Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase.
Son 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.
Cherniack
A D
AD
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
Klarlund
J K
JK
Czech
M P
MP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis; D007301:Insect Hormones / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020664:Son of Sevenless Proteins; D013379:Substrate Specificity; D014162:Transfection; D020663:ras Guanine Nucleotide Exchange Factors
D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D007301:Insect Hormones; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020664:Son of Sevenless Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases