Metaphase arrest by cyclin E-Cdk2 requires the spindle-checkpoint kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.     

Activation of G protein-coupled receptor kinase 1 involves interactions between its N-terminal region and its kinase domain

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its ～20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.     

Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.     

cAMP-dependent positive control of cyclin A2 expression during G1/S transition in primary hepatocytes.

cAMP positively and negatively regulates hepatocyte proliferation but its molecular targets are still unknown. Cyclin A2 is a major regulator of the cell cycle progression and its synthesis is required for progression to S phase. We have investigated whether cyclin A2 and cyclin A2-associated kinase might be one of the targets for the cAMP transduction pathway during progression of hepatocytes through G1 and G1/S. We show that stimulation of primary cultured hepatocytes by glucagon differentially modulated the expression of G1/S cyclins. Glucagon indeed upregulated cyclin A2 and cyclin A2-associated kinase while cyclin E-associated kinase was unmodified. In conclusion, our study identifies cyclin A2 as an important effector of the cAMP transduction network during hepatocyte proliferation.     

Activation of p34cdc2 kinase by cyclin A

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.     

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.     

Skp2 contains a novel cyclin A binding domain that directly protects cyclin A from inhibition by p27Kip1

Skp2 is well known as the F-box protein of the SCF(Skp2) x Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpression, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A interaction is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins. Furthermore, a short peptide derived from the mapped cyclin A binding sequences of Skp2 can block Skp2-cyclin A interaction but not p27-cyclin A interaction, whereas a previously identified RXL peptide can block p27-cyclin A interaction but not Skp2-cyclin A interaction. Functionally, Skp2-cyclin A interaction is separable from Skp2 ability to mediate p27 ubiquitylation. Rather, Skp2-cyclin A interaction serves to directly protect cyclin A-Cdk2 from inhibition by p27 through competitive binding. Finally, we show that disruption of cyclin A binding with point mutations in the cyclin A binding domain of Skp2 compromises the ability of overexpressed Skp2 to counter cell cycle arrest by a p53/p21-mediated cell cycle checkpoint without affecting its ability to cause degradation of cellular p27 and p21. These findings reveal a new functional mechanism of Skp2 and a new regulatory mechanism of cyclin A.     

Tylophorine arrests carcinoma cells at G1 phase by downregulating cyclin A2 expression

Tylophorine, a representative phenanthroindolizidine alkaloid from Tylophoraindica plants, exhibits anti-inflammatory and anti-cancerous growth activities. However, the underlying mechanisms of its anti-cancer activity have not been elucidated and its effects on cell cycle remain ambiguous. Here, we reveal by asynchronizing and synchronizing approaches that tylophorine not only retards the S-phase progression but also dominantly arrests the cells at G1 phase in HepG2, HONE-1, and NUGC-3 carcinoma cells. Moreover, tylophorine treatment results in down regulated cyclin A2 expression and overexpressed cyclin A2 rescues the G1 arrest by tylophorine. Thus, we are the first to report that the downregulated cyclin A2 plays a vital role in G1 arrest by tylophorine in carcinoma cells.     

Human TopBP1 participates in cyclin E/CDK2 activation and preinitiation complex assembly during G1/S transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.