Rho-associated kinase在phorbol ester誘發細胞凋亡之角色探討

Abstract

在本實驗室過去的研究中發現Rho-associated kinase (ROCK)在phorbol-12-myristate-13-acetate (PMA)誘發D2骨髓血球細胞凋亡的過程中扮演決定性的角色。為了瞭解在這個過程中有哪些分子受到ROCK的調控而改變，我利用蛋白質體學的研究方法，發現heterogeneous nuclear ribonucleoprotein C1 and C2 (hnRNP C1/C2)這兩個pre-mRNA結合蛋白質在PMA誘發的凋亡前期細胞中，從細胞核位移到細胞質；這個hnRNP C1/C2位移現象並不是因為該細胞的核膜破損所造成，也無關於caspase的作用；而是透過ROCK調控的細胞骨架改變所引發的核轉出。在TNF-alpha所誘發NIH3T3凋亡細胞中也可觀察到這個現象。我在HEK293T細胞中過度表現活化態的ROCK就足以造成hnRNP C1/C2的核轉出；同時也證實hnRNP C1/C2的C端40個氨機酸序列是一個可受到ROCK調控的核轉出訊號。此外，我發現ROCK所造成的細胞骨架改變也影響了細胞核中RNA的生合成速率。這些結果指出在PMA誘發的凋亡細胞中，透過ROCK活化所產生的訊息會傳遞到細胞核，造成hnRNP C1/C2的核轉出、核結構改變以及整體性的基因表現降低。 由於促進細胞附著(adhesion)可以阻斷PMA誘發的細胞凋亡，說明細胞剝離(de-adhesion)作用在PMA誘發的細胞凋亡扮演重要角色。我發現當懸浮的D2細胞附著於fibronectin (FN)基質上時，其RhoA的活性會明顯地降低。利用具有能通透細胞膜的活化態TAT-RhoAV14重組蛋白質處理已附著於FN基質的細胞，透過ROCK的作用，伴隨著MLC磷酸化增加及細胞膜表面lipid rafts的重新分佈，D2細胞從FN基質剝離而呈現懸浮狀態。有趣的是這個現象可以藉由抑制protein tyrosine phosphatase (PTP)的活性，或表現抑制型態的Shp-2(C/S)所干擾。利用ROCK kinase活性的測定，我證實表現抑制型態的Shp-2(C/S)會抑制ROCK的活性；進一步發現若將ROCK的Serine 1134及1137位置突變成alanine時，則失去其被Shp-2(C/S)調控的特性。由於Shp-2基因在血液細胞生成的過程中是必須的，從我的研究中推測Shp-2可能在該過程中藉由影響RhoA/ROCK所調控的細胞附著機制而扮演重要角色。

In D2 cells, a myeloid leukemia cell line, Rho-associated kinase (ROCK) is required for phorbol-12-myristate-13- acetate (PMA)-induced apoptosis. Using a proteomic approach, I found that heterogeneous nuclear ribonucleoprotein C1 and C2 (hnRNP C1/C2), two nuclear restricted pre-mRNA binding proteins, are translocated to the cytoplasm in a ROCK-dependent manner in PMA-induced pro-apoptotic cells, where nuclear envelopes remain intact. The subcellular localization change of hnRNP C1/C2 appears to be dependent on ROCK-mediated cytoskeletal change and independent of caspase execution and new protein synthesis. This phenomenon is also observed in TNFalpha-induced apoptotic NIH3T3 cells. Over-expression of dominant active form of ROCK is sufficient to induce translocation of hnRNP C1/C2 in HEK293T cells. I also defined that C-terminal 40-amino-acid region of hnRNP C1/C2 is a novel nuclear export signal responsive to ROCK-activation. Besides, I found that ROCK-mediated cell contraction also induces down-regulation of general RNA synthesis. Here I conclude that a novel nuclear export is activated by the ROCK signaling pathway to exclude hnRNP C1/C2 from nucleus, by which the compartmentalization of specific hnRNP components is disturbed in apoptotic cells. Since PMA-induced apoptosis could be inhibited by cell adhesion, here I also investigated regulation of ROCK signaling in cell adhesion. The results showed that the RhoA activity was down-regulated while D2 cells were adherent to fibronectin (FN) matrix. Treatment of cell-permeable dominant active form of TAT-RhoAV14 induces cell de-adhesion from FN matrix, accompanying by lipid rafts redistribution and myosin light chain (MLC) phosphorylation. Interestingly, inhibition of tyrosine phosphatase or co-expression of catalytically inactive tyrosine phosphatase Shp-2(C/S) abrogated the RhoAV14-induced cell de-adhesion. By in vitro kinase analysis, I demonstrated that ROCK kinase activity was repressed in the cells co-expressing dominant negative Shp-2(C/S). Interestingly, mutation on S1134A/S1137A of ROCK abrogated its Shp-2(C/S)-responsive down-regulation. Given that the gene targeted mice experiment has shown that Shp-2 gene is essential for hematopoiesis. The results of my study suggest that Shp-2 may play a pivotal role in the myelopoiesis through regulating RhoA/ROCK-mediated cell de-adhesion.