Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is downregulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the ␤-transducin repeat-containing protein 1͞2 (␤-TrCP1͞2) F-box protein-containing SKP1͞Cul1͞F-box protein (SCF) complex (SCF ␤-TrCP1͞2 ) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for ␤-TrCP, recognition of Wee1A by ␤-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for ␤-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by ␤-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCF ␤-TrCP1͞2 in vitro. Mutation of these residues or depletion of ␤-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that ␤-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide.

The c-mos proto-oncogene product, pp39mos, is present in unfertilized Xenopus eggs, and disappears on fertilization. Microinjection of synthetic mos RNA into two-cell embryos induces cleavage arrest at metaphase. By contrast, egg cytosol extracts, when immunodepleted of endogenous pp39mos, lose their cleavage-arresting activity in injected embryos. These results demonstrate that Mos protein is the cytostatic factor CSF, long known as an endogenous meiotic inhibitor in vertebrate eggs.

Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome and, consequently, regulates metaphase-anaphase transition and exit from mitosis. Here we report that Cdc2-cyclin B-activated Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses APC activity. PKA is superior to Plk in its regulation of APC, and Plk activity peaks whereas PKA activity is falling at metaphase. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity.

Most nonmalignant cells are anchorage-dependent; they require substrate attachment for growth and, in some instances, survival. This requirement is lost on oncogenic transformation. The cyclin E-CDK2 complex, which is required for the G1-S transition of the cell cycle, was activated in late G1 phase in attached human fibroblasts, but not in fibroblasts maintained in suspension. In transformed fibroblasts the complex was active regardless of attachment. The lack of cyclin E-CDK2 activity in suspended cells appeared to result from increased expression of CDK2 inhibitors and a concomitant decrease in phosphorylation of CDK2 on threonine-160. Suppression of cyclin E-CDK2 activity may thus underlie the anchorage dependence of cell growth.

In Xenopus the c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation, for the progression from meiosis I to meiosis II and for the second meiotic metaphase arrest, acting as an essential component of the cytostatic factor CSF. Its function in mouse oocytes is unclear, however, as is the biological significance of c-mos mRNA expression in testes and several somatic tissues. We have generated c-mos-deficient mice by gene targeting in embryonic stem cells. These mice grew at the same rate as their wild-type counterparts and reproduction was normal in the males, but the fertility of the females was very low. The c-mos-deficient female mice developed ovarian teratomas at a high frequency. Oocytes from these females matured to the second meiotic metaphase both in vivo and in vitro, but were activated without fertilization. The results indicate that in mice Mos plays a role in the second meiotic metaphase arrest, but does not seem to be essential for the initiation of oocyte maturation, spermatogenesis or somatic cell cycle.

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is downregulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF ␤-TrCP . The F-box protein ␤-TrCP (␤-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to ␤-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved ␤-TrCPbinding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in ␤-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to ␤-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of ␤-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second ␤-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.polo-like kinase 1 ͉ ubiquitin ͉ cell cycle ͉ CK2 ͉ ␤-TrCP

The c‐mos proto‐oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N‐terminal residue, or by a rule referred to here as the ‘second‐codon rule’. We demonstrate that unstable Mos is degraded by the ubiquitin‐dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N‐terminus of Mos from being recognized by a ubiquitin‐protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N‐terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.

The Xenopus c-mos proto-oncogene product, pp39mos, accumulates in the unfertilized egg during maturation, is hyperphosphorylated and exhibits protein kinase activity. On fertilization, or soon after the completion of meiosis, the accumulated pp39mos undergoes selective proteolysis. Using an in vitro protease assay system, we show here that this specific proteolysis is caused by the calcium-dependent cysteine protease, calpain.