Identification of a 95-kDa WEE1-like tyrosine kinase in HeLa cells.
Human WEE1 (WEE1Hu) was cloned on the basis of its ability to rescue wee1+ mutants in fission yeast [Igarashi, M., Nagata, A., Jinno, S., Suto, K. & Okayama, H. (1991) Nature (London) 353, 80-83]. Biochemical studies carried out in vitro with recombinant protein demonstrated that WEE1Hu encodes a tyrosine kinase of approximately 49 kDa that phosphorylates p34cdc2 on Tyr-15 [Parker, L. L. & Piwnica-Worms, H. (1992) Science 257, 1955-1957]. To study the regulation of WEE1Hu in human cells, two polyclonal antibodies to bacterially produced p49WEE1Hu were generated. In addition, a peptide antibody generated against amino acids 361-388 of p49WEE1Hu was also used. Unexpectantly, these antibodies recognized a protein with an apparent molecular mass of 95 kDa in HeLa cells, rather than one of 49 kDa. Immunoprecipitates of p95 phosphorylated p34cdc2 on Tyr-15, indicating that p95 is functionally related to p49WEEIHu, and mapping studies demonstrated that p95 is structurally related to p49WEE1Hu. In addition, the substrate specificity of p95 was more similar to that of fission yeast p107wee1 than to that of human p49WEE1. Finally, the kinase activity of p95 toward p34cdc2/cyclin B was severely impaired during mitosis. Taken together, these results indicate that the original WEE1Hu clone isolated in genetic screens encodes only the catalytic domain of human WEE1 and that the authentic human WEE1 protein has an apparent molecular mass of approximately 95 kDa.
Parker
L L
LL
Department of Molecular Genetics, Pfizer Central Research, Groton, CT 06340, USA.
Sylvestre
P J
PJ
Byrnes
M J
MJ
Liu
F
F
Piwnica-Worms
H
H
D000918:Antibody Specificity; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins; D003429:Cross Reactions; D016213:Cyclins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D009687:Nuclear Proteins; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology; D029702:Schizosaccharomyces pombe Proteins; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D016213:Cyclins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D016203:CDC2 Protein Kinase