Ligation of cell surface-associated glucose-regulated protein 78 by receptor-recognized forms of alpha 2-macroglobulin: activation of p21-activated protein kinase-2-dependent signaling in murine peritoneal macrophages

J Immunol. 2005 Aug 15;175(4):2525-33. doi: 10.4049/jimmunol.175.4.2525.

Abstract

Previous studies of the plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) demonstrated that alpha2M-proteinase complexes (alpha2M*) modulate immune responses and promotes macrophage locomotion and chemotaxis. Alpha2M* 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 alpha2M* to activate PAK-1 and PAK-2. Upon macrophage stimulation with alpha2M*, PAK-2 is autophosphorylated, resulting in increased kinase activity; however, PAK-1 is negligibly affected. Alpha2M*-stimulated macrophages showed a marked elevation in the levels of Rac x GTP. Receptor tyrosine phosphorylation upon binding of alpha2M* 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 alpha2M* stimulation. We further demonstrate that phosphorylation of Lin-11, Isl-1, Mec-3 (LIM) kinase and cofilin is promoted by treating macrophages with alpha2M*. Thus, alpha2M* regulates activation of the PAK-2-dependent motility mechanism in these cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Cell Fractionation
  • Cell Membrane / enzymology
  • Cell Membrane / metabolism
  • Cofilin 1 / biosynthesis
  • Dose-Response Relationship, Immunologic
  • Enzyme Activation / physiology
  • Heat-Shock Proteins / antagonists & inhibitors
  • Heat-Shock Proteins / biosynthesis
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism*
  • Immunoprecipitation
  • Ligands
  • Lim Kinases
  • Macrophages, Peritoneal / enzymology*
  • Macrophages, Peritoneal / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Molecular Chaperones / antagonists & inhibitors
  • Molecular Chaperones / biosynthesis
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Oncogene Proteins / metabolism
  • Phosphorylation
  • Protein Binding / physiology
  • Protein Kinases / biosynthesis
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Serine-Threonine Kinases / physiology*
  • RNA Interference
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Signal Transduction* / genetics
  • Tyrosine / metabolism
  • Up-Regulation
  • alpha-Macroglobulins / metabolism*
  • alpha-Macroglobulins / physiology
  • p21-Activated Kinases
  • rac1 GTP-Binding Protein / metabolism
  • ras GTPase-Activating Proteins / biosynthesis

Substances

  • Adaptor Proteins, Signal Transducing
  • Cofilin 1
  • Heat-Shock Proteins
  • Ligands
  • Molecular Chaperones
  • Nck protein
  • Oncogene Proteins
  • alpha-Macroglobulins
  • ras GTPase-Activating Proteins
  • Tyrosine
  • Protein Kinases
  • Receptor Protein-Tyrosine Kinases
  • Lim Kinases
  • Limk1 protein, mouse
  • Pak2 protein, mouse
  • Protein-Serine-Threonine Kinases
  • p21-Activated Kinases
  • rac1 GTP-Binding Protein
  • molecular chaperone GRP78