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 approximate to 49 kDa that phosphorylates p34(cdc2) 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 p34(cdc2) On Tyr-15, indicating that p95 is functionally related to p49WEE1Hu, 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 p107(wee1) than to that of human p49WEE1. Finally, the kinase activity of p95 toward p34(cdc2)/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 approximate to 95 kDa.