Originally, WEE1 was isolated as a gene responsible for the “wee” phenotype in fission yeast (Russell and Nurse 1987). WEE1 from fission yeast has been shown to autophosphorylate at serine and tyrosine residues, although the exact role of serine phosphorylation is still unknown. WEE1 from fission yeast and other species has been shown to phosphorylate cyclin-associated CDK at Tyr 15; this residue is located near the ATP-binding pocket of CDK. Another WEE1 family member, Myt1, can phosphorylate Thr 14 and Tyr 15 of CDKs. However, Myt1 preferentially phosphorylates Thr 14; thus, WEE1 is considered to be principally responsible for Tyr 15 phosphorylation.
Substrates for the Protein Kinase Activity
WEE1 phosphorylates CDK proteins at Tyr 15, which is near the ATP-binding pocket, but only when the CDK is associated with cyclin. Among the cyclin-associated CDKs, WEE1 efficiently phosphorylates CDK1 (Cdc2) and CDK2, but not CDK4, in vitro (Watanabe et al. 1995). Phosphorylation of cyclin-associated CDKs during S or G2 inactivates them, delaying the onset of mitosis. Specifically, WEE1 localizes to the nucleus during interphase of the cell cycle, where it is thought to protect the nucleus from premature mitotic onset by phosphorylating the CDK1 complex. Cyclin A-/E-associated CDK2 is phosphorylated at Tyr 15 in vivo as well, but the exact role of the phosphorylation on the cell cycle progression is not clear.
Orthologs and Paralogs
WEE1 (or WEE1A) is expressed in somatic cells of mammalian cells, while its closest homolog, WEE1B, is expressed only in embryonic cells (Nakanishi et al. 2000). In contrast, only an ortholog of Myt1is reported and is expressed in both somatic and embryonic cells. In Xenopus, the embryonic form of WEE1 (or WEE1A) was isolated first, and the somatic form (named WEE2 or WEE1B) was isolated later. Thus, the nomenclature of Xenopus Wee1 differs from that of mammalian WEE1. Fission yeast has two WEE1 family kinases, WEE1 and MIK1, both of which has redundant role on each other. Among these two, MIK1 is reported to be more similar to mammalian WEE1 (Watanabe et al. 1995). Budding yeast has only one WEE1, “Swe1,” and there seems to be one WEE1 protein (Dwee1) in addition to Dmyt1 in Drosophila as well. Three WEE1-like genes are encoded in the genome of C. elegans (Wee-1.1, Wee-1.2, Wee-1.3). Transient expression of Wee-1.1 during embryogenesis is observed, while no expression of Wee-1.2 is reported. Wee-1.3 is more similar to MYT1 than WEE1 of other species in the sequence revel.
Regulation of Activity
The activity of WEE1 is regulated both by protein phosphorylation and degradation. During S and G2 phase, WEE1 is phosphorylated at a serine residue (S642) near its carboxyl terminus (C terminus). A mutation in WEE1 in which the serine at position 642 is replaced with alanine (S642A) cannot bind to 14-3-3 scaffolding proteins, indicating that the phosphorylation of S642 is required for 14-3-3 binding (Wang et al. 2000; Rothblum-Oviatt et al. 2001; Katayama et al. 2005). While Chk1 was shown to phosphorylate the Xenopus Wee1 (embryonic type, homolog of mammalian WEE1B) at its 14-3-3 binding site (Lee et al. 2001), the protein kinase that phosphorylates S642 of mammalian WEE1 is not known. However, WEE1/14-3-3 binding is maintained following UCN-01 treatment (an inhibitor of some protein kinases such as Chk1), suggesting that a kinase other than Chk1 is responsible for phosphorylation of mammalian WEE1 at S642 (Rothblum-Oviatt et al. 2001).
Using two-hybrid screening methods, 14-3-3ζ and 14-3-3β were separately isolated by two groups as molecules that interact with the C terminus of WEE1 (Honda et al. 1997; Wang et al. 2000). These interactions were confirmed in cultured cells. Binding of exogenously expressed 14-3-3σ to WEE1 was also shown in HeLa cells. 14-3-3 binding has been shown to function as a positive regulator of WEE1 through protein stabilization or activation of intrinsic kinase activity (Wang et al. 2000; Rothblum-Oviatt et al. 2001). However, in contrast to these reports, another group has reported that AKT/PKB phosphorylates Ser 642, and 14-3-3θ binds to the phosphorylated Ser 642 residue in human cells, but 14-3-3β and 14-3-3σ do not. The authors suggest that AKT suppresses the kinase activity of WEE1 through 14-3-3θ binding and subsequent translocation to the cytoplasm (Katayama et al. 2005).
During M phase, WEE1 is phosphorylated at the amino (N)-terminal non-catalytic domain (Watanabe et al. 1995). This phosphorylation event inactivates WEE1, but the mechanism by which it occurs is still not clear. Phosphorylation at Thr 186 of Xenopus somatic Wee1 (equivalent to Thr 239 in mouse and human WEE1) creates a binding site for Pin1; this association is responsible for the inactivation of WEE1 during M phase (Okamoto and Sagata 2007).
Regulation of Protein Level
The residues flanking serine 123 (Ser-Pro-Val-Lys) are a good consensus sequence for CDK phosphorylation. Cyclin B/CDK1 phosphorylates Ser 123 of WEE1 efficiently in vitro (Watanabe et al. 2004). In vivo, phosphorylation of Ser 123 occurs during S, G2, and M phase possibly by CDK2 bound to cyclin A/E and cyclin B/CDK1 (Watanabe et al. 2005). As phosphorylation of Ser 123 induces the phosphorylation of Ser 121 by CK2 (an abundant and constitutively active protein kinase), WEE1 singly phosphorylated at Ser 123 may be very transient. Phosphorylation of Ser 123 also creates the binding site for the Plk1 PBD domain and accelerates the phosphorylation of Ser 53 (Ser 52 in mouse) by Plk1 (Watanabe et al. 2005).
The expression level of WEE1 mRNA is high in G1/S phase in antigen-stimulated murine T cells and is low in M phase in regenerating mouse liver cells. In the latter system, the transcription of Wee1 is shown to be directly regulated by the molecular components of the circadian clock (Matsuo et al. 2003).
The subcellular localization of WEE1 has predominately been analyzed in human cells. During interphase, WEE1 localizes in the nucleus and is thought to prevent premature mitotic entry by phosphorylating and inactivating CDK1-containing complexes that have originated in the cytoplasm. Just before mitosis, WEE1 is redistributed into the cytoplasm. During cell division, WEE1 locates at the mitotic equator and mid-body bridges. As described in the Regulation of Activity section, the binding of 14-3-3 proteins to pS642 of Wee1 may induce its translocation to the cytoplasm. In the cells, WEE1 expression is ubiquitous according to Unigene (http://www.ncbi.nlm.nih.gov/UniGene).
Small interfering (si) RNA-mediated depletion of WEE1 induces apoptotic cell death in human cells. Wee1 homozygous knockout mice have a defective G2/M cell cycle checkpoint and die before embryonic day 3.5 (before reaching the blastocyst stage) (Tominaga et al. 2006). Furthermore, pharmacologic inhibition of Wee1 by its specific inhibitor (PD0166285) abrogates the G2/M checkpoint and induced apoptosis (Wang et al. 2001).
Overexpression of WEE1 induces cell cycle arrest at the G2 phase in human cells (McGowan and Russell 1993; Watanabe et al. 2004). Overexpression of Dwee1 in Drosophila induces abnormal organ development, which is caused by the inhibition of temporally and spatially regulated cell division (Price et al. 2002).
Wee1i, a splice valiant isolated from rat, contains an open reading frame that starts at the second methionine (Met215) of Wee1. Wee1i is expressed in rat brain, thymus, and kidney as well as normal rat kidney (NRK) and PC12 cells lines at the RNA level. The 49 kDa protein products from Wee1i are detected in rat brain and murine cell lines; however, in the same analyses, 49 kDa protein products could not be detected in human cells (Yu et al. 2005).
WEE1 phosphorylates cyclin-associated CDK proteins at Tyr 15. Phosphorylation of CDKs during S or G2 inactivates them, delaying the onset of mitosis. WEE1 (or WEE1A) is expressed in somatic cells of mammalian cells, while its closest homolog, WEE1B, is expressed only in embryonic cells. The activity of WEE1 is regulated both by protein phosphorylation and degradation. During M phase, WEE1 is phosphorylated at the amino (N)-terminal non-catalytic domain. This phosphorylation inactivates WEE1, but the mechanism by which it occurs is still not clear. Using two-hybrid screening methods, 14-3-3 proteins are reported to be as molecules that interact with the C terminus of WEE1. These interactions were confirmed in cultured cells and seem to have roles on subcellular localization and stability of WEE1. WEE1 degradation is heightened at M phase, which is important for the rapid activation of CDK1 at the onset of M phase. The E3 ubiquitin ligase that is responsible for the WEE1 ubiquitination is the β-TrCP-containing SCF (Skp, Cullin, F-box) complex. β-TrCP binds to residues surrounding phosphorylated Ser 53 (pS53) and pS121 of human WEE1 (pS52 and pS121 of mouse WEE1), which are phosphorylated by Plk1 and CK2, respectively. Wee1 homozygous knockout mice have a defective G2/M cell cycle checkpoint and die before embryonic day 3.5.