Involvement of PKCζ and GSK3β in the stability of the metaphase spindle.
Involvement of PKCζ and GSK3β in the stability of the metaphase spindle.
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PKCγ 和 GSK3β 参与中期纺锤体的稳定性。
DOI：
10.1007/s11626-011-9476-6
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Capco,DavidG
中科院分区：
文献类型：
--
作者：
Kalive,Madhavi;Baluch,DPage;Capco,DavidG
在体细胞中，有丝分裂纺锤体是中心体，蛋白激酶C （PKC）的几种同工型与有丝分裂纺锤体有关，但它们在稳定有丝分裂纺锤体中的作用尚不清楚。其他蛋白激酶如糖原合成酶激酶3β (GSK3β)也被证明与有丝分裂纺锤体有关。在这项研究中，我们发现3T3细胞中期纺锤体中心体区域有活性（磷酸化）PKCζ的富集。为了了解PKC和GSK3β两种激酶是否与有丝分裂纺锤体相关，首先，在中期细胞的两极研究了磷酸化PKC同工型与GSK3β的共定位。荧光共振能量转移（FRET）分析表明磷酸化pkc ζ与磷酸化（ser9）GSK3β分子接近。其次，在3T3细胞中，无活性的GSK3β参与维持完整的有丝分裂纺锤体。第三，本研究还表明，在细胞中添加磷酸化pkc ζ特异性抑制剂可以破坏有丝分裂纺锤体微管和与之相关的一些蛋白质。小鼠成纤维细胞中期的有丝分裂纺锤体似乎是由PKCζ通过GSK3β作用维持的。Phospho-PKCζ在分子上接近GSK3β，而PKC的其他同工异构体如pPKCβII， pPKCγ， pPKCμ和pPKCθ的距离不足以产生显著的FRET读数。这种紧密的分子接近性支持了GSK3β可能是PKCζ的底物的观点。
In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.