ABSTRACT
Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcepsilonRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, W(sh)/W(sh) mast cell-deficient mice locally reconstituted with Pak1(-/-) bone marrow-derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1(-/-) BMMCs exhibited a reduction in FcepsilonRI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcepsilonRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.
Subject(s)
Calcium Signaling/physiology , Cytoskeleton/ultrastructure , Mast Cells/metabolism , p21-Activated Kinases/physiology , Actins/metabolism , Adoptive Transfer , Animals , Antigens, CD/genetics , Antigens, CD/physiology , Biological Transport , Biopolymers , Bone Marrow Cells/cytology , Calcimycin/pharmacology , Calcium Signaling/drug effects , Cytoskeleton/metabolism , Enzyme Activation , Female , Immunoglobulin E/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Passive Cutaneous Anaphylaxis/immunology , Platelet Membrane Glycoproteins , Radiation Chimera , Receptors, IgE/physiology , Recombinant Fusion Proteins/physiology , Secretory Vesicles/drug effects , Secretory Vesicles/metabolism , Signal Transduction , Tetraspanin 30 , beta-N-Acetylhexosaminidases/metabolism , p21-Activated Kinases/deficiency , p21-Activated Kinases/geneticsABSTRACT
Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.
