Regulation of ROCKI by tyrosine phosphorylation
碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 97 === RhoA-regulated cell contractility plays important roles in variety of cellular processes. Rho-associated coiled–coil kinase (ROCK) is one of the downstream effectors of RhoA signaling and generates cell contraction force by promoting myosin light chain (MLC...
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ndltd-TW-097NTU051040092016-05-04T04:31:32Z http://ndltd.ncl.edu.tw/handle/62513161876573925004 Regulation of ROCKI by tyrosine phosphorylation Tyrosine磷酸化修飾對ROCKI活性調控之探討 Jheng-Guang Jhong 鍾政光 碩士 國立臺灣大學 生物化學暨分子生物學研究所 97 RhoA-regulated cell contractility plays important roles in variety of cellular processes. Rho-associated coiled–coil kinase (ROCK) is one of the downstream effectors of RhoA signaling and generates cell contraction force by promoting myosin light chain (MLC) phosphorylation. Two isoforms of ROCK, ROCKI and ROCKII, have been characterized. Previously, our laboratory has demonstrated that RhoA-dependent ROCKII activation is negatively regulated by tyrosine phosphorylation of ROCKII, which is controlled by c-Src and Shp2. In this study, I investigated whether ROCKI activation is also regulated by tyrosine phosphorylation. First, I detected tyrosine phosphorylation of ROCKI in HEK293T cells after treatment with pervanadate, a phosphatase inhibitor, and this phosphorylation event requires Src kinase. By expressing of dominant active SrcY527F and performing in vitro assay with c-Src kinase, I demonstrated that c-Src phosphorylates ROCKI in vivo and in vitro. Furthermore, I generated several deletion and YF substitution mutants of flag-ROCKI, and found that Tyr913 residue is one of Src-mediated phosphorylation sites of ROCKI. However, expression of flag-ROCKI(Y913F) had little effect on nocodazole-stimulated RhoA-mediated MLC phosphorylation in NIH3T3 cells, indicating that phosphorylation of Tyr913 may not participate in negative regulation of ROCKI. In summary, I may conclude that in addition to Tyr913, ROCKI has another tyrosine phosphorylation sites, and the tyrosine phosphorylation on regulating ROCKI activity should be investigated further. Zee-Fen, Chang 張智芬 2009 學位論文 ; thesis 37 zh-TW |
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碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 97 === RhoA-regulated cell contractility plays important roles in variety of cellular
processes. Rho-associated coiled–coil kinase (ROCK) is one of the downstream
effectors of RhoA signaling and generates cell contraction force by promoting myosin
light chain (MLC) phosphorylation. Two isoforms of ROCK, ROCKI and ROCKII,
have been characterized. Previously, our laboratory has demonstrated that
RhoA-dependent ROCKII activation is negatively regulated by tyrosine
phosphorylation of ROCKII, which is controlled by c-Src and Shp2. In this study, I
investigated whether ROCKI activation is also regulated by tyrosine phosphorylation.
First, I detected tyrosine phosphorylation of ROCKI in HEK293T cells after
treatment with pervanadate, a phosphatase inhibitor, and this phosphorylation event
requires Src kinase. By expressing of dominant active SrcY527F and performing in
vitro assay with c-Src kinase, I demonstrated that c-Src phosphorylates ROCKI in
vivo and in vitro. Furthermore, I generated several deletion and YF substitution
mutants of flag-ROCKI, and found that Tyr913 residue is one of Src-mediated
phosphorylation sites of ROCKI. However, expression of flag-ROCKI(Y913F) had
little effect on nocodazole-stimulated RhoA-mediated MLC phosphorylation in
NIH3T3 cells, indicating that phosphorylation of Tyr913 may not participate in
negative regulation of ROCKI.
In summary, I may conclude that in addition to Tyr913, ROCKI has another
tyrosine phosphorylation sites, and the tyrosine phosphorylation on regulating ROCKI
activity should be investigated further.
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author2 |
Zee-Fen, Chang |
author_facet |
Zee-Fen, Chang Jheng-Guang Jhong 鍾政光 |
author |
Jheng-Guang Jhong 鍾政光 |
spellingShingle |
Jheng-Guang Jhong 鍾政光 Regulation of ROCKI by tyrosine phosphorylation |
author_sort |
Jheng-Guang Jhong |
title |
Regulation of ROCKI by tyrosine phosphorylation |
title_short |
Regulation of ROCKI by tyrosine phosphorylation |
title_full |
Regulation of ROCKI by tyrosine phosphorylation |
title_fullStr |
Regulation of ROCKI by tyrosine phosphorylation |
title_full_unstemmed |
Regulation of ROCKI by tyrosine phosphorylation |
title_sort |
regulation of rocki by tyrosine phosphorylation |
publishDate |
2009 |
url |
http://ndltd.ncl.edu.tw/handle/62513161876573925004 |
work_keys_str_mv |
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