Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis.
Journal - Oncogene (ENGLAND )
Sam68 (Src-associated in mitosis) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein which associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. We now show that Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated on threonine residues exclusively during mitosis as well as on serine residues during both interphase and mitosis. Recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, was phosphorylated on threonine and serine residues after incubation with mitotic lysates several-fold more extensively than after incubation with unsynchronized lysates. Cdc2 was identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of its biological effects during mitosis.
|ISSN : ||0950-9232|
|Mesh Heading : ||3T3 Cells Adaptor Proteins, Signal Transducing Animals CDC2 Protein Kinase Cell Cycle Cell Extracts Cyclin B DNA-Binding Proteins Enzyme Inhibitors Hela Cells Humans Interphase Kinetin Mice Phosphorylation Proto-Oncogene Proteins pp60(c-src) Purines RNA-Binding Proteins Recombinant Proteins Serine Threonine metabolism antagonists & inhibitors physiology metabolism pharmacology drug effects metabolism pharmacology genetics genetics metabolism metabolism metabolism|
|Mesh Heading Relevant : ||Mitosis metabolism metabolism metabolism|
Stimulation of yeast adenylyl cyclase activity by lysophospholipids and fatty acids. Implications for the regulation of Ras/effector function by lipids.
Journal - The Journal of biological chemistry (UNITED STATES )
A reconstituted system containing membranes prepared from various Saccharomyces cerevisiae strains (CYR1 ras1 ras2) and a recombinant RAS2 protein was used to evaluate the effect of lipids on adenylyl cyclase activity. Incubation of wild-type membranes with lysophosphatidylinositol, lysophosphatidylserine, or lysophosphatidylcholine stimulated adenylyl cyclase activity in the absence and presence of RAS between 2-10-fold depending upon the individual lipid. Unsaturated fatty acids preferentially increased activity 2-3-fold in the presence of RAS. Other phospholipids as well as several detergents had only marginal effects on adenylyl cyclase activity. Lipids had no effect on either the binding or hydrolysis of GTP by RAS. LysoPI decreased both the Km for ATP and the amount of RAS required for enzyme activation. Four catalytically active deletion mutants of the CYR1 protein including one containing only the C-terminal 417 amino acids were similarly responsive to lysoPI when compared to the wild-type enzyme. These data suggest that lysophospholipids and fatty acids, metabolites of the mitogenically responsive enzyme phospholipase A2, may represent a novel mechanism for modulating the activity of downstream effector molecules and their interaction with Ras proteins.
|ISSN : ||0021-9258|
|Mesh Heading : ||Adenosine Triphosphate Adenylate Cyclase Detergents Enzyme Activation Fatty Acids Fungal Proteins GTP Phosphohydrolases Guanosine Triphosphate Lysophospholipids Protein Binding Saccharomyces cerevisiae metabolism metabolism metabolism metabolism|
|Mesh Heading Relevant : ||Saccharomyces cerevisiae Proteins ras Proteins metabolism metabolism metabolism enzymology|