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Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability. [ABSTRACT FROM AUTHOR]

CLIP-170 is implicated in the formation of kinetochore–microtubule attachments through direct interaction with the dynein/dynactin complex. However, whether this important function of CLIP-170 is regulated by phosphorylation is unknown. Herein, we have identified polo-like kinase 1 (Plk1) and casein kinase 2 (CK2) as two kinases of CLIP-170 and mapped S195 and S1318 as their respective phosphorylation sites. We showed that a CK2 unphosphorylatable mutant lost its ability to bind to dynactin and to localize to kinetochores during prometaphase, indicating that the CK2 phosphorylation of CLIP-170 is involved in its dynactin-mediated kinetochore localization. Furthermore, we provide evidence that Plk1 phosphorylation of CLIP-170 at S195 enhances its association with CK2. Finally, we detected defects in the formation of kinetochore fibres in cells expressing the CLIP-S195A and -S1318A, but not the CLIP-S195E and -S1318D, confirming that Plk1- and CK2-associated phosphorylations of CLIP-170 are involved in the timely formation of kinetochore–microtubule attachments in mitosis. [ABSTRACT FROM AUTHOR]

Nuclear envelope breakdown (NEBD) is an essential step during the G2/M transition in higher eukaryotic cells. Increasing evidence supports the notion that both microtubules and microtubule- associated motor proteins are critical regulators of NEBD. Although it has been described that pl500Iued the major component of the dyneinldynactin complex, localizes in the nuclear envelope during prophase, the exact role of pl50GIued and its regulation during NEBD are largely elusive. Polo-like kinase 1 (Plkl), the best characterized Ser/Thr kinase, is involved in mitotic entry in several systems; how- ever, the targets of Plkl during NEBD are unknown. Herein, we show that in mammalian cells both Plkl and pisoelued regulate NEBD and that PIki interacts with and phosphoryates p150GIued dur- ing NEBD at prophase. Using various approaches, we showed that PIki phosphorylates p1 50GIued at Ser-179 and that the p5179 epitope is generated at the nuclear envelope of prophase cells. Significantly. Plkl-mediated phosphorylation of pisoolued at Ser-179 positively regulates its accumulation at the nuclear envelope during prophase. Finally, we found that cells expressing the Plkl-unphosphorylatable mutant (p15OdS179A) arrest at G2. as indicated by reduced NEBD, increased levels of cyclin B and phospho-H3, but a decreased level of Cdc2 kinase activity. Taking these data together, we conclude that Plkl phosphorylation of pisoolued might be one major pathway of NEBD regulation. [ABSTRACT FROM AUTHOR]

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known Gs-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes. 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]