14-3-3 Binding to Cyclin Y contributes to cyclin Y/CDK14 association

Cyclin Y is a highly conserved cyclin among eumetazoans, yet its function and regulation are poorly understood. To search for Cyclin Y-interacting proteins, we screened a yeast two-hybrid library using human Cyclin Y （CCNY） as a bait and identified the following interactors： CDK14 and four members of the 14-3-3 family （ε,β,η,τ）. The interaction between CCNY and 14-3-3 proteins was confirmed both in vitro and in vivo. The results showed that Ser-100 and Ser-326 residues in CCNY were crucial for 14-3-3 binding. Interestingly, binding of CCNY to 14-3-3 significantly enhanced the association between CCNY and CDK14. Our findings may add a new layer of regulation of CCNY binding to its kinase partner.     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4–6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest.The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase. That, however, appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling and not a consequence of the G₂ arrest as can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest.Additionally, our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.Key words: fibroblast growth factor, chondrocyte, G₂/M arrest, Myt1, cyclin B1, CDK1     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.     

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.     

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.     

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 B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actin

Substantial actin remodelling occurs prior to mitosis as cells alter their shape in preparation for cytokinesis. In mammalian cells, mitosis is initiated by a heterodimer of cyclin B1 and the cyclin dependent kinase 1 (Cdk1) serine/threonine kinase. In this report. we show that human cyclin B1 binds the actin cross-linking protein Filamin-A (FLNa). The proteins co-immunoprecipitate and co-localize in mitotic human cells. We find that cyclin B1/Cdk1 can phosphorylate FLNa in vitro and reduce its ability to gelate actin. We have also identified serine 1436 as one FLNa residue phosphorylated by cyclin B1/Cdk1 in vitro. Our results suggest a role for cyclin B1/Cdk1 in FLNa-dependent actin remodelling.     

Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.     

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.     

Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication

In animal cells, duplication of centrosomes and DNA is coordinated. Since CDK2/cyclin E triggers initiation of both events, activation of CDK2/cyclin E is thought to link these two events. We identified nucleophosmin (NPM/B23) as a substrate of CDK2/cyclin E in centrosome duplication. NPM/B23 associates specifically with unduplicated centrosomes, and NPM/B23 dissociates from centrosomes by CDK2/cyclin E-mediated phosphorylation. An anti-NPM/B23 antibody, which blocks this phosphorylation, suppresses the initiation of centrosome duplication in vivo. Moreover, expression of a nonphosphorylatable mutant NPM/ B23 in cells effectively blocks centrosome duplication. Thus, NPM/B23 is a target of CDK2/cyclin E in the initiation of centrosome duplication.     

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.