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.

Complex formation and cooperation of protein kinase C theta and Akt1/protein kinase B alpha in the NF-kappa B transactivation cascade in Jurkat T cells.

Protein kinase C theta (PKC theta) is known to induce NF-kappa B, an essential transcriptional element in T cell receptor/CD28-mediated interleukin-2 production but also T cell survival. Here we provide evidence that PKC theta is physically and functionally coupled to Akt1 in this signaling pathway. First, T cell receptor/CD3 ligation was sufficient to induce activation as well as plasma membrane recruitment of PKC theta. Second, PKC theta selectively cooperated with Akt1, known to act downstream of CD28 co-receptor signaling, in activating a NF-kappa B reporter in T cells. Third, Akt1 function was shown to be required for PKC theta-mediated NF-kappa B transactivation. Fourth, PKC theta co-immunoprecipitated with Akt1; however, neither Akt1 nor PKC theta served as a prominent substrate for each other in vitro as well as in intact T cells. Finally, plasma membrane targeting of PKC theta and Akt1 exerted synergistic transactivation of the I-kappa B kinase beta/inhibitor of NF-kappa B/NF-kappa B signaling cascade independent of T cell activation. Taken together, these findings suggest a direct cross-talk between PKC theta and Akt1 in Jurkat T cells.

Membrane proximal ERK signaling is required for M-calpain activation downstream of epidermal growth factor receptor signaling.

Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as de-adhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesion-related substrates.

Photo-induced inactivation of protein kinase calpha by dequalinium inhibits motility of murine melanoma cells.

Dequalinium (DECA) is a potent antitumor agent and inhibitor of protein kinase C (PKC). Previously it was shown that PKCalpha activity in vitro could be irreversibly inhibited when treated with DECA at low micromolar concentrations and irradiated with 366 nm of light. This approach was used to probe the role of intracellular PKC activity in the motility of metastatic murine melanoma B16 F10 cells and as a target for DECA analogs with increasing PKC inhibitory potencies. Pretreatment of a monolayer of B16 F10 cells with 250 nM of a DECA analog in the presence of UV irradiation for 5 min resulted in 1) complete inhibition of cell motility for up to 4 h in a time-lapse motility assay and 40 to 60% inhibition of cell migration in a Boyden chamber, and 2) inhibition by 40 to 60% of intracellular phosphatidylserine/Ca(2+)-dependent PKC catalytic activity, signifying inactivation of a conventional PKC isoform. Because PKCalpha is the only conventional PKC isoform detected in B16 F10 cells, a stably transfected clone expressing a kinase-defective mutant of PKCalpha was developed that exhibited a substantial loss of adhesion and motility and was refractory to further inhibition by DECA. These findings identify PKCalpha catalytic activity both as a mechanistic component of cell motility and adhesion and as a critical intracellular target of DECA. These studies further suggest that the combined use of UV with nanomolar concentrations of DECA offers an effective chemotherapeutic approach to inhibit metastatic behavior of melanoma cells.

Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota.

Atypical protein kinase C-iota (aPKCiota) plays an important role in mitogenic signaling, actin cytoskeleton organization, and cell survival. Apart from the differences in the regulatory domain, the catalytic domain of aPKCiota differs considerably from other known kinases, because it contains a modification within the glycine-rich loop motif (GXGXXG) that is found in the nucleotide-binding fold of virtually all nucleotide-binding proteins including PKCs, Ras, adenylate kinase, and the mitochondrial F1-ATPase. We have used site-directed mutagenesis and kinetic analysis to investigate whether these sequence differences affect the nucleotide binding properties and catalytic activity of aPKCiota. When lysine 274, a residue essential for ATP binding and activity conserved in most protein kinases, was replaced by arginine (K274R mutant), aPKCiota retained its normal kinase activity. This is in sharp contrast to results published for any other PKC or even distantly related kinases like phosphoinositide 3-kinase gamma, where the same mutation completely abrogated the kinase activity. Furthermore, the sensitivity of aPKCiota for inhibition by GF109203X, a substance acting on the ATP-binding site, was not altered in the K274R mutant. In contrast, replacement of Lys-274 by tryptophan (K274W) completely abolished the kinase activity of PKCiota. In accordance with results obtained with other kinase-defective PKC mutants, in cultured cells aPKCiota-K274W acted in a dominant negative fashion on signal transduction pathways involving endogenous aPKCiota, whereas the effect of the catalytically active K274R mutant was identical to the wild type enzyme. In summary, aPKCiota differs from classical and novel PKCs also in the catalytic domain. This information could be of significant value for the development of specific inhibitors of aPKCiota as a key factor in central signaling pathways.

Nuclear mitogen-activated protein kinase activation by protein kinase czeta during reoxygenation after ischemic hypoxia.

We examined the upstream kinases for mitogen-activated protein kinase (MAPK) activation during ischemic hypoxia and reoxygenation using H9c2 cells derived from rat cardiomyocytes. Protein kinase C (PKC)zeta, an atypical PKC isoform mainly expressed in rat heart, has been shown to act as an upstream kinase of MAPK during ischemic hypoxia and reoxygenation by analyses with PKC inhibitors, antisense DNA, a dominant negative kinase defective mutant, and constitutively active mutants of PKCzeta. Immunocytochemical observations show PKCzeta staining in the nucleus during ischemic hypoxia and reoxygenation when phosphorylated MAPK is also detected in the nucleus. This nuclear localization of PKCzeta is inhibited by treatment with wortmannin, a phosphoinositide 3-kinase inhibitor that also inhibits MAPK activation in a dose-dependent manner. This is supported by the inhibition of MAPK phosphorylation by another blocker of phosphoinositide 3-kinase, LY294002. An upstream kinase of MAPK, MEK1/2, is significantly phosphorylated 15 min after reoxygenation and observed mainly in the nucleus, whereas it is present in the cytoplasm in serum stimulation. The phosphorylation of MEK is blocked by PKC inhibitors and phosphoinositide 3-kinase inhibitors, as observed in the case of MAPK phosphorylation. These observations indicate that PKCzeta, which is activated by phosphoinositide 3-kinase, induces MAPK activation through MEK in the nucleus during reoxygenation after ischemic hypoxia.

T cell expressed PKCtheta demonstrates cell-type selective function.

T lymphocyte stimulation leading to interleukin-2 (IL-2) expression requires activation of protein kinase C (PKC); however, the relevant PKC isoform(s) have not yet been systematically defined. Here we examine seven major T cell expressed PKC isoforms (PKCalpha, delta, epsilon, zeta, nu, theta and iota) and identify PKCtheta to be essential for IL-2 expression (via the critical NF-AT and NF-kappaB enhancer) in Jurkat T cells. Employing a conditionally activated PKCtheta estrogen-receptor fusion mutant, a de novo synthesis-independent transactivation of JNK2 was established. Based on mRNA in situ hybridization to mouse whole body sections, PKCtheta was found to be highly expressed in lymphoid organs but also skeletal muscle and the nervous system. PKCtheta function appears to be cell-type specific, since its isoenzyme-selective function was not observed in ectopic expression studies, employing COS-1 or NIH3T3 cells. These results confirm PKCtheta to be the prime target for the activating effect of phorbol ester in T cell signaling and suggest that gene expression as well as gene function of PKCtheta is strictly controlled by the cell type.

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.

Synergistic action of protein kinase C theta and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells.

Deletion of activated peripheral T cell clones by apoptosis requires the regulated expression of Fas ligand (FasL) and sensitization of these cells to CD95-mediated signaling. To investigate the signaling pathways responsible for FasL expression in T cells, we tested-besides subfamily-selective protein kinase C (PKC) inhibitors - the effect of constitutively active mutants of representatives of all PKC subfamilies, i.e. PKCalpha,epsilon,theta,iota, on FasL luciferase promoter reporter constructs. In synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCtheta, but not PKCalpha,epsilon,iota, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCtheta AE-estrogen receptor fusion mutant led to a CaN-dependent and rapid FasL reporter activity detected as early as 4 h after addition of 4-hydroxytamoxifen, incidating a direct effect of PKCtheta action on FasL expression. Consistently, in Jurkat T cells, expression of PKCtheta AE / CaN significantly enhanced FasL protein expression and apoptosis in a CD95-dependent manner since cell death was not observed in T cells co-expressing the caspase-8 inhibitor crmA. Taken together, our results support the notion that PKCtheta and CaN are sufficient to regulate apoptosis through FasL expression.

Involvement of adenylate cyclase and p70(S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1.

Our previous results show that recombinant gp41 (aa565-647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-kappaB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-kappaB binding activity in as much as no NF-kappaB bound to the main NF-kappaB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70(S6)-kinase (rapamycin), and Gi protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-kappaB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive Gi/Go protein to effect p70(S6)-kinase activation.

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.

Protein kinase Ctheta, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells.

The predominant expression of protein kinase C (PKC) theta in T cells (J. Biol. Chem. 1993. 268: 4997-5004), its isoenzyme-specific ability to stimulate AP-1 transcriptional activity (Mol. Cell. Biol. 1996. 16: 1842-1850) and the recent discovery of its selective and antigen-dependent colocalization with the contact region between T cells and antigen-presenting cells (Nature 1997. 385: 83-89) suggest that, among the PKC family members, PKCtheta plays a specialized role in T cell activation. By investigating the downstream effectors of PKCtheta we now demonstrate a direct and isoenzyme-specific contribution of PKCtheta to c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not extracellular regulated kinase (ERK) activation. Expression of a constitutively active (CA) form of PKCtheta (but not CA-PKCalpha, epsilon and lambda/iota) resulted in strong activation of JNK/SAPK and expression of a dominant-negative form of PKCtheta interfered with the endogenous activation signal for JNK/SAPK. Importantly, Ca2+ ionophore and CA-PKCtheta (but not CA-PKCalpha, epsilon and lambda/iota) caused synergistic activation of the IL-2 promoter. Together, these data establish that PKCtheta is required for activation of JNK/SAPK signaling leading to IL-2 promoter transcription in T lymphocytes.

Epidermal growth factor (EGF) receptor blockade inhibits the action of EGF, insulin-like growth factor I, and a protein kinase A activator on the mitogen-activated protein kinase pathway in prostate cancer cell lines.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are potent mitogens that regulate proliferation of prostate cancer cells via autocrine and paracrine loops and promote tumor metastasis. They exert their action through binding to the corresponding cell surface receptors that initiate an intracellular phosphorylation cascade, leading to the activation of mitogen-activated protein kinases (MAPKs), which recruit transcription factors. We have studied the effects of EGF, IGF-I, and the protein kinase A (PKA) activator forskolin on the activation of p42/ extracellular signal-regulated kinase (ERK)2, which is a key kinase in mediation of growth factor-induced mitogenesis in prostate cancer cells. The activity of p42/ERK2 was determined by immune complex kinase assays and by immunoblotting using a phospho p44/p42 MAPK-specific antibody. EGF, IGF-I, and forskolin-induced PKA activity stimulate intracellular signaling pathways converging at the level of p42/ERK2. In the androgen-insensitive DU145 cell line, there is a constitutive basal p42/ ERK2 activity that is not present in androgen-sensitive LNCaP cells. Constitutive p42/ERK2 activity is abrogated by blockade of the EGF receptor. Hence, it is obviously caused by an autocrine loop involving this receptor. The effects of EGF on p42/ERK2 are potentiated by forskolin in both cell lines. The blockade of PKA by the specific inhibitor H89 attenuates this synergism. This finding is in contrast to those obtained in several other systems studied thus far, in which PKA activators inhibited MAPKs. p42/ERK2 in DU145 cells is highly responsive to IGF-I stimulation, whereas no effect of IGF-I on p42/ERK2 can be measured in LNCaP cells. Moreover, our results demonstrate that selective blockade of the EGF receptor in prostate cancer cells does not only inhibit the action of EGF, but also IGF-I-induced activation of the MAPK pathway and the interaction with the PKA pathway. In conclusion, these findings offer new possibilities for a therapeutical intervention in prostate cancer by targeting signaling pathways of growth factors and PKA.

Functional granulocyte/macrophage colony stimulating factor receptor is constitutively expressed on neoplastic plasma cells and mediates tumour cell longevity.

It has been shown that granulocyte/macrophage colony stimulating factor (GM-CSF) is able to support myeloma cell propagation in cooperation with interleukin (IL)-6, the major growth factor for malignant plasma cells, although the biological mechanisms involved remain unknown. Therefore we investigated (i) the expression levels of the GM-CSF receptor (GM-CSFR) constituents in three malignant plasma cell lines and in native malignant plasma cells, (ii) the ability of the receptor to mediate common signalling pathways regulating proliferation and cell survival in malignant plasma cell lines, and (iii) the effects of GM-CSF on tumour cell biology. The GM-CSFRalpha subunit was detected in the malignant plasma cell lines RPMI-8226, MC/CAR, IM-9 as well as 6/6 native myeloma cell samples derived from the bone marrow of patients with overt disease. Furthermore, GM-CSFR expression was also detected in the CD19+ fraction from 2/3 bone marrow samples and 5/8 peripheral blood samples derived from patients with malignant plasma cell disorders, but not in the CD19+ fraction of peripheral blood from healthy donors. The expressed cytokine receptor alpha-subunit was able to constitute a functional signalling complex with the ubiquitously expressed GM-CSFRbeta subunit, as demonstrated by the fact that GM-CSF induced the p21-ras/mitogen-activated protein kinase (MAPK) signalling cascade in malignant plasma cell lines. Since this signalling cascade plays an essential role in the mediation of both proliferation and cell survival, we investigated the impact of GM-CSF on these two events. Application of GM-CSF led to an increase of DNA-synthesis in MC/CAR, IM-9 and RPMI-8226 cells. Furthermore, it increased longevity of these malignant plasma cell lines by reducing the rates of spontaneous apoptosis. We conclude that (i) the functional GM-CSFR is commonly expressed on malignant plasma cells and that (ii) GM-CSF promotes the clonal expansion of myeloma cells by inhibiting spontaneous apoptosis and promoting DNA synthesis.

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.