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ABSTRACT: Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.
The Journal of Cell Biology 08/2012; 198(4):491-9. · 10.26 Impact Factor
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ABSTRACT: The Hedyotis diffusa Willd herbal compounds (HDWHCs) are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic
efficacy in China. However, the underlying mechanisms remain to be elucidated. In this study, we used freeze-dried powder
of the water extracts of HDWHCs to investigate the potential mechanisms of HDWHCs in cancer treatment. HDWHCs treatment significantly
inhibited vascular endothelial growth factor (VEGF) mRNA levels and VEGF transcriptional activation in cancer cells. HDWHCs
also had a remarkable inhibitory effect on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha). Forced expression
of HIF-1α restored VEGF transcriptional activation inhibited by HDWHCs, indicating that HDWHCs suppressed VEGF expression
through decreasing HIF-1alpha expression. Moreover, HDWHCs inhibited cyclooxygenase-2 (COX-2) expression, and overexpression
of HIF-1alpha restored HDWHCs’ inhibitory effect on COX-2 at transcriptional level. These findings may provide better understanding
of HDWHCs’ anti-cancer mechanism in cancer treatment.
Keywords
Hedyotis diffusa Willd
-Chinese medicine-vascular endothelial growth factor (VEGF)-hypoxia-inducible factor 1alpha (HIF-1alpha)-cyclooxygenase-2 (COX-2)
04/2012; 5(4):361-368.
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ABSTRACT: Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.
Molecular cell 05/2010; 38(3):383-92. · 14.61 Impact Factor
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ABSTRACT: Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore-microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1-PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.
The Journal of Cell Biology 03/2010; 188(6):809-20. · 10.26 Impact Factor
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ABSTRACT: Accurate segregation of chromosomes in mitosis requires that spindle microtubules attach sister kinetochores to opposite poles of the mitotic spindle (biorientation). To achieve biorientation of all chromosomes, incorrect attachments are selectively destabilized, providing a fresh opportunity to biorient, whereas correct attachments are stabilized. Tension across the centromere may be the signal that distinguishes different attachment states, as spindle microtubules pull bioriented sister kinetochores in the opposite direction. Destabilization of incorrect attachments requires the Ipl1/Aurora B kinase, which phosphorylates kinetochore substrates that directly interact with microtubules. The present review focuses on how Aurora B regulates attachments in response to centromere tension.
Biochemical Society Transactions 10/2009; 37(Pt 5):976-80. · 3.71 Impact Factor
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ABSTRACT: Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using fluorescence resonance energy transfer-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.
Science 02/2009; 323(5919):1350-3. · 31.20 Impact Factor
