JCB logo
amgmicro.com
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents
This Article
Right arrow Full Text
Right arrow Full Text (PDF, 5733K)
Right arrow PPT slides of all figures
Right arrow Alert me when this article is cited
Right arrow Citation Map
Services
Right arrow Email this article
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new content in the JCB
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pomiès, P.
Right arrow Articles by Beckerle, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pomiès, P.
Right arrow Articles by Beckerle, M. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

© The Rockefeller University Press, 0021-9525/1997//157 $5.00
The Journal of Cell Biology, Volume 139, Number 1, , 1997 157-168

Article

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with {alpha}-Actinin



Pascal Pomiès*, Heather A. Louis*, and Mary C. Beckerle*

* Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is {alpha}-actinin. We have shown that the CRP1–{alpha}-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1–{alpha}-actinin interaction is 1.8 ± 0.3 µM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and {alpha}-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that {alpha}-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin–binding domain of {alpha}-actinin. In reciprocal mapping studies, we showed that {alpha}-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the {alpha}-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the {alpha}-actinin–binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with {alpha}-actinin may be critical for its role in muscle differentiation.

Abbreviations used in this paper: aa, amino acids; CEF, chicken embryo fibroblasts; CRP, cysteine-rich protein; HBB, Hepes binding buffer; GST, glutathione-S-transferase.

Address all correspondence to Mary Beckerle, Department of Biology, 201 South Biology Building, University of Utah, Salt Lake City, UT 84112-0840. Tel.: (801) 581-4485. FAX: (801) 581-4668. E-mail: beckerle{at}bioscience.utah.edu

This research was supported by grants from the National Institutes of Health and the American Cancer Society to M.C. Beckerle and from the Philippe Foundation, Inc. to P. Pomiès. M.C. Beckerle is the recipient of a Faculty Research Award from the American Cancer Society.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:



  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents