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Mol Cell Biol, May 1998, p. 3044-3058, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Activation of Rho-Dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein

Zeynep F. Altun-Gultekin,1,dagger Sanjay Chandriani,2 Cecile Bougeret,2 Toshimasa Ishizaki,3 Shuh Narumiya,3 Petra de Graaf,4 Paul Van Bergen en Henegouwen,4 Hidesaburo Hanafusa,2 John A. Wagner,1 and Raymond B. Birge2,*

Department of Neurology and Neuroscience, Cornell University Medical College,1 and Laboratory of Molecular Oncology, The Rockefeller University,2 New York, New York; Second Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto, Japan3; and Department of Molecular Cell Biology, Utrecht University, Utrecht, The Netherlands4

Received 1 October 1997/Returned for modification 12 November 1997/Accepted 2 February 1998

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha 3-integrin, and a higher-molecular-weight form of beta 1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

* Corresponding author. Mailing address: The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7412. Fax: (212) 327-7943. E-mail: birger{at}rockvax.rockefeller.edu.

dagger Present address: Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635.

Mol Cell Biol, May 1998, p. 3044-3058, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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