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S Kampfer

Publications and source records attributed to S Kampfer.

11 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

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.

3T3 Cells↗
PubMed1999

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.

3T3 Cells↗
PubMed1999

Isolation and characterization of human DNA from mosquitoes (Culicidae).

Human DNA was prepared from mosquitoes (Culicidae) which were collected in a room shared by four human individuals. Several insects did not contain human blood and DNA preparation from them was not successful. However, high molecular weight human genomic DNA could be isolated from four insects. HLA-DQalpha and D1S80 analysis showed that the blood from one insect was a mixture from two persons, whereas the others contained blood from single individuals. Human DNA isolated 26 h after ingestion was still suitable for typing. These results showed that DNA isolated from mosquitoes is qualitatively and quantitatively sufficient for DNA typing and could be helpful to identify individuals involved in certain cases of body violence or captivity.

Alleles↗
PubMed1998

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Animals↗