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Cell cycle progression from G2 phase into mitosis is regulated by a complex network of mechanisms, all of which finally control the timing of Cyclin B/CDK1 activation. PLK1 regulates a network of events that contribute to regulating G2/M phase progression. Here we have used a proteomics approach to identify proteins that specifically bind to the Polobox domain of PLK1. This identified a panel of proteins that were either associated with PLK1 in G2 phase and/or mitosis, the strongest interaction being with the MAPK scaffold protein JIP4. PLK1 binding to JIP4 was found in G2 phase and mitosis, and PLK1 binding was self-primed by PLK1 phosphorylation of JIP4. PLK1 binding is required for JIP4-dependent p38MAPK activation in G2 phase during normal cell cycle progression, but not in either G2 phase or mitotic stress response. Finally, JIP4 is a target for caspase-dependent cleavage in mitotically arrested cells. The role for the PLK1-JIP4 regulated p38MAPK activation in G2 phase is unclear, but it does not affect either progression into or through mitosis.

Whereas many components regulating the progression from S phase through G2 phase into mitosis have been identified, the mechanism by which these components control this critical cell cycle progression is still not fully elucidated. Cyclin A/Cdk2 has been shown to regulate the timing of Cyclin B/Cdk1 activation and progression into mitosis although the mechanism by which this occurs is only poorly understood. Here we show that depletion of Cyclin A or inhibition of Cdk2 during late S/early G2 phase maintains the G2 phase arrest by reducing Cdh1 transcript and protein levels, thereby stabilizing Claspin and maintaining elevated levels of activated Chk1 which contributes to the G2 phase observed. Interestingly, the Cyclin A/Cdk2 regulated APC/CCdh1 activity is selective for only a subset of Cdh1 targets including Claspin. Thus, a normal role for Cyclin A/Cdk2 during early G2 phase is to increase the level of Cdh1 which destabilises Claspin which in turn down regulates Chk1 activation to allow progression into mitosis. This mechanism links S phase exit with G2 phase transit into mitosis, provides a novel insight into the roles of Cyclin A/Cdk2 in G2 phase progression, and identifies a novel role for APC/CCdh1 in late S/G2 phase cell cycle progression.

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin-dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. Results. In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N- and C-terminal regions of CDC25B and requires CDC25B binding to its CDK–cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. Conclusions. Our results therefore suggest that CDC25B associates with a Ctn2-containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.

Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-beta-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis.

Cytokinesis involves two phases: (1) membrane ingression followed by (2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLC(S19)) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.

Cyclin A/cdk2 has a role in progression through S phase, and a large pool is also activated in G2 phase. Here we report that this G2 phase pool regulates the timing of progression into mitosis. Knock down of cyclin A by siRNA or addition of a specific cdk2 small molecule inhibitor delayed entry into mitosis by delaying cells in G2 phase. The G2 phase-delayed cells contained elevated levels of inactive cyclin B/cdk1. However, increased microtubule nucleation at the centrosomes was observed, and the centrosomes stained for markers of cyclin B/cdk1 activity. Both microtubule nucleation at the centrosomes and the phosphoprotein markers were lost with short-term treatment of the cdk1/2 inhibitor roscovitine but not the Mek1/2 inhibitor U0126. Cyclin A/cdk2 localized at the centrosomes in late G2 phase after separation of the centrosomes but before the start of prophase. Thus G2 phase cyclin A/cdk2 controls the timing of entry into mitosis by controlling the subsequent activation of cyclin B/cdk1, but also has an unexpected role in coordinating the activation of cyclin B/cdk1 at the centrosome and in the nucleus.