TTK/hMps1在異常細胞分裂中調控p53蛋白之機制探討

The role of the spindle checkpoint kinase TTK/hMps1 in p53-dependent post mitotic checkpoint

黃億富(Yi-Fu Huang)

指導教授 ： 張大慈 ； 謝小燕

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2009年)

摘要

在一些不良的條件下，細胞被迫長時間停在有絲分裂期而無法走出，最終細胞會嘗試適應這樣的環境，而在沒有細胞分裂之下就離開了有絲分裂期。這樣的過程會產生含有四倍數染色體的細胞，如此的細胞是不正常的，可藉由細胞凋亡的機制來消除這些不正常的細胞。此外，透過紡錘絲檢查點以及p53蛋白的作用，也可以讓那些不正常的細胞停在G1期而無法繼續增生。p53是一個抑癌蛋白，也是一個轉錄因子，透過轉錄活化它的下游基因，p53不但能阻止癌細胞的生長也能促使癌細胞走向死亡。Nocodazole和Taxol (紫杉醇)這一類破壞紡錘絲的藥物會活化p53，但是它們怎麼去活化p53的機制仍然不清楚。本研究發現在紡錘絲被破壞時，紡錘絲檢查點中的蛋白質激酶TTK/hMps1有活化p53 的功能。在試管中，TTK/hMps1磷酸化p53第15和第20位置的絲胺酸 (Serine) 以及第18位置的酥胺酸 (Threonine)。在細胞內，少了TTK/hMps1會減少因紡錘絲被破壞所引起的p53酥胺酸18的磷酸化; 相反的，在細胞內過表現TTK/hMps1會增加p53的磷酸化。這樣因TTK/hMps1所造成的p53磷酸化破壞了MDM2和p53之間的結合，也減少了MDM2所造成的p53降解，使得p53更為穩定。此外，紡錘絲被破壞時TTK/hMps1會和p53結合。當細胞長時間受到破壞紡錘絲的藥物攻擊時，少了TTK/hMps1如同少了p53一樣，都會讓細胞走向含有多倍數染色體的狀態，而這些可能都與TTK/hMps1所調控的p53第18位置的磷酸化加強了p53活化它的二個下游 p21以及Lats2的能力有關。進一步分析此磷酸化的功能，若是將p53第18位置的酥胺酸置換成天冬胺酸 (Aspartic acid) 以模擬磷酸化，或是將酥胺酸以丙胺酸 (Alanine)取代達到阻斷磷酸化的反應，前者模擬磷酸化的p53更能夠抑制含有四倍數染色體的細胞增生。綜合前面研究的結論，在紡錘絲被破壞時，透過磷酸化p53，紡錘絲檢查點中的蛋白質激酶TTK/hMps1和p53能夠共同去阻止細胞走向含有多倍數染色體的狀態，使得基因體的穩定性得以維持。

關鍵字

細胞分裂 ； 檢查點 ； 抑癌蛋白p53

並列摘要

Upon prolonged arrest in mitosis, cells undergo adaptation and exit mitosis without cell division. These tetraploid cells are arrested in the subsequent G1 phase in a spindle checkpoint and p53-dependent manner. p53 has long been known to be activated by spindle poison such as nocodazole and taxol, although the underlying mechanism is still unclear. In this study, evidence was presented which demonstrated that stabilization and activation of p53 by spindle disruption required the spindle checkpoint kinase TTK/hMps1. Down regulation of TTK/hMPS1 diminishes p53 response after spindle damage. In vitro, TTK/hMPS1 phosphorylates p53 on Ser15, Thr 18, and Ser20. Ablation of TTK/hMPS1 in vivo reduces spindle damage-induced p53 Thr18 phosphorylation and p53 response. Conversely, overexpression of TTK/hMPS1 enhances Thr18 phosphoylation and stabilizes p53 by disrupting the interaction with MDM2 and by abrogating MDM2-mediated p53 ubiquinylation. In addition, TTK/hMPS1 coimmunoprecipitates with p53 after spindle damage. Upon prolonged treatment with spindle poisons, down regulation of TTK/hMPS1 leads to polyploidy, a phenomenon closely mimicked by p53 ablation. TTK/hMPS1-mediated Thr18 phosphorylation enhances p53-dependent activation of not only p21 but also Lats2, two mediators of the post-mitotic checkpoint. Furthermore, a phospho-mimicking substitution at Thr18 (T18D) is more capable than the phospho-deficient mutant (T18A) in rescuing the tetraploid checkpoint defect of the p53-depleted cells. Our data indicate that by phosphorylating p53 Thr18, TTK/hMPS1 works with p53 to prevent polyploidy after spindle damage.

並列關鍵字

TTK/hMps1 ； p53 ； spindle checkpoint

參考文獻

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