Identification of a 95-kDa WEE1-like tyrosine kinase in HeLa cells.

Abstract

Human WEEl (WEEJHu) was cloned on the basis of its ability to rescue weel+ mutants in fission yeast [Igarashi, M., Nagata, A., Science 257, 1955Science 257, -1957. To study the regulation of WEEJHu 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 p49WEElHu, and mapping studies demonstrated that p95 is structurally related to p49WEElHu. In addition, the substrate specificity of p95 was more similar to that of fission yeast pl07weel 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 WEEIHu 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 -95 kDa.In Schizosaccharomyces pombe, weel+ functions as a dosedependent inhibitor of mitosis (1). Biochemical studies demonstrated that weel+ encodes a protein kinase of 107 kDa, which autophosphorylates on serine, threonine, and tyrosine residues (2-4). The physiologic target of plo7weel is p34cdc2, a serine/threonine-specific protein kinase whose activity is required for the entry of cells into mitosis. p107weel phosphorylates p34cdc2 on Tyr-15, a modification that is sufficient for the inactivation of the p34cdc2/cyclin B complex (4). Tyr-15 phosphorylation is also catalyzed by Mikl, a tyrosine kinase that functionally compensates for loss of weel + in fission yeast (5,6). Acting in opposition to wee] + and mikl + is the mitotic inducer cdc25+. cdc25+ encodes a protein phosphatase that dephosphorylates Tyr-15 of p34cdc2, thereby activating the p34cdc2/cyclin B complex (7-12). Functional homologs of weel + and cdc25+ have been isolated from higher eukaryotic cells.A human homolog of weel + (WEElHu) was isolated on the basis of its ability to functionally compensate for loss of weel + function in fission yeast (13). WEElHu encodes a tyrosine kinase of 49 kDa, denoted p49WEE1Hu (14-16). Structurally, p49WEElHu consists primarily of a catalytic domain, lacking the large amino-terminal regulatory domain present in its fission yeast counterpart. The kinase domain of p49WEE1Hu is 29% identical to that of the S. pombe protein plO7weel. Like plo7weel, recombinant p49WEE1Hu phosphorylates p34cdc2 on Tyr-15 and inactivates the p34cdc2/cyclin B complex (15, 16). However, there are notable differences between the human and yeast enzymes. Whereas p107weel shows strict substrate specificity for the cyclin-bound form of p34cdc2 (4), p49WEE1Hu will also phosphorylate monomeric p34cdc2 and a peptide containing Tyr-15.All of the above-mentioned studies were carried ou...

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

“…In mammalian somatic cells, the fate of wee1 is less clear: some studies found that wee1 protein was reduced during mitosis (Baldin and Ducommun, 1995;Watanabe et al, 1995), whereas others found that it was stable (McGowan and Russell, 1995;Parker et al, 1995). In Xenopus egg extracts, immunoblots of endogenous wee1 show that overall wee1 protein levels are essentially constant across the early embryonic cell cycles (Walter et al, 1997), a point that we confirm here.…”

Knockdown of Chk1, Wee1 and Myt1 by RNA Interference Abrogates G2 Checkpoint and Induces Apoptosis

“…8 The inhibitory phosphorylations of threonine 14 (T14) and tyrosine 15 (Y15) at the ATP-binding pocket are mediated by Wee1 and Myt1 kinases. [9][10][11][12][13] Wee1 is the major kinase phosphorylating the Y15 site. While Myt1 preferentially phosphorylates the T14 site, it can also phosphorylate the Y15 site.…”

ΔMEKK3:ER* activation induces a p38α/β2-dependent cell cycle arrest at the G2 checkpoint

“…The loss of these controlling subunits would certainly account for both the reduction in CDK activity and the lack of any CDK1 Y 15 inhibitory phosphorylation, since Wee1/Myt1 can only phosphorylate this inhibitory site once the cyclin B/CDK1 complex is assembled (Parker et al, 1995;Booher et al, 1997). There are several reported precedents for this.…”