The role of RhoA regulation for nuclear translocation of phosphorylated extracellular signal-regulated kinase and activation of focal adhesion kinase during phorbol ester stimulation.

• Main Authors: Chia-Ling Hsieh, 謝嘉凌
• Other Authors: Zee-Feng Chang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/89621064868485350578

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 91 === In D2 cells, an erythroblastic cell line, PMA treatment induces a portion of cells displayed adhesion, others remained in suspension to apoptosis. Recently, we have shown that PMA-induced phosphorylation of ERK via the PKC/Raf-1/MEK cascade is similar in these two populations of D2 cells; however, nuclear distribution of phospho-ERK is not observed in PMA-induced pro-apoptotic cells. In this study, however, we found that phospho-p38, another MAPK, is predominantly present in the nuclei of pro-apoptotic cells. By transfection with pEBV-Rta-NLS-GFP, we further showed that NLS-dependent nuclear transport is still intact in PMA-induced pro-apoptotic cells. In addition, the phosphorylation patterns of phospho-ERK did not seem to be different in these two populations of cells. Here, we concluded that cytosolic retention of phospho-ERK in pro-apoptotic cells is neither a result of general defect in nuclear translocation, nor due to a specific alteration in phosphorylation status. In this study, PMA-induced activation of focal adhesion kinase (FAK) is observed in the adhesion, but not in the suspension cells. Overexpression of the constitutive activated RhoAV14 could override CD2-FAK-mediated cell survival following PMA treatment. Endogenous RhoA activity was found to remain elevated in suspension cells, and to become decreased in the adhesion cells. Interestingly, we also observed that the phosphotyrosine phosphatase (PTP) activity that dephosphorylates of FAK was specifically present in the cells containing elevated level of RhoA, and that inhibition of RhoA could abolish this activity independent of adhesion. Given that inhibition of Rho-associated kinase could revert the pro-apoptotic cells to display adhesion and nuclear localization of phospho-ERK, we proposed that integrin-mediated adhesion induces FAK activation and down-regulates RhoA-dependent pathway in the adhesion cells, thus providing a cellular context allowing nuclear translocation of phospho-ERK. As for the suspension cells, elevated level of RhoA prohibits cell adhesion and FAK activation with a concurrent activation of Rho-associated kinase for interference with ERK nuclear translocation.