Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-rel...Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.展开更多
Objective： The mucus production is an indicator for the histological grade of mucinous epithelial ovarian cancer （mEOC）. In our previous study, Crk expression was targeted in the human ovarian mucinous adenocarcino...Objective： The mucus production is an indicator for the histological grade of mucinous epithelial ovarian cancer （mEOC）. In our previous study, Crk expression was targeted in the human ovarian mucinous adenocarcinoma cell line MCAS through RNA interference, resulting in the establishment of Crk knock down cells. These cells exhibited decreased tumorigenic potential both in vitro and in vivo. The purpose of this study was to investigate if there is any change in the capability of forming mucus in these Crk knock down cells. Methods： Cytoplasmic periodic acid Schiff （PAS） staining and particle excluding assay were conducted to assess the mucus formation within and around cells, respectively. Additionally, the amount of mucus formed in tumor lumps from nude mice model was measured following HE and PAS staining. Results： The increased mucus production in Crk knockdown mEOC cells （MCAS） was manifested by increased number of enlarged cells filled with vacuoles-like mucus observed by phase-contrast microscope and cytoplasmic PAS staining; and enhanced mucus secretion was represented by the assembly of pericellular matrix in particle excluding assay and increased mucus area in tumor lumps from nude mice models. Conclusion： The course of carcinogenesis in mEOC is associated with the altered pattern of mucus production and secretion. The adaptor protein Crk is implicated in both pathways.展开更多
Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is...Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.展开更多
文摘Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.
基金a grant from the National Natural Science Foundation of China(No.C30672432,No.30772330)the Natural Science Foundation of Chongqing City(No.2007BB5319)the Japan-China Sasakawa Medical Fellowship
文摘Objective： The mucus production is an indicator for the histological grade of mucinous epithelial ovarian cancer （mEOC）. In our previous study, Crk expression was targeted in the human ovarian mucinous adenocarcinoma cell line MCAS through RNA interference, resulting in the establishment of Crk knock down cells. These cells exhibited decreased tumorigenic potential both in vitro and in vivo. The purpose of this study was to investigate if there is any change in the capability of forming mucus in these Crk knock down cells. Methods： Cytoplasmic periodic acid Schiff （PAS） staining and particle excluding assay were conducted to assess the mucus formation within and around cells, respectively. Additionally, the amount of mucus formed in tumor lumps from nude mice model was measured following HE and PAS staining. Results： The increased mucus production in Crk knockdown mEOC cells （MCAS） was manifested by increased number of enlarged cells filled with vacuoles-like mucus observed by phase-contrast microscope and cytoplasmic PAS staining; and enhanced mucus secretion was represented by the assembly of pericellular matrix in particle excluding assay and increased mucus area in tumor lumps from nude mice models. Conclusion： The course of carcinogenesis in mEOC is associated with the altered pattern of mucus production and secretion. The adaptor protein Crk is implicated in both pathways.
基金Acknowledgments We thank M Hamaguchi （Nagoya Univ., Japan） and T Iwahara （Osaka Bioscience Institute, Japan） forJak null MEFs, and N Gotoh （Tokyo Univ., Japan）, H Higashi （Hokkaido Univ., Japan）, N Mochizuki （National Cardiovascular Cent. Res. Inst., Japan）, H Hanafusa （Prof. emeritus, The Rockefeller Univ., USA and Direc- tor em., OBI, Japan）, SK Hanks （Vanderbilt Univ., USA）, and M Matsuda （Kyoto Univ., Japan） for plasmids. We also thank K Sasai （Hokkaido Univ., Japan） and Y Ohba （Hokkaido Univ., Japan） for valuable discussion. This work was supported in part by grants-in- aid from the Ministry of Education, Science, Culture, and Sports, and the Ministry of Health, Labor, and Welfare, Japan, as well as Suhara Memorial Foundation （Sapporo, Japan）, and the Mochida Medical Science Foundation （Tokyo, Japan）. The work of SF and TK is supported by grants from Cancer Research UK and the Brit- ish Cancer Charity ＂Heads Up＂. We dedicate this work to our great mentor Hidesaburo Hana- fusa, professor emeritus of the Rockefeller University, who passed away on March 15, 2009 at the age of 79. He devoted his life to science and in particular to creating the oncogene research field, to teaching and to providing profound affection to his students and postdocs. All of the alumni of Saburo＇s laboratory pride them- selves in having been his apprentices and we would like to hereby express our deeply felt gratitude to Saburo.
文摘Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.
