p120 catenin (p120) binds and stabilizes classical cadherins, making it a critical regulator of cell-cell adhesion. Here, we report an efficient technique (designated ReCLIP for Reversible Cross-Link Immuno-Precipitation) for identifying novel p120 binding partners that provides evidence of an interaction between p120 and the RhoA substrate p160 Rho Kinase (ROCK1). Briefly, proteins are covalently crosslinked in situ using thiol-cleavable DSP (Dithiobis[succinimidyl propionate) or DTME (Dithio-bismaleimidoethane) chemistries and then recovered by immunoprecipitating p120. Binding partners are then selectively eluted and identified by single-dimension liquid-chromatography tandem mass spectrometry. Crosslinking dramatically improved the efficiency of p120 co-immunoprecipitation with other members of the cadherin complex and revealed several new putative binding partners. Interestingly, one of these was the RhoA substrate p160 Rho Kinase (ROCK1). Using immunofluorescence and immunoprecipitation-based analyses, we showed that a fraction of ROCK1 associates with E-cadherin-bound p120 at cell-cell junctions. ROCK1 depletion by shRNAt led to disorganization of the adherens junction, but did not effect p120 or E-cadherin expression levels. Instead, disruption of the cadherin complex appeared to be due to a disruption in junctional actin. Using an in vitro kinase assay and phospho-specific p120 antibodies, we demonstrated that ROCK1 phosphorylates p120 on serine 268. These data reveal that ROCK1 can phosphorylate and interact with p120, and implies a role for this interaction in regulating cadherin stability.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03222011-133954 |
| Date | 02 April 2011 |
| Creators | Smith, Andrew Leslie |
| Contributors | Alissa M. Weaver |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03222011-133954/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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