Binding of Activated α2-Macroglobulin to Its Cell Surface Receptor GRP78 in 1-LN Prostate Cancer Cells Regulates PAK-2-dependent Activation of LIMK

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 signaling 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*-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, α2-macroglobulin. These studies suggest a mechanism by which α2M* enhances the metastatic potential of these cells.