Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15.

Abstract

In fission yeast, the M‐phase inducing kinase, a complex of p34cdc2 and cyclin B, is maintained in an inhibited state during interphase due to the phosphorylation of Cdc2 at Tyr15. This phosphorylation is believed to be carried out primarily by the Wee1 kinase. In human cells the negative regulation of p34cdc2/cyclin B is more complex, in that Cdc2 is phosphorylated at two inhibitory sites, Thr14 and Tyr15. The identities of the kinases that phosphorylate these sites are unknown. Since fission yeast Wee1 kinase behaves as a dual‐specificity kinase in vitro, a popular hypothesis is that a human Wee1 homolog might phosphorylate p34cdc2 at both sites. We report here that a human gene, identified as a possible Wee1 homologue, blocks cell division when overexpressed in HeLa cells. This demonstrates functional conservation of the Wee1 mitotic inhibitor. Contrary to the dual‐specificity kinase hypothesis, purified human Wee1 phosphorylates p34cdc2 exclusively on Tyr15 in vitro; no Thr14 phosphorylation was detected. Human and fission yeast Wee1 also specifically phosphorylate synthetic peptides at sites equivalent to Tyr15. Mutation of a critical lysine codon (Lys114) believed to be essential for kinase activity abolished both the in vivo mitotic inhibitor function and in vitro kinase activities of human Wee1. These results conclusively prove that Wee1 kinases inhibit mitosis by directly phosphorylating p34cdc2 on Tyr15, and strongly indicate that human cells have independent kinase pathways directing the two inhibitor phosphorylations of p34cdc2.

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

“…We showed a morphological cell enlargement and a broad distribution of¯uorescence intensities with an increased DNA content in SAG knockout cells (Figure 4). Several recent ®ndings also support this notion: (1) Xenopus Cdc34, an E2 that binds to SAG/ROC/Rbx/Hrt Ohta et al, 1999;Seol et al, 1999;Skowyra et al, 1999;Tan et al, 1999;Tyers and Willems, 1999) promoted degradation of Wee1 (Michael and Newport, 1998), a key G2/M negative regulator (McGowan and Russell, 1993;Parker and Piwnica Worms, 1992); (2) SCF components, Skp1 and Cul1 were localized to the centrosome and regulated the centrosome duplication cycle in mammalian cells (Freed et al, 1999); and (3) Cdc4, an F-box protein in SCF complex was required for the onset of S phase as well as anaphase in Saccharomyces cerevisiae (Goh and Surana, 1999).…”

A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast

“…Cdc25p accumulates in a flp1 null S. pombe mutant In S. pombe cells, as well as in most eukaryotes, entry into mitosis is controlled by the balance between the activities of the inhibitory kinase Wee1p and the opposing effect of the phosphatase Cdc25p in regulating the activity of Cdc2p (Russell and Nurse, 1986;Russell and Nurse, 1987a; Gould Flp1p controls degradation of Cdc25p and Norbury et al, 1991;Norbury and Nurse, 1992;McGowan et al, 1993), the catalytic subunit of CDKcomplexes. We therefore examined the consequences of mutating the flp1 gene upon the stability and phosphorylation state of Cdc25p and Wee1p.…”

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

“…9, May 1998coded by wee1 ϩ negatively regulates G 2 /M-phase transition by phosphorylating Cdc2 (McGowan and Russell, 1993). Cells harboring the deleted wee1 allele are viable, but timing of the initiation of mitosis is advanced.…”