Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase
UMass Chan AffiliationsProgram in Molecular Medicine
Calcium-Calmodulin-Dependent Protein Kinases
Guanine Nucleotide Exchange Factors
Hemagglutinin Glycoproteins, Influenza Virus
Molecular Sequence Data
Polymerase Chain Reaction
Recombinant Fusion Proteins
Son of Sevenless Proteins
ras Guanine Nucleotide Exchange Factors
Medicine and Health Sciences
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AbstractSon of sevenless-1 and -2 (Sos-1 and -2) are guanosine nucleotide exchange factors implicated in the activation of Ras by both the insulin and epidermal growth factor signal transduction pathways. Ras appears to function by initiating the activation of cellular protein kinases including mitogen-activated protein (MAP) kinases. Sos proteins contain numerous sequences in their carboxyl-terminal regions which correspond to consensus sites for MAP kinase phosphorylation. To examine whether these sites are substrates for MAP kinases, the cDNA encoding Drosophila Sos (dSos) was tagged with sequences encoding the major antigenic epitope of the influenza virus hemagglutinin (HA) to create a dSosHA fusion construct. dSosHA was transiently expressed in COS-1 cells and immunoprecipitated with anti-HA antibodies. When immune complexes were incubated with purified MAP kinase and [gamma-32P]ATP, a phosphorylated band of 180 kDa was observed when analyzed by SDS-polyacrylamide gel electrophoresis. This band was not present in immunoprecipitations from cells transfected with vector alone. No phosphorylation of the 180 kDa band was seen when immunoprecipitates were incubated with [gamma-32P]ATP in the absence of MAP kinase. Two dimensional analysis of tryptic peptides from dSosHA phosphorylated by MAP kinase in vitro revealed two major phosphorylated species that were also found in dSosHA isolated from COS-1 cells labeled with 32Pi. These results are consistent with the hypothesis that a feedback loop exists wherein growth factor-activated MAP kinases phosphorylate and regulate Sos proteins.
SourceJ Biol Chem. 1994 Feb 18;269(7):4717-20.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/42480
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