The role of the hCDC14B-HIPK2-MeCP2 axis in mitotic cell fate determination.

CDC14是一种保守的磷酸酶，在模式生物出芽酵母中，它在有丝分裂退出（有丝分裂向G1过渡）的调控中起重要作用。人类细胞有三种CDC14类似物，其中只有hCDC14A和hCDC14B广泛表达。在过去，我们分析了hCDC14A和hCDC14B的功能，并确定了这些磷酸酶的底物。hCDC14A与中心体和肌动蛋白细胞骨架有关，我们已经显示了在肌动蛋白组织和纤毛长度控制中的功能。相反，hCDC14B在间期与核仁结合，在有丝分裂中与中心体和染色质结合。最近，我们通过对hCDC14B过表达细胞的磷酸化蛋白质组分析鉴定了hCDC14B底物。MeCP2（甲基CpG结合蛋白2）与三甲基化组蛋白H3结合，作为转录抑制因子，被鉴定为hCDC14B底物。对hCDC14B基因缺失的未转化的人RPE1细胞的分析显示，细胞周期蛋白B1蛋白水平（从而cdk1 -细胞周期蛋白B1活性）在这些细胞中增加，与CDC14B KO小鼠胚胎成纤维细胞的数据相似。我们的数据表明，与hCDC14B野生型细胞相比，细胞周期蛋白B1的增加导致有丝分裂纺锤体受到干扰的细胞在有丝分裂中停留的时间更长。我们的初步数据表明，细胞周期蛋白B1受hCDC14B和激酶HIPK2（同源结构域相互作用蛋白激酶2）对MeCP2S92的拮抗活性控制。磷酸化的MeCP2S92可能通过翻译控制促进细胞周期蛋白B1的积累。该模型得到了大量初步数据的支持，并提出了一种新的调控机制，该机制通过有丝分裂和细胞命运决定（滑入G1或有丝分裂细胞死亡）来控制有丝分裂干扰的进展。这里我们将进一步测试这个模型。我们将研究hCDC14B水平的改变如何影响细胞命运以响应有丝分裂干扰，这如何影响细胞周期蛋白B1，最后我们将测试由HIPK2和hCDC14B决定的MeCP2磷酸化状态以未知机制控制细胞周期蛋白B1的翻译的概念。第二部分重点关注HIPK2在有丝分裂胁迫下的调控，HIPK2修饰的后果，纺锤体组装检查点是否调控HIPK2，以及HIPK2如何在有丝分裂胁迫条件下变得稳定。总之，这个项目将帮助我们了解细胞如何调节细胞周期蛋白B1水平以响应有丝分裂干扰，以及这如何影响细胞命运的决定。由于有丝分裂滑入G1伴随着四倍体细胞的形成，而四倍体细胞是肿瘤转化的中间状态，因此该建议将有助于理解这一有害步骤的机制原理以及如何预防这一过程。

CDC14 is a conserved phosphatase that in the model organism budding yeast has an essential function in the regulation of mitotic exit, the transition from mitosis into G1. Human cells have three CDC14 paralogues of which only hCDC14A and hCDC14B are widely expressed. In the past we have analysed the functions of hCDC14A and hCDC14B and have identified substrates of these phosphatases. hCDC14A is associated with centrosomes and the actin cytoskeleton and we have shown functions in actin organisation and cilia length control. In contrast, hCDC14B associates with the nucleolus during interphase and binds to centrosomes and chromatin in mitosis. Recently, we have identified hCDC14B substrates through phospho-proteome analysis of hCDC14B overexpressing cells. The protein MeCP2 (Methyl CpG binding Protein 2) that binds to tri-methylated histone H3 where it functions as transcriptional repressor was identified as hCDC14B substrate.Analysis of untransformed human RPE1 cells with genomic deletion in hCDC14B revealed that cyclin B1 protein levels (and thereby CDK1-cyclin B1 activity) are increased in these cells, similar to published data from CDC14B KO mouse embryonic fibroblasts. Our data suggest that this increase in cyclin B1 has the consequence that cells with disturbances that affect the mitotic spindle stay longer in mitosis in comparison to hCDC14B wild type cells. Our preliminary data suggest that cyclin B1 is controlled by the antagonistic activities of hCDC14B and the kinase HIPK2 (Homeodomain Interacting Protein Kinase 2) on MeCP2S92. Phosphorylated MeCP2S92 then promotes, probably via translation control, enhanced accumulation of cyclin B1. This model is supported by numerous preliminary data and suggests a novel regulatory mechanism that controls progression through mitosis and cell fate decision (slippage into G1 versus mitotic cell death) in response to mitotic disturbances. Here we will test this model further. We will study how altered hCDC14B levels impact cell fate in response to mitotic disturbances, how this impacts cyclin B1 and finally we will test the notion that the phosphorylation status of MeCP2 as determined by HIPK2 and hCDC14B controls translation of cyclin B1 by an unknown mechanism. The second part is focused on the regulation of HIPK2 in response to mitotic stress, the consequences of HIPK2 modifications, whether the spindle assembly checkpoint regulates HIPK2 and how HIPK2 becomes stabilized under mitotic stress conditions. Together, this project will help us to understand how cells adjust cyclin B1 levels in response to mitotic disturbances and how this impacts cell fate decision. Since mitotic slippage into G1 is accompanied with the formation of tetraploid cells that are an intermediate state in neoplastic transformation, this proposal will help understanding the mechanistic principals of this deleterious step and how it can be prevented.