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1 Department of Biochemistry and Protein Function Discovery Program, Queen's University, Kingston, Ontario, Canada
* To whom correspondence should be addressed. E-mail: maka{at}post.queensu.ca.
Cortactin, a predominant substrate of Src family kinases, plays an important role in the Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles, and has been shown recently to be enriched in podosomes induced by either c-Src or phorbol ester. The mechanisms by which cortactin regulates podosome-formation have not been determined, however. In this study, we showed that cortactin is required for podosome-formation using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Either treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Tyr 421 and 466, the Src-phosphorylation sites on cortactin. The Src kinase inhibitor, SU6656, significantly inhibits formation of podosomes induced by phorbol ester and phosphorylation of cortactin; whereas, PKC
inhibitor did not affect podosome-formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F or Y466D mutated sites did not affect podosome-formation or cortactin translocation to the podosomes, although endogenous Tyr-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in the phosphorylation of cortactin and podosome-formation in smooth muscle cells; 2) expression of cortactin is essential for the genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; 4) Tyr-phosphorylation of cortactin may be involved in the regulation of the stability and turn-over of podosomes rather than targeting this protein to the site of podosome-formation.
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