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 w ee1(+) 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 th at WEE1Hu encodes a tyrosine kinase of approximate to 49 kDa that phos phorylates p34(cdc2) on Tyr-15 [Parker, L. L. & Piwnica-Worms, H. (199 2) Science 257, 1955-1957]. To study the regulation of WEE1Hu in human cells, two polyclonal antibodies to bacterially produced p49WEE1Hu we re generated. In addition, a peptide antibody generated against amino acids 361-388 of p49WEE1Hu was also used. Unexpectantly, these antibod ies recognized a protein with an apparent molecular mass of 95 kDa in HeLa cells, rather than one of 49 kDa, Immunoprecipitates of p95 phosp horylated p34(cdc2) On Tyr-15, indicating that p95 is functionally rel ated to p49WEE1Hu, and mapping studies demonstrated that p95 is struct urally 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 s creens encodes only the catalytic domain of human WEE1 and that the au thentic human WEE1 protein has an apparent molecular mass of approxima te to 95 kDa.