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Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Podosome Formation and Degradation of Extracellular Matrix in src-transformed Fibroblasts¹

Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan [K. M., H. M., T. T.]; PRESTO [H. M.] and CREST [T. T.], Japan Science and Technology Corporation, Saitama 332-0012 Japan; and Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Victoria 3050, Australia [H. H., H. Ma.]

Transformation of cells by the src oncogene causes dramatic changes in adhesive structures. In v-src-transformed 3Y1 rat fibroblasts (3Y1-src), there are actin-rich protrusive structures called podosomes by which attachment to the extracellular matrix is thought to occur. In this study, we found that neural Wiskott-Aldrich syndrome protein (N-WASP) colocalizes with filamentous actin (F-actin) in podosomes. Expression of dominant-negative mutants of N-WASP, cof N-WASP and VPH N-WASP, both of which are incapable of activating the Arp2/3 complex, suppressed podosome formation, suggesting that N-WASP is essential in this process. Localization of N-WASP in podosomes appears to be attributable to interaction between N-WASP and the SH3 domain of cortactin. Indeed, microinjection of the cortactin SH3 domain suppressed podosome formation. We also observed that 3Y1-src cells cultured on fibronectin degrade the fibronectin primarily at the podosomes and that the inhibition of podosome formation by cof N-WASP abolishes the fibronectin degradation. These results suggest the importance of N-WASP in podosome formation and extracellular matrix degradation, which are processes thought to underlie the invasive phenotype of 3Y1-src cells.

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