochemistry of these compounds was established by means of NMR spectroscopy. Antiproliferative activity was determined in a panel of human tumor cell lines (CCRF-CEM, Burkitt's lymphoma, HeLa, ZR-75-1, HT-29, and MEXF 276L) in vitro. Generally, the new compounds were found to be more potent (IC50 = 0.011-0.436 microM) than the ribonucleotide reductase inhibitor hydroxyurea (IC50 = 140 microM). Most of the compounds exhibited the highest activity against Burkitt's lymphoma with an IC50 of 0.011-0.035 microM. [14C]Cytidine incorporation into DNA was quantitated for selected hydrazones (Z-A, E-1, Z-3, Z-4, E-5, Z-5, E-13, E-18, Z-19, Z-24, and E-26) as a measure of the inhibition of ribonucleotide reductase in Burkitt's lymphoma cells. The E-configurated compounds were found to inhibit [14C]cytidine incorporation to a greater extent (IC50 = 0.67-5.05 microM) than the Z-isomers (IC50 = 7.20 to > 10 microM). Principal component analysis of the IC50 values obtained for inhibition of cell proliferation revealed that the cell lines tested can be grouped into three main families showing different sensitivities toward the compounds in our series [(i) CCRF-CEM, Burkitt's lymphoma, and Hela; (ii) HT-29; and (iii) MEXF 276 L].

Differential Ca2+ signaling induced by activation of the epidermal growth factor and nerve growth factor receptors.

Stimulation by epidermal growth factor (EGF) of NIH3T3 cells overexpressing the EGF receptor (EGFR) results in a release of Ca2+ from internal stores. Ca2+ release is followed by an influx of extracellular calcium which can be recorded by the influx of the calcium surrogate Mn2+. Both Ca2+ release and Mn2+/Ca2+ influx are inhibited by expression of the dominant negative Asn17-Ras mutant and abrogated by microinjected neutralizing anti-Ras antibody Y13-259, whereas microinjection of the anti-Ras antibody Y13-238 which does not interact with the effector binding domain of Ras is without any effect on the EGF-induced Ca2+ transient. Neither Asn17-Ha-Ras nor the Y13-259 antibody interferes with the thapsigargin-induced Mn2+/Ca2+ influx. The nerve growth factor receptor (Trk)-mediated Ca2+ transient was found to be unaffected by the dominant negative Ras mutant or microinjected neutralizing anti-Ras antibodies. Substitution of the phospholipase Cgamma1 (PLCgamma1) binding site of the EGFR by the PLCgamma binding domain of Trk renders the EGFR-induced Ca2+ influx insensitive to the expression of Asn17-Ha-Ras, whereas the Ca2+ signal induced by Trk carrying the PLC binding site of EGFR is Ras-dependent and abrogated by the dominant negative Ras mutant. It is concluded that the Ca2+ transient induced by the activated EGFR, not, however, the Ca2+ transient elicited by the activated NGFR/Trk, is a Ras-mediated phenomenon and that the role of Ras in regulating EGFR-induced Ca2+ influx depends on the structure of the PLCgamma binding domain.

Cellular signalling as a target in cancer chemotherapy. Phospholipid analogues as inhibitors of mitogenic signal transduction.

Mitogenic signalling mechanisms emerged as novel targets for tumor chemotherapy. Current strategies for pharmacological interventions are briefly discussed. Phospholipid analogues are treated in greater detail. It is shown here that this new class of antitumor agents acts as inhibitors of mitogenic signal transduction. The common target of all phospholipid analogues studied so far is the phosphatidylinositol (PI)-specific phospholipase C (PLC). This results in an attenuated formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The reduction in IP3-levels leads to a depressed release of Ca2+ from internal stores, and the reduced formation of DAG interferes with the growth factor-induced activation of protein-kinase C (PKC). In addition to the effect on PI-specific PLC, most phospholipid analogues inhibit PKC directly by interacting with the regulatory domain of the enzyme. This effect, however, is not observed with all phospholipid analogues. Some potent growth inhibitory representatives from this group like hexadecylphosphoserine or hexadecylphosphonoserine do not affect PKC in cell-free extracts. It is concluded, therefore, that the direct inhibition of PKC is not required for the growth-inhibitory activity of these agents. The ability of phospholipid analogues to interact with PKC was also not found to be correlated the occurrence of unwanted side effects. Phospholipid analogues have also been found to act as inhibitors of phospholipase D (PLD). However, in this case the correlation to the growth inhibitory potency of various phospholipid analogues was less clear, so that the contribution of the PLD inhibition to the growth inhibitory effect of these agents still remains to be established. The inhibition of the thrombin-induced rise in cytosolic free Ca2+ by phospholipid analogues is reversible by washing the cells in phospholipid-free medium. These findings suggest that phospholipid analogues do not cause persistent membrane damage and may act as cytostatic rather than cytotoxic agents.

Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation.

The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites resulted in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhanced its inositol trisphosphate (IP3) and calcium signals and resulted in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibited a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impaired the IP3/calcium signal and attenuated the MAP kinase activation and receptor dephosphorylation, but resulted in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. Our findings establish the significance of PLC gamma affinity for signal definition, the role of this receptor tyrosine kinase substrate as a negative feedback regulator and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations.

Activation of Ca2+ influx by transforming Ha-ras.

The effect of an induction of transforming Ha-ras on Ca2+ influx into NIH3T3 cells was studied employing Fura-2 quenching by Mn2+. The expression of transforming p21Ha-ras caused a significant increase in Mn2+ influx which was blocked by Cd2+, La3+, niguldipine and the Ca(2+)-channel blocker SK&F96365. This effect was specific for transforming Ha-ras and was not seen after overexpression of the Ha-ras proto-oncogene or v-mos. In addition to the enhanced Mn2+ influx, transforming p21Ha-ras elicited an increased efflux of the K(+)-congener 86Rb+ which was inhibitable by Ca(2+)-channel blockers and charybdotoxin, a selective inhibitor of high and intermediate conductance Ca(2+)-dependent K+ channels. Charybdotoxin did not reduce the increase in Mn2+ influx by ras, demonstrating that the activation of Ca(2+)-dependent K+ channels was not required for the sustained Mn2+/Ca2+ influx in the presence of transforming Ha-ras. In ras-expressing cells, the bradykinin-induced Mn2+ influx and charybdotoxin sensitive 86Rb+ efflux were markedly potentiated. The increase in the inositol- 1,4,5-trisphosphate and inositol-1,3,4,5-tetrakisphosphate levels by ras is not sufficient to explain the elevated Mn2+ influx. The mitogenic response to an expression of transforming Ha-ras was inhibited by the Ca(2+)-channel blockers not, however, by charybdotoxin. These data suggest the existence of an agonist-independent activation of a receptor- or second messenger-operated Ca2+ channel by transforming Ha-ras which is necessary for the mitogenic response to the activation of the oncogene.

Interference of new alkylphospholipid analogues with mitogenic signal transduction.

The interference of several new hexadecylphosphocholine analogues with mitogenic signal transduction was investigated in NIH3T3 fibroblasts by studying the effects of these agents on thrombin-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation and the subsequent Ca2+ release, on protein kinase C (PKC) in cell-free extracts, on the PKC-mediated activation of the Na+/H+ antiporter and on c-fos induction. The compounds investigated include hexadecylphosphocholine (HePC), octadecyl-[2-(N-methyl-piperidinio)-ethyl]-phosphate (D20133), octadecyl-(N,N-dimethyl-piperidinio-4-yl)-phosphate (D21266); octadecyl-[2-(trimethyl-arsonio)-ethyl]-phosphate (D21805) and hexadecylphospho-L-serine (HePS). The data indicate that (i) all compounds inhibit the thrombin-induced progression of growth-arrested NIH3T3 cells into S phase with similar IC50 values; (ii) the common denominator of all compounds is a reduction of Ins(1,4,5)P3 formation, resulting in an attenuation of Ca2+ release; (iii) the direct interaction with PKC does not significantly contribute to the antitumor activity of these agents; (iv) the new HePC congeners D21266, D21133 and D21805 affect the same targets as HePC, i.e. PKC and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC). The lower toxicities of these compounds cannot be explained by a less pronounced inhibition of PKC or PLC, respectively.

Neopterin derivatives together with cyclic guanosine monophosphate induce c-fos gene expression.

We have previously shown that neopterin enhances hydrogen peroxide and chloramine T activity in a luminol-dependent chemiluminescence assay and strengthens toxicity of these agents against bacteria at slightly alkaline pH (pH 7.5), while 7,8-dihydroneopterin was shown to be a scavenger independent of the pH value. Besides various oxidants, phenolic antioxidants were shown to specifically induce expression of the c-fos and c-jun mRNAs. Using an inducible cfosCAT reporter transactivation system we studied the function of the pteridine derivatives on c-fos transactivation. For the first time, we demonstrate that neopterin and 7,8-dihydroneopterin, particularly together with cyclic guanosine monophosphate, induce c-fos gene expression. In humans, interferon-gamma induces the release of neopterin and 7,8-dihydroneopterin and also the synthesis of nitric oxide radical which in turn stimulate the formation of cGMP. Thus, in certain situations all three substances, namely neopterin, 7,8-dihydroneopterin and cGMP, may be present locally and even in the circulation at the same time. Based on our findings this constellation would significantly enhance the risk of c-fos gene expression and therefore promote tumour growth and development.

Activation of c-fos expression by transforming Ha-ras in HC11 mouse mammary epithelial cells is PKC-dependent and mediated by the serum response element.

The mechanism by which transforming Ha-ras induces c-fos expression in HC11 mouse mammary epithelial cells was investigated with regard to controversial data concerning the role of protein kinase C (PKC) and the required promoter elements of the fos gene. HC11 cells carrying a glucocorticoid-inducible Ha-ras (val12) construct were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of a human fos promoter which includes the serum response element (SRE), the adjacent c-fos AP-1 site (FAP) and the cAMP response element (CRE). Induction of the Ha-ras gene by dexamethasone lead to a transactivation of expression of the transfected fos promoter construct which was inhibited by the PKC inhibitor BM41440 and abrogated in PKC-'depleted' cells. A similar transactivation was observed when the fos promoter construct was co-transfected with a constitutively active ras expression vector. Again, this effect was depressed by the PKC inhibitor and abolished in PKC-'depleted' cells. 'PKC-depletion' was achieved by long-term exposure to 12-O-tetradecanoylphorbol-13-acetate. This procedure was shown to deplete cells of PKC alpha and to reduce significantly PKC epsilon. Long-term exposure to bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT (TK: thymidine kinase) construct by Ha-ras. In order to delineate the promoter elements mediating the transcriptional activation, constructs which lack the FAP and the CRE sites but contain an intact SRE were co-transfected with the ras construct. Elimination of the FAP and CRE sequences did not affect the transcriptional activation by Ha-ras (val12). It is concluded that in HC11 cells, transforming Ha-ras activates c-fos expression in a PKC-dependent manner, presumably implying PKC epsilon, and that the SRE is sufficient to mediate transcriptional activation.

Role of protein kinase C in ras-mediated fos-expression.

HC-11 mouse mammary epithelial cells stably transfected with a glucocorticoid-inducible Ha-ras construct encoding a transforming (val12) p21Ha-ras were cotransfected with a c-fos-CAT construct containing the human c-fos promoter up to position -711 and the CAT reporter gene. Expression of Ha-ras by dexamethasone leads to a transcriptional activation of the fos-CAT construct which was found to be sensitive to the PKC inhibitor ilmofosine (BM41440) and abrogated by PKC depletion following long-term exposure to TPA. The responsiveness to Ha-ras is retained if only the portion of the fos promoter covering the serum response element (SRE) and the adjacent fos AP-1 (FAP) site are put in front of a CAT gene linked to a thymidine kinase (TK) promoter. Further depletion of the FAP-site does not affect the inducibility by Ha-ras. Transcriptional activation of the SRE-FAP-TK-CAT as well as the SRE-TK-CAT constructs by Ha-ras is sensitive to the PKC-inhibitor ilmofosine (BM41440) and blocked by long-term exposure to TPA. Long-term exposure to TPA depletes cells of PKC alpha and significantly reduces the PKC epsilon levels. Long-term exposure in bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT-construct by Ha-ras. It is concluded that (i) transforming Ha-ras induces c-fos in HC-11 cells via PKC (presumably epsilon), (ii) the signal is mediated to the serum response element (SRE) of the fos promoter and (iii) the fos AP-1 (FAP) site is not required for this mechanism.