Cyclin B and Cyclin A Confer Different Substrate Recognition Properties on CDK2

The transitions of the cell cycle are regulated by the cyclin dependent protein kinases(CDKs). The cyclins activate their respective CDKs and confer substrate recognitionproperties. We report the structure of phospho-CDK2/cyclin B and show that cyclin Bconfers M phase-like properties on CDK2, the kinase that is usually associated with S phase.Cyclin B produces an almost identical activated conformation of CDK2 as that produced bycyclin A. There are differences between cyclin A and cyclin B at the recruitment site, whichin cyclin A is used to recruit substrates containing an RXL motif. Because of sequencedifferences this site in cyclin B binds RXL motifs more weakly than in cyclin A. Despitesimilarity in kinase structures, phospho-CDK2/cyclin B phosphorylates substrates, such asnuclear lamin and a model peptide derived from p107, at sequences SPXX that differ fromthe canonical CDK2/cyclin A substrate recognition motif, SPXK. CDK2/cyclin Bphosphorylation at these non-canonical sites is not dependent on the presence of a RXLrecruitment motif. The p107 peptide contained two SP motifs each followed by a noncanonicalsequence of which only one site (Ser640) is phosphorylated by pCDK2/cyclin Awhile two sites are phosphorylated by pCDK2/cyclin B. The second site is too close to theRXL motif to allow the cyclin A recruitment site to be effective, as previous work has shownthat there must be at least 16 residues between the catalytic site serine and the RXL motif.Thus the cyclins A and B in addition to their role in promoting the activatory conformationalswitch in CDK2, also provide differential substrate specificity.     

A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. In this study we provide evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, we show that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, we demonstrate that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. We propose a model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop.     

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyse whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase ? dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesised cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132. Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.     

Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

Meiotic inactivation of Xenopus Myt1 by CDK/XRINGO, but not CDK/cyclin, via site-specific phosphorylation

Cell-cycle progression is regulated by cyclin-dependent kinases (CDKs). CDK1 and CDK2 can be also activated by noncyclin proteins named RINGO/Speedy, which were identified as inducers of the G2/M transition in Xenopus oocytes. However, it is unclear how XRINGO triggers M phase entry in oocytes. We show here that XRINGO-activated CDKs can phosphorylate specific residues in the regulatory domain of Myt1, a Wee1 family kinase that plays a key role in the G2 arrest of oocytes. We have identified three Ser that are major phosphoacceptor sites for CDK/XRINGO but are poorly phosphorylated by CDK/cyclin. Phosphorylation of these Ser inhibits Myt1 activity, whereas their mutation makes Myt1 resistant to inhibition by CDK/XRINGO. Our results demonstrate that XRINGO-activated CDKs have different substrate specificity than the CDK/cyclin complexes. We also describe a mechanism of Myt1 regulation based on site-specific phosphorylation, which is likely to mediate the induction of G2/M transition in oocytes by XRINGO.     

Nitrogen-containing flavonoids as CDK1/Cyclin B inhibitors: design, synthesis, and biological evaluation

A novel series of nitrogen-containing flavonoids 5a-l, 6a,b, and 7a,b were designed and synthesized as cyclin-dependent kinases (CDKs) inhibitors. The representative compounds 5a, 5b, 5e, and 5g showed potent CDK1/Cyclin B inhibitory activities. All compounds displayed a significant growth inhibitory action in vitro against Bel-7402, PC-3, ECA-109, A-549, HL-60, and MCF-7 cancer cell lines. Flow cytometry analysis showed that 5b induced apoptosis in PC-3 cells.     

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.     

Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.     

Phosphorylation of SAMHD1 by Cyclin A2/CDK1 Regulates Its Restriction Activity toward HIV-1

1. Download : Download full-size image

     

Cyclin A and Cks1 promote kinase consensus switching to non-proline-directed CDK1 phosphorylation

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

     

The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCF(SKP2) to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2(-/-) knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2(-/-) cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In?vivo, Cks2(-/-) cortical progenitor cells are limited in their capacity to differentiate into mature neurons,?a phenotype akin to animals lacking p27. We propose that?the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.     

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

Cyclin B2/cyclin-dependent kinase1 dissociation precedes CDK1 Thr-161 dephosphorylation upon M-phase promoting factor inactivation in Xenopus laevis cell-free extract

Cyclin-dependent kinase 1 (CDK1) is the enzymatic subunit of M-phase Promoting Factor (MPF). It is positively regulated by phosphorylation on Thr-161 and association with a cyclin B molecule. The role of Thr-161 dephosphorylation upon MPF inactivation remains unclear; nevertheless, degradation of cyclin B is thought to be a direct cause of MPF inactivation. However, MPF inactivation actually precedes cyclin B degradation in Xenopus cell-free extracts. Here we study in details the temporal relationship between histone H1 kinase (reflecting MPF activity) inactivation, Thr-161 dephosphorylation, CDK1-cyclin B2 dissociation and cyclin B2 proteolysis in such extracts. We show an asynchrony between inactivation of histone H1 kinase and degradation of cyclin B2. CDK1 dephosphorylation on Thr 161 is an even later event than cyclin B2 degradation, reinforcing the hypothesis that cyclin B dissociation from CDK1 is the key event inactivating MPF. Cyclins synthesized along with MPF inactivation could deliver shortly living active MPF molecules, potentially increasing the asynchrony between histone H1 kinase inactivation and cyclin B2 degradation. We confirm this by showing that in the absence of protein synthesis, such a tendency is lower, but nevertheless, still detectable. Finally, to characterise better CDK1/cyclin B dissociation, we show that CDK1 begins to dissociate from cyclin B2 before the very beginning of cyclin B2 degradation and that the diminution in CDK1-associated cyclin B2 is faster than the decline of its total pool. Thus, neither cyclin B2 degradation nor Thr-161 dephosphorylation participates directly in CDK1 inactivation as measured by histone H1 kinase decline upon the exit from mitotic M-phase in Xenopus embryo extract.     

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His₆-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni²⁺-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs. Published: August 18, 2004.     

Homology model of the CDK1/cyclin B complex

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.