[show abstract][hide abstract] ABSTRACT: The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.
Molecular and Cellular Biology 03/1997; 17(2):732-41. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two-hybrid screen of a mouse-embryo cDNA library we detected an interaction between A-Raf and CK2beta subunit. This binding was specific, as no interaction between CK2beta and B-Raf or c-Raf-1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A-Raf kinase domain. A-Raf kinase activity was enhanced 10-fold upon coexpression with CK2beta in Sf9 cells. The alpha subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf-isoform-specific activation and a regulatory role for CK2beta independent of the CK2alpha subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.
[show abstract][hide abstract] ABSTRACT: PC12 pheochromocytoma cells possess four known MEK activators: A-, B-, c-Raf-1 and MEKK. In order to examine whether differentiation factors or growth factors have a Raf isozyme preference for activation of the mitogenic cytoplasmic Raf-MEK-MAPK protein kinase cascade, the activation kinetics of these enzymes in response to epidermal growth factor (EGF) and nerve growth factor (NGF) were compared. An initial activation of all three Raf kinases was noticed, but only A- and B-Raf showed sustained activation by NGF, which was not seen after EGF treatment. Furthermore, expression of oncogenic versions of all three Raf kinases as well, as a potentially Raf-independent MEK activator, v-Mos, leads to activation of MAPK and to differentiation of PC12 cells. These data suggest a differential regulation of Raf kinases and that probably no alternative Raf substrates are involved in differentiation processes of PC12 cells.
[show abstract][hide abstract] ABSTRACT: NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.
Molecular and Cellular Biology 04/1996; 16(3):868-76. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: The frequency of H-2Kk and HPRT-deficient T cells was measured in the H-2Kb, kDd,k genotype mouse 8-10 weeks after X-ray exposure at doses up to 6 Gy to compare the mutant frequency (MF) of an autosomal gene with that of an X-chromosomal gene. H-2K mutants were enriched by magnetic cell separation (MACS) using the H-2Kk-specific monoclonal antibody H100.5/28 and were isolated by limiting dilution cloning. Finally, the mutant phenotype was verified by flow cytometric analysis in a representative number of clones. The frequency of HPRT-deficient T cells rises from 2.5 x 10(-6) at 0 Gy to a maximum of 1.13 x 10(-4) at 4 Gy, and decreases to 2.9 x 10(-5) at 6 Gy. The H-2K- MF in the non-irradiated mouse was 8.4 x 10(-7). It increases with dose to a maximum of 8.1 x 10(-6) at 4 Gy and declines to 3.3 x 10(-6) at 6 Gy. The H-2K- MF measured depends on the monoclonal antibody used for the isolation of mutants. In a pilot study with another H-2Kk-specific monoclonal antibody (11.4.1), the spontaneous MF was four times higher than in experiments with the H100.5/28 monoclonal antibody. The expression of other class 1 antigens was investigated in H-2K- clones. The H-2Dd antigen had also disappeared in six of 41 clones from irradiated animals. This gene is situated at a distance of 1500 kb from the K-locus. The H-2Kb antigen was present in every investigated clone. In the discussion a model is presented that explains the shape of the dose-response curve of MF by selection against mutants in vivo systems under homeostasis. The results of the present investigation indicate that observed X-ray mutagenicity depends on many factors and that several genes have to be explored before reliable risk estimates are possible.
International Journal of Radiation Biology 05/1995; 67(4):421-30. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: A method is described for determining the frequency of cells with a mutation in an autosomal gene coding for a membrane protein. Using a monoclonal antibody to H-2Kk surface antigen and magnetic cell separation (MACS) more than 10(4)-fold enrichment of the H-2Kk negative population was achieved, as tested with artificial mixtures containing a known number of antigen-negative cells. After a second magnetic sorting mutant frequencies as low as 10(-6) could be measured. The number of clonogenic mutants was evaluated by limiting dilution cloning and verification of the mutant phenotype by FACScan (flow cytometry) analysis in a representative number of clones. The spontaneous frequency of H-2Kk deficient mutants was 0.80 x 10(-6), and this increased after irradiation with 6 Gy X rays to 3.38 x 10(-6) within the next 8 weeks. About 50 mutant clones were screened for the presence of other class 1 antigens on the cell surface by FACScan analysis. All mutants continued to express other class 1 antigens.
Journal of Immunological Methods 06/1994; 171(1):121-30. · 2.23 Impact Factor