Two characteristics of highly malignant cells are their increased motility and secretion of proteinases allowing these cells to penetrate surrounding basement membranes and metastasize. Activation of 21-kDa activated kinases (PAKs) is an important mechanism for increasing cell motility. Recently, we reported that binding of receptor-recognized forms of the proteinase inhibitor ␣ 2 -macroglobulin (␣ 2 M*) to GRP78 on the cell surface of 1-LN human prostate cancer cells induces mitogenic signaling and cellular proliferation. In the current study, we have examined the ability of ␣ 2 M* to activate PAK-1 and PAK-2. Exposure of 1-LN cells to ␣ 2 M* caused a 2-to 3-fold increase in phosphorylated PAK-2 and a similar increase in its kinase activity toward myelin basic protein. By contrast, the phosphorylation of PAK-1 was only negligibly affected. Silencing the expression of the GRP78 gene, using either of two different mRNA sequences, greatly attenuated the appearance of phosphorylated PAK-2 in ␣ 2 M*-stimulated cells. Treatment of 1-LN cells with ␣ 2 M* caused translocation of PAK-2 in association with NCK to the cell surface as evidenced by the coimmunoprecipitation of PAK-2 and NCK in the GRP78 immunoprecipitate from plasma membranes. ␣ 2 M*-induced activation of PAK-2 was inhibited by prior incubation of the cells with specific inhibitors of tyrosine kinases and phosphatidylinositol 3-kinase. PAK-2 activation was accompanied by significant increases in the levels of phosphorylated LIMK and phosphorylated cofilin. Silencing the expression of the PAK-2 gene greatly attenuated the phosphorylation of LIMK. In conclusion, we show for the first time the activation of PAK-2 in 1-LN prostate cancer cells by a proteinase inhibitor, ␣ 2 -macroglobulin. These studies suggest a mechanism by which ␣ 2 M* enhances the metastatic potential of these cells.
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