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J. Biol. Chem., Vol. 280, Issue 8, 6879-6889, February 25, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/8/6879    most recent M412119200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosenberger, G. Articles by Kutsche, K. Search for Related Content PubMed PubMed Citation Articles by Rosenberger, G. Articles by Kutsche, K. Social Bookmarking                      What's this?

PIX Associates with Calpain 4, the Small Subunit of Calpain, and Has a Dual Role in Integrin-mediated Cell Spreading^*

Georg Rosenberger, Andreas Gal, and Kerstin Kutsche

From the Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, D-22529 Hamburg, Germany

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that PIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to -parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that PIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases µ-calpain and m-calpain, as a novel binding partner of PIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The PIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, PIX colocalized with µ- and m-calpain, integrin-linked kinase, and 1 integrin in early integrin-containing clusters. Overexpression of PIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that PIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of PIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that PIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas PIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

Received for publication, October 26, 2004 , and in revised form, December 10, 2004.

^* This work was supported by Grant SFB444 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Butenfeld 42, D-22529 Hamburg, Germany. Tel.: 49-40-42803-4597; Fax: 49-40-42803-5138; E-mail: kkutsche{at}uke.uni-hamburg.de.

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