位於抗乳癌基因BRCA1的蛋白質磷酸水解酶結合位點之臨床相關突變會干擾DNA修復機制

Abstract

目前已知抗乳癌基因BRCA1會透過[RK]-x(0,1)-[VI]-x-[FW]這個第一型磷酸水解酶PP1共同結合基序與PP1進行互動，結合位點在BRCA1的898~901殘基之間，序列為898KVTF901。 先前的研究顯示改造而得的突變F901A會消除BRCA1與PP1之間的結合，並會干擾由BRCA1調節的DNA損傷訊息傳導與修復機制。 剔除整個KVTF序列也被發現會引起類似的表型，顯示BRCA1與PP1的結合對於BRCA1的正常運作具有重要的意義。 有趣的是，乳癌信息中心資料庫列有一個K898E突變，原先是在亞甚肯納茲猶太裔的患者身上被發現。 我們試著檢視這個突變對於BRCA1-PP1結合能力以及BRCA1所調節的DNA損傷反應機制之影響。 我們的結果顯示BRCA1K898E和PP1之間的結合能力顯著降低，且BRCA1K898E也無法減輕缺乏BRCA1的細胞對於游離輻射的高敏感性，同時這些細胞也被觀察到在接受游離輻射後DNA修復機制有所不足。 免疫染色分析更顯示缺乏BRCA1的細胞以BRCA1K898E重組之後，在接受游離輻射後的Rad51焦點形成能力明顯的低於以野生型BRCA1重組的細胞。 綜合這些結果可以發現PP1結合基序的完整性對於PP1和BRCA1的結合非常重要；PP1和BRCA1的互動也對於BRCA1所調節的DNA修復機制扮演關鍵的調控角色。 這也顯示BRCA1和PP1的結合與互動有機會發展為癌症治療的生物標記或藥物標靶。

BRCA1 is known to bind and interact with PP1 through an [RK]-x(0,1)-[VI]-x-[FW] PP1-binding consensus motif located at residues 898~901 of the BRCA1 protein, with the sequence 898KVTF901. Previous studies have shown that an engineered F901A mutation can abolish binding between BRCA1 and PP1, and disrupt BRCA1-mediated DNA damage signaling and repair. Deletion of the entire KVTF sequence was also found to induce a similar phenotype, suggesting that the binding interaction between BRCA1 and PP1 is critical for the normal functioning of BRCA1. Interestingly, the Breast Cancer Information Core database lists a K898E mutation, originally identified in a patient of Ashkenazi Jewish descent. We sought to examine the impact of this mutation upon BRCA1-PP1 binding and the BRCA1-mediated DNA damage response. Our results demonstrated that binding between BRCA1K898E and PP1 was significantly reduced. Furthermore, BRCA1K898E was unable to mitigate IR hypersensitivity in BRCA1-deficient cells, and inadequate post-IR DNA repair was also observed. Immunostaining analysis revealed that BRCA1-deficient cells reconstituted with BRCA1K898E had significantly lower rates of Rad51 foci formation post-IR, as compared to cells reconstituted with wild-type BRCA1. Taken together, these results indicate that the integrity of the PP1-binding motif is crucial for the binding interaction between PP1 and BRCA1, which in turn plays a key role in regulating BRCA1-mediated DNA repair. This suggests that the BRCA1-PP1 binding interaction may have potential as a biomarker and therapeutic target for the treatment of cancer.