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M K Owada

Publications and source records attributed to M K Owada.

41 records · Page 3Linked to original sources
PubMed1981

Association of the transformation-specific protein pp60src with the membrane of an avian sarcoma virus.

The transformation-specific protein pp60(src) coded for by avian sarcoma viruses and its associated protein kinase activity is present in virus particles of Rous sarcoma virus, Schmidt-Ruppin strain, subgroup D. Quantitative comparison of the immunoglobulin G-phosphorylating activity in Schmidt-Ruppin D virus and Schmidt-Ruppin D virus-transformed fibroblasts indicated that there was two- to fourfold less activity in the virus particles. Disruption of virus particles with nonionic detergent demonstrated that the protein kinase activity fractionated together with the viral membrane protein gp85. Therefore, viral membranes were isolated by floating detergent-disrupted virus through a discontinuous sucrose density gradient. At a characteristic density corresponding to 26% sucrose, viral membranes were identified by the radioactively labeled viral glycoprotein and furthermore by the membrane marker enzyme Na(+)-K(+)-stimulated, Mg(2+)-activated ATPase and were visualized by electron microscopy. Contamination by cell membranes could be ruled out, since (i) the virus preparation was free of cell membrane contaminants as judged from electron microscopy, (ii) floating of intact virus did not release membraneous material, and (iii) virus-free tissue culture fluid from Schmidt-Ruppin D virus-transformed nonproducer cells (which potentially contain cell membranes) did not contribute any immunoglobulin G-phosphorylating activity after mixing with nontransforming virus and pelleting it. Both pp60(src) and the protein kinase activity were found to be associated with the viral membrane. Solubilization of virus by detergent released two phosphoproteins, with molecular weights of 42,000 and 45,000 which reacted with sera specific for pp60(src) and revealed protein kinase activity but which were not membrane bound and may have represented degradation products of pp60(src). Surface iodination of intact virus particles (harvested at 3-h intervals) did not result in radioactive labeling of pp60(src), whereas collection at 24-h intervals allowed iodination of pp60(src). In contrast to the viral glycoprotein gp85, the iodinated virion-associated pp60(src) was insensitive to mild proteolytic treatment. Binding to tumorbearing-rabbit serum, immunoglobulin G phosphorylation, and endogenous phosphorylation of 60,000-, 45,000-and 42,000-dalton proteins required lysed virus and were not possible with intact virus. These results indicated that pp60(src) was embedded within the viral membrane. Membrane proteins phosphorylated in vitro were analyzed for their phosphoamino acid composition. Eight polypeptides exhibited phosphorylation in tyrosine and were absent in nontransforming viral controls.

Animals↗
PubMedDate not supplied

A tyrosine-specific protein kinase inhibitor, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide, blocks the phosphorylation of tyrosine kinase substrate in intact cells.

Inhibition by alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) of tyrosine-specific protein kinase was examined using epidermal growth factor (EGF)-treated A431 cells at the concentration of 25 to 100 microM. ST 638 had negligible effects on the growth and morphology of A431 cells and on EGF binding to its receptor, and subsequent down-regulation of the receptor. ST 638 specifically inhibited EGF-induced phosphorylation of tyrosine residues of whole cell proteins in a dose-dependent manner without affecting the phosphorylation of serine and threonine residues. ST 638 greatly inhibited the EGF-induced phosphorylation of lipocortin I at 25 microM, and yet had a negligible effect on the EGF-induced phosphorylation of EGF receptor. Neither the amount of [35S]methionine-labeled lipocortin I nor the serine/threonine phosphorylation level of fodrin beta-subunit was affected by the same concentration of ST 638. These results indicate that the phosphorylation of lipocortin I is not relevant to the transformation of A431 cells. In cell lines transformed by src or fgr oncogene encoding tyrosine kinase, ST 638 also inhibited phosphorylation of calpactin I (p36) without affecting that of the oncogene products. Two-dimensional polyacrylamide gel electrophoresis showed that ST 638 specifically inhibited the EGF-induced phosphorylation and dephosphorylation of cellular proteins in A431 cells.

Animals↗