A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G₂/M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G₂/M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.Despite broad progress in studies of cell cycle control in eukaryotes, advanced models are lacking for the regulation of mitotic entry in human cells. This regulation is pivotal in cell cycle control, and a better understanding of it may be crucial to improving cytotoxic cancer chemotherapy, the mainstay of cancer treatment. Models of mitotic entry in higher eukaryotes revolve around activation of the cyclin B/Cdk1 (cyclin-dependent kinase 1 or Cdc2) complex, which drives the major events of mitosis. A rise in the cyclin B level triggers mitotic entry in Xenopus egg extracts but not in mammalian cells (15, 47). Inhibitory phosphorylation of Cdk1 on the ATP-binding site residue tyrosine 15 (Y15) has been recognized as a key constraint throughout eukaryotes (29, 42). Wee1 and Myt kinases perform this phosphorylation in vertebrate cells, where Wee1 appears to be dominant (34). Kim and Ferrell and others have recently developed an elegant model for ultrasensitive, switch-like inactivation of Wee1 by cyclin B/Cdk1 in a positive feedback loop that contributes to mitotic entry in Xenopus egg extracts (27).Although cyclin A(A2)/Cdk2 is traditionally omitted from models of mitotic entry, accumulating evidence from several different approaches suggests that cyclin A/Cdk complexes play roles. Cyclin A levels rise during S phase and peak in G₂ before falling abruptly in prometaphase of mitosis (60). Microinjection of cyclin A/Cdk2 complexes in human G₂ phase cells was observed to drive mitotic entry (14). Conversely, microinjection of antibodies directed against cyclin A in S-phase cells inhibited mitotic entry without an apparent effect on bulk DNA synthesis (45). In complementary approaches that supported biochemical analyses, cyclin A RNA interference (RNAi) or induction of a dominant negative mutant of Cdk2 (Cdk2-dn), the major cyclin A binding partner, inhibited mitotic entry (13, 15, 21, 37). In these settings, cyclin B/Cdk1 complexes accumulated in inactive, Y15-phosphorylated forms (13, 21, 37). Cdc25 phosphatases, which can reverse this phosphorylation, show reduced activity in this context (37), but increased Cdc25 activity could not readily overcome the arrest (13). RNAi-mediated knockdown of Wee1 was found capable of overriding the arrest mediated by cyclin A RNAi, suggesting that Wee1 is a key rate-limiting factor (13). However, whether and by what mechanisms cyclin A complexes might regulate Wee1 and drive Cdk1 dephosphorylation and mitotic entry have remained unclear.Recently, genetic studies in mice have reinforced these observations while providing evidence for some cell type differences (24). Although Cdk2 is not essential, in its absence Cdk1 binds more cyclin A and E and provides redundant functions (4, 25, 44). Deletion of the cyclin A gene is lethal for embryos and adults (24). Gene deletion in fibroblasts in vitro did not completely abrogate their proliferation but caused S and G₂/M delays. In this setting cyclin E was upregulated, and combined deletion of cyclin E yielded arrest in G₁, S, and G₂/M phases. Cyclin A gene deletion was alone sufficient to block proliferation of hematopoietic stem cells, suggesting that cyclin A is essential for their proliferation.Wee1 is regulated on multiple levels, including inhibitory phosphorylation in the amino-terminal regulatory domain (NRD), residues 1 to 292. This region is predicted to be intrinsically disordered (56), and few functional elements have been identified in it. The cyclin B/Cdk1 complex has been thought to be the principal or exclusive kinase responsible for NRD phosphorylation (18, 27, 28). Two sites in the Xenopus embryonic Wee1 NRD, Thr 104 and Thr 150 (referred to here by the homologous residue, T239, in human somatic Wee1), have been identified as Cdk phosphorylation sites that inhibit Wee1 activity (28). Recent studies of Xenopus somatic Wee1 suggest that T239 phosphorylation may antagonize the function of a surrounding motif, dubbed the Wee box (43). This small, conserved region appears to augment the activity of the carboxy-terminal kinase domain.We show here that cyclin A/Cdk2 complexes directly bind Wee1 as a substrate in human cells. In particular, a conserved cyclin A/Cdk binding RXL motif in the Wee1 NRD is required for efficient T239 phosphorylation. Further analysis revealed that RXL1 is located within a Crm1 binding site that mediates Wee1 export during S and G₂ phases. Cyclin A/Cdk2 activity appears to foster Wee1 export, but this export is not essential for mitotic entry. These findings further define roles of cyclin A/Cdk complexes in regulating Wee1 and mitotic entry in human cells and dissect the mechanisms and consequences of Wee1 redistribution during the run-up to mitosis.     

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation.     

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.     

Concerted mechanism of Swe1/Wee1 regulation by multiple kinases in budding yeast

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Here, we show that Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this modification served as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. We propose that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events.     

Novel Positive Feedback Loop between Cdk1 and Chk1 in the Nucleus during G2/M Transition

Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G₂/M transition. We reported recently that Chk1 is phosphorylated at Ser²⁸⁶ and Ser³⁰¹ by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser²⁸⁶ and Ser³⁰¹ to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B₁-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B₁ and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.     

Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis

Activation of the cyclin-dependent kinase (Cdk1) cyclin B (CycB) complex (Cdk1:CycB) in mitosis brings about a remarkable extent of protein phosphorylation. Cdk1:CycB activation is switch-like, controlled by two auto-amplification loops--Cdk1:CycB activates its activating phosphatase, Cdc25, and inhibits its inhibiting kinase, Wee1. Recent experimental evidence suggests that parallel to Cdk1:CycB activation during mitosis, there is inhibition of its counteracting phosphatase activity. We argue that the downregulation of the phosphatase is not just a simple latch that suppresses futile cycles of phosphorylation/dephosphorylation during mitosis. Instead, we propose that phosphatase regulation creates coherent feed-forward loops and adds extra amplification loops to the Cdk1:CycB regulatory network, thus forming an integral part of the mitotic switch. These network motifs further strengthen the bistable characteristic of the mitotic switch, which is based on the antagonistic interaction of two groups of proteins: M-phase promoting factors (Cdk1:CycB, Cdc25, Greatwall and Endosulfine/Arpp19) and interphase promoting factors (Wee1, PP2A-B55 and a Greatwall counteracting phosphatase, probably PP1). The bistable character of the switch implies the existence of a CycB threshold for entry into mitosis. The end of G2 phase is determined by the point where CycB level crosses the CycB threshold for Cdk1 activation.     

Monitoring the Cell Cycle by Multi-Kinase-Dependent Regulation of Swe1/Wee1 in Budding Yeast

In eukaryotes, G2/M transition is induced by the activation of cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. Recent advances in our understanding of mitotic entry in budding yeast has revealed that these cells utilize the level of Swe1 (Wee1 ortholog) phosphorylation as a means of monitoring cell cycle progression and of coordinating morphogenetic events with mitotic entry. Swe1 is phosphorylated by at least three distinct kinases at different stages of the cell cycle. This cumulative phosphorylation leads to the hyperphosphorylation and degradation of Swe1 through ubiquitin-mediated proteolysis. Thus, Swe1 functions as an important cell cycle modulator that integrates multiple upstream signals from prior cell cycle events before its ultimate degradation permits passage into mitosis.     

Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.     

Spatiotemporal regulations of Wee1 at the G2/M transition

Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.     

Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.     

Two Distinct Controls of Mitotic Cdk1/Cyclin B1 Activity Requisite for Cell Growth Prior to Cell Division

Cell growth prior to cell division is restricted by the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complexes. Recently, we identified that the death-effector domain (DED) containing protein, DEDD, acts as a novel inhibitor of mitotic Cdk1/cyclin B1, influencing cell size. Like cyclin B1, DEDD protein levels specifically peak during the G2/M phase. In the nucleus, DEDD associates with Cdk1/cyclin B1 complexes, via direct binding to cyclin B1, and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared to that in DEDD+/+ cells. This accelerates mitotic progression in DEDD-/- cells, with a shortened G2/M phase, reduced rRNA, and diminished cell volume. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Interestingly, the DED domain is not involved in the association of DEDD with Cdk1/cyclin B1, but is indispensable for the cell sizing function of DEDD. Together, in addition to the well-established machinery for activation of Cdk1 through dephosphorylation of its inhibitory-residues, we propose a novel mechanism for impeditive regulation of mitotic Cdk1/cyclin B1 mediated by DEDD within the nucleus, which allows sufficient cell growth prior to cell division.     

The overlooked greatwall: a new perspective on mitotic control

The role of the dual specificity protein phosphatase, Cdc25, in activating the cyclin-dependent kinase-cyclin B complex (Cdk1-CycB) by overcoming the inhibitory Wee1 kinase is a long-established principle for mitotic entry. Recently, however, evidence has emerged of a regulatory network that facilitates Cdk1-CycB activity by inhibiting the form of protein phosphatase 2A having a B55 regulatory subunit (PP2A-B55). Here, I review the genetic and biochemical evidence for Greatwall kinase and its substrate Endosulphine as the key components of this previously obscure regulatory network. Not only is the inhibition of PP2A-B55 by phospho-endosulphine required to prevent dephosphorylation of Cdk1-CycB substrates until mitotic exit, but it is also required to promote Cdc25 activity and inhibit Wee1 at mitotic entry. I discuss how these alternating states of preferential PP2A-B55 or Cdk1-CycB activity can have an impact upon the regulation of Polo kinase and its ability to bind different partner proteins as mitosis progresses.     

Identification of Drosophila Myt1 kinase and its role in Golgi during mitosis

Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. Here, we present the identification of Drosophila Myt1 (dMyt1) kinase and examine the relationship of Myt1 and Wee1 activities in the context of cdc2 phosphorylation. dMyt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells resulted in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) resulted in an increased rate of proliferation. In addition, loss of dMyt1 alone or in combination with Drosophila Wee1 (dWee1) resulted in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of dMyt1 alone resulted in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of cdc2 on the tyrosine-15 (Tyr-15) residue was only observed when both dMyt1 and dWee1 expression was reduced via RNAi and not by Wee1 alone. Most strikingly, in the absence of dMyt1, Golgi fragmentation during mitosis was incomplete. Our findings suggest that dMyt1 and dWee1 have distinct roles in the regulation of cdc2 phosphorylation and the regulation of mitotic events.     

Maturation promoting factor destabilization facilitates postovulatory aging‐mediated abortive spontaneous egg activation in rat

The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging‐mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee‐1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging‐mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post‐hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr‐14/Tyr‐15 and dephosphorylation of Thr‐161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging‐mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging‐mediated abortive SEA in rat eggs.     

Maturation promoting factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes

Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3′,5′‐cyclic monophosphate (cAMP), guanosine 3′,5′‐cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase‐promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin‐dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase‐II (M‐II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M‐II arrest in rat oocytes.     

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2A(Cdc55)) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2A(Cdc55) sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2A(Cdc55) and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength.     

Analysis of β3-endonexin mutants for their ability to interact with cyclin A

We have recently identified beta(3)-endonexin as a molecule that interacts with cyclin A-associated kinase. In this study, beta(3)-endonexin mutants were constructed by PCR-based site-directed mutagenesis, and characterized. Beta(3)-endonexin has a cyclin binding motif, RxL, in its N-terminal region, and two SP sequences which resemble a known target site for cyclin-dependent kinases (Cdks). The R5A/L7A mutant of beta(3)-endonexin, in which the RxL motif has been changed to AxA, is unable to bind to cyclin A, as revealed by two-hybrid experiments and in vitro pull-down assays. A GST-beta(3)-endonexin fusion, but not the corresponding R5A/L7A mutant, inhibits phosphorylation of Rb protein by cyclin A/Cdk2 in vitro. A cyclin A/Cdk2 kinase complex produced in, and purified from, insect cells phosphorylated GST-beta(3)-endonexin in vitro. The S33A or S46A mutant is partially phosphorylated by cyclin A/Cdk2, whereas no phosphorylation of the S33A/S46A double mutant is detectable. This demonstrates that these two serine residues, each of which is followed by a proline residue, are target sites for phosphorylation by cyclin A-associated kinase. The R5A/L7A mutant form of beta(3)-endonexin, which is defective for binding to cyclin A, is also not phosphorylated by cyclin A/Cdk2, confirming that the phosphorylation requires binding to cyclin A in the kinase complex. The neutralizing effect of beta(3)-endonexin on the toxicity associated with the expression of full-length human cyclin A in budding yeast is correlated with its ability to bind to cyclin A. Taken together, these data suggest that beta(3)-endonexin is phosphorylated by cyclinA/Cdk2 in vitro and that cyclin A-associated kinase activity is inhibited by the binding of beta(3)-endonexin to the kinase complex.     

Identification of Clb2 residues required for Swe1 regulation of Clb2-Cdc28 in Saccharomyces cerevisiae

Wee1 kinases regulate the cell cycle through inhibitory phosphorylation of cyclin-dependent kinases (CDKs). Eukaryotic cells express multiple CDKs, each having a kinase subunit (Cdk) and a regulatory "cyclin" subunit that function at different stages of the cell cycle to regulate distinct processes. The cyclin imparts specificity to CDK-substrate interactions and also determines whether a particular CDK is subject to Wee1 regulation. Saccharomyces Wee1 (Swe1) inhibits Cdc28 (Cdk1) associated with the mitotic cyclin, Clb2, but not with the G(1) (Cln1, -2, and -3) or the S-phase (Clb5 and -6) cyclins. Here, we show that this specificity depends on two amino acids associated with a conserved "hydrophobic patch" (HP) motif on the cyclin surface, which mediates specificity of CDK-substrate interactions. Mutation of Clb2 residues N260 and K270 largely abrogates Clb2-Cdc28 regulation by Swe1, and reciprocal mutation of the corresponding residues in Clb5 can subject Clb5-Cdc28 to regulation by Swe1. Swe1 phosphorylation by Clb2-Cdc28, which is thought to activate Swe1 kinase, depends on N260 and K270, suggesting that specific regulation of Clb2-Cdc28 by Swe1 derives from the specific ability of Clb2 to target Swe1 for activating phosphorylation. The stable association of Swe1 with Clb2-Cdc28 also depends on these residues, suggesting that Swe1 may competitively inhibit Clb2-Cdc28 interactions with substrates, in addition to its well-known function as a regulator of CDK activity through tyrosine phosphorylation.