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 α2-macroglobulin (α2M∗) to GRP78 on the cell surface of 1-LN human prostate cancer cells induces mitogenic signalling and cellular proliferation. In the current study, we have examined the ability of α2M∗ to activate PAK-1 and PAK-2. Exposure of 1-LN cells to α2M∗ 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 α2M∗-stimulated cells. Treatment of 1-LN cells with α2M∗ 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. α2M∗-indueed 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 inhibiter, α2-macroglobullin, These studies suggest a mechanism by which α2M∗ enhances the metastatic potential of these cells. [ABSTRACT FROM AUTHOR]