Ligation of Cell Surface-Associated Glucose-Regulated Protein 78 by Receptor-Recognized Forms of α2-Macroglobulin: Activation of p21-Activated Protein Kinase-2-Dependent Signaling in Murine Peritoneal Macrophages

Abstract

Previous studies of the plasma proteinase inhibitor α2-macroglobulin (α2M) demonstrated that α2M-proteinase complexes (α2M*) modulate immune responses and promotes macrophage locomotion and chemotaxis. α2M* binds to cell surface-associated glucose-regulated protein 78 (GRP78), which activates downstream signaling events. The role of p21-activated protein kinase-1 and -2 (PAK-1 and -2) in promoting cellular motility is well documented. In the current study, we examined the ability of α2M* to activate PAK-1 and PAK-2. Upon macrophage stimulation with α2M*, PAK-2 is autophosphorylated, resulting in increased kinase activity; however, PAK-1 is negligibly affected. α2M*-stimulated macrophages showed a marked elevation in the levels of Rac·GTP. Receptor tyrosine phosphorylation upon binding of α2M* to GRP78, recruits PAK-2 to the plasma membrane via the adaptor protein NCK. Consistent with this hypothesis, silencing of GRP78 gene expression greatly attenuated the levels of membrane-associated PAK-2 and NCK. PAK-2 activity was markedly decreased by inhibition of tyrosine kinases and PI3K before α2M* stimulation. We further demonstrate that phosphorylation of Lin-11, Isl-1, Mec-3 (LIM) kinase and cofilin is promoted by treating macrophages with α2M*. Thus, α2M* regulates activation of the PAK-2-dependent motility mechanism in these cells.