Marion E. Pesenti

The Polo-box domain (PBD) localizes Polo-like kinase 1 (PLK1) near mitotic substrates required for chromosome biorientation. Recent work on mitotic kinetochores showed PLK1 docking begins hierarchically at master docking motifs on BUB1 and CENP-U. Whether master docking motifs have common molecular features remains poorly understood. Presence on CENP-U of two neighbouring motifs generated by initial CDK1 priming and subsequent PLK1 phosphorylation led us to hypothesize PBD dimerization might be involved. Using biochemical, biophysical, and modelling approaches, we gathered strong evidence that CENP-U contains a single master docking motif. The motif is very high affinity and sufficient to form extensive interactions with the PBD, engaging multiple pockets on its surface without obvious added benefits from dimerization. Comparisons with motifs in BUB1, BUBR1, and PRC1 suggest commonalities of master PLK1 docking motifs. We discuss the implications of our observations for the mechanism of PLK1 activation.

Activation of PLK1, a master mitotic kinase, requires phosphorylation of its activation segment on Thr210, within a basic consensus sequence for Aurora kinases. Aurora B-dependent phosphorylation of Thr210 has been reported, but other evidence identified a strict requirement for the Aurora A partner Bora for Thr210 phosphorylation. Here, we investigate the elusive mechanistic basis for this requirement. We show that Aurora A:Bora phosphorylates Thr210 of PLK1 in vitro. On the contrary, T210 was not phosphorylated by isolated Aurora A, additional Aurora A:activator complexes, or Aurora B:INCENP, even when used at high kinase/substrate ratios. A transient interaction of Bora and PLK1, identified by structural modeling and probed mutationally, is uniquely required for Thr210 phosphorylation. Dependency on Bora for Thr210 phosphorylation is eliminated after mutating Lys208, in the Aurora consensus, into arginine. This conservative mutation turns PLK1 into a substrate of nearly all tested active Aurora kinases, including Aurora B. Collectively, these results shine a new light on the specificity of the PLK1 activation mechanism.

Activation of PLK1, a master mitotic kinase, requires phosphorylation of its activation segment on Thr210, within a basic consensus sequence for Aurora kinases. Aurora B-dependent phosphorylation of Thr210 has been reported, but other evidence identified a strict requirement for the Aurora A partner Bora for Thr210 phosphorylation. Here, we investigate the elusive mechanistic basis for this requirement. We show that Aurora A:Bora phosphorylates Thr210 of PLK1 in vitro, while Aurora A, other Aurora A:activator complexes, and Aurora B:INCENP fail to target T210 even at high kinase/substrate ratios. A transient interaction of Bora and PLK1, identified by structural modelling and probed mutationally, is uniquely required for Thr210 phosphorylation. Dependency on Bora for Thr210 phosphorylation is eliminated after mutating Lys208, in the Aurora consensus, into arginine. This conservative mutation turns PLK1 into an excellent substrate of nearly all tested active Aurora kinases, especially Aurora B. Collectively, these results shine a new light on the specificity of the PLK1 activation mechanism.

The Polo-box domain (PBD) localizes Polo-like kinase 1 (PLK1) near mitotic substrates required for chromosome biorientation. Recent work on mitotic kinetochores showed PLK1 docking begins hierarchically at master docking motifs on BUB1 and CENP-U. Whether master docking motifs have common molecular features remains poorly understood. Presence on CENP-U of two neighbouring motifs generated by initial CDK1 priming and subsequent PLK1 phosphorylation led us to hypothesize PBD dimerization might be involved. Using biochemical, biophysical, and modelling approaches, we gathered strong evidence that CENP-U contains a single master docking motif. The motif is very high affinity and sufficient to form extensive interactions with the PBD, engaging multiple pockets on its surface without obvious added benefits from dimerization. Comparisons with motifs in BUB1, BUBR1, and PRC1 suggests apparent commonalities of master PLK1 docking motifs. We discuss the implications of our observations for the mechanism of PLK1 activation.

The FAcilitates Chromatin Transcription (FACT) complex is a dimeric histone chaperone that operates on chromatin during transcription and replication. FACT also interacts with a specialized centromeric nucleosome containing the histone H3 variant centromere protein A (CENP-A) and with CENP-TW, two subunits of the constitutive centromere–associated network (CCAN), a 16-protein complex associated with CENP-A. The significance of these interactions remains elusive. Here, we show that FACT has multiple additional binding sites on CCAN. The interaction with CCAN is strongly stimulated by casein kinase II phosphorylation of FACT. Mitotic localization of FACT to kinetochores is strictly dependent on specific CCAN subcomplexes. Conversely, CENP-TW requires FACT for stable localization. Unexpectedly, we also find that DNA readily displaces FACT from CCAN, supporting the speculation that FACT becomes recruited through a pool of CCAN that is not stably integrated into chromatin. Collectively, our results point to a potential role of FACT in chaperoning CCAN during transcription or in the stabilization of CCAN at the centromere during the cell cycle.

During cell division, the microtubule cytoskeleton undergoes dramatic cell cycle-driven reorganizations of its architecture. Coordinated by changes in the phosphorylation patterns of a multitude of microtubule associated proteins, the mitotic spindle first self-assembles to capture the chromosomes and then reorganizes in anaphase as the chromosomes are segregated. A key protein for this reorganization is PRC1 which is differentially phosphorylated by the mitotic kinases CDK1 and PLK1. How the phosphorylation state of PRC1 orchestrates spindle reorganization is not understood mechanistically. Here, we reconstitute in vitro the transition between metaphase and anaphase-like microtubule architectures triggered by the changes in PRC1 phosphorylation. We find that whereas PLK1 regulates its own recruitment by PRC1, CDK1 controls the affinity of PRC1 for antiparallel microtubule binding. Dephosphorylation of CDK1-phosphorylated PRC1 is required and sufficient to trigger the reorganization of a minimal anaphase midzone in the presence of the midzone length controlling kinesin KIF4A. These results demonstrate how phosphorylation-controlled affinity changes regulate the architecture of active microtubule networks, providing new insight into the mechanistic underpinnings of the cell cycle-driven reorganization of the central spindle during mitosis.

The centromere, a chromosome locus defined by the histone H3–like protein centromeric protein A (CENP-A), promotes assembly of the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G ₁ phase of the cell cycle to compensate for its dilution after DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside of early G ₁ . Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G ₁ , but the molecular details of this process are still unknown. We reveal here a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings clarify how PLK1 contributes to the epigenetic maintenance of centromeres.

The FAcilitates Chromatin Transcription (FACT) complex is a dimeric histone chaperone that operates on chromatin during transcription and replication. FACT also interacts with a specialized centromeric nucleosome containing the histone H3 variant CENP-A and with CENP-TW, two subunits of CCAN, a 16-protein complex associated with CENP-A. The significance of these interactions remains elusive. Here, we show that FACT has multiple additional binding sites on CCAN. The interaction with CCAN is strongly stimulated by casein kinase II (CK2) phosphorylation of FACT. Mitotic localization of FACT to kinetochores is strictly dependent on specific CCAN subcomplexes. Unexpectedly, we also find that DNA readily displaces FACT from CCAN, suggesting that FACT becomes recruited through a pool of CCAN that is not stably integrated into chromatin. Collectively, our results point to a potential role of FACT in chaperoning CCAN during transcription or in the stabilization of CCAN at the centromere during the cell cycle. Teaser: DNA-sensitive, direct interactions with multiple inner kinetochore subunits deliver FACT to the kinetochore.

The centromere, a chromosome locus defined by the histone H3-like protein CENP-A, seeds the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 cell-cycle phase, compensating for its two-fold dilution following DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details are still unknown. We reveal a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings solve the long-standing question of how PLK1 contributes to the epigenetic maintenance of centromeres. One-Sentence Summary PLK1 licenses epigenetic maintenance of centromeres by regulating a conformational switch on the MIS18α protein.

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2’s chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.