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
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Animals
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Cell Fractionation
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Cell Membrane / enzymology
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Cell Membrane / metabolism
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Cofilin 1 / biosynthesis
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Dose-Response Relationship, Immunologic
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Endoplasmic Reticulum Chaperone BiP
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Enzyme Activation / physiology
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Heat-Shock Proteins / antagonists & inhibitors
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Heat-Shock Proteins / biosynthesis
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Heat-Shock Proteins / genetics
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Heat-Shock Proteins / metabolism*
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Immunoprecipitation
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Ligands
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Lim Kinases
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Macrophages, Peritoneal / enzymology*
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Macrophages, Peritoneal / metabolism
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Mice
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Mice, Inbred C57BL
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Molecular Chaperones / antagonists & inhibitors
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Molecular Chaperones / biosynthesis
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Molecular Chaperones / genetics
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Molecular Chaperones / metabolism*
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Oncogene Proteins / metabolism
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Phosphorylation
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Protein Binding / physiology
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Protein Kinases / biosynthesis
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Protein Serine-Threonine Kinases / metabolism
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Protein Serine-Threonine Kinases / physiology*
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RNA Interference
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Receptor Protein-Tyrosine Kinases / metabolism*
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Signal Transduction* / genetics
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Tyrosine / metabolism
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Up-Regulation
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alpha-Macroglobulins / metabolism*
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alpha-Macroglobulins / physiology
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p21-Activated Kinases
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rac1 GTP-Binding Protein / metabolism
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ras GTPase-Activating Proteins / biosynthesis
Substances
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Adaptor Proteins, Signal Transducing
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Cofilin 1
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Endoplasmic Reticulum Chaperone BiP
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Heat-Shock Proteins
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Hspa5 protein, mouse
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Ligands
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Molecular Chaperones
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Nck protein
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Oncogene Proteins
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alpha-Macroglobulins
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ras GTPase-Activating Proteins
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Tyrosine
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Protein Kinases
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Receptor Protein-Tyrosine Kinases
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Lim Kinases
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Limk1 protein, mouse
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Pak2 protein, mouse
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Protein Serine-Threonine Kinases
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p21-Activated Kinases
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rac1 GTP-Binding Protein