Florian Überall

Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

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.

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

The predominant expression of protein kinase C (PKC) θ 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 (Nature1997. 385: 83 – 89) suggest that, among the PKC family members, PKCθ plays a specialized role in T cell activation. By investigating the downstream effectors of PKCθ we now demonstrate a direct and isoenzyme‐specific contribution of PKCθ 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 PKCθ (but not CA‐PKCα, ϵ and λ / ι) resulted in strong activation of JNK/SAPK and expression of a dominant‐negative form of PKCθ interfered with the endogenous activation signal for JNK/SAPK. Importantly, Ca2+ ionophore and CA‐PKCθ (but not CA‐PKCα, ϵ and λ / ι) caused synergistic activation of the IL‐2 promoter. Together, these data establish that PKCθ is required for activation of JNK/SAPK signaling leading to IL‐2 promoter transcription in T lymphocytes.

Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-α or novel nPKC-ε increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-η significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-η as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-α, nPKC-ε, and nPKC-η in vitro Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-α K368R or (dominant negative) PKC-ε K436R. The fact, that the constitutively active PKC-λ A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-α and novel nPKC-ε, but not atypical aPKC-λ are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.

Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-λ, ϵ and ζ

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.

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 ε, λ, and ζ, but not cPKC-α. 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 ε and ζ, but not λ are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-ε or aPKC-ζ 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-ζ, indicating that aPKC-ζ 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-ε, 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 ε, λ, and ζ, but not cPKC-α. Evidence is presented that aPKC-λ acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes ε and ζ act downstream of Rac and are required for the activation of ERKs.

Synergistic action of protein kinase C θ 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. PKCα,ϵ,θ,ι, on FasL luciferase promoter reporter constructs. In synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCθ, but not PKCα,ϵ,ι, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCθ 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 PKCθ action on FasL expression. Consistently, in Jurkat T cells, expression of PKCθ 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 PKCθ and CaN are sufficient to regulate apoptosis through FasL expression.

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.

ABSTRACT 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-fosinduction 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.

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‐γ 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.