Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis.
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.
Resnick
R J
RJ
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Taylor
S J
SJ
Lin
Q
Q
Shalloway
D
D
D016475:3T3 Cells / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D002457:Cell Extracts; D019926:Cyclin B / Q000378:metabolism; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007399:Interphase; D007701:Kinetin; D051379:Mice; D008938:Mitosis; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002457:Cell Extracts; D019926:Cyclin B; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C489559:KHDRBS1 protein, human; C513253:Khdrbs1 protein, mouse; D011687:Purines; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; C090046:olomoucine; D016392:Proto-Oncogene Proteins pp60(c-src); D016203:CDC2 Protein Kinase; D007701:Kinetin