title: Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin.
abstract: The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis(4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)β(2) integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)β(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Konrad
authors.initials: K
authors.lastname: Pazdrak
authors.forename: Travis W
authors.initials: TW
authors.lastname: Young
authors.forename: Christof
authors.initials: C
authors.lastname: Straub
authors.forename: Susan
authors.initials: S
authors.lastname: Stafford
authors.forename: Alexander
authors.initials: A
authors.lastname: Kurosky
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D004804:Eosinophils / Q000187:drug effects* / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010797:Phthalimides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C117146:4,5-bis(4-fluoroanilino)phthalimide; D051339:Actin Depolymerizing Factors; D016023:Integrins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D051419:Paxillin; D010455:Peptides; D010750:Phosphoproteins; D010797:Phthalimides; D020543:Proteome; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; C026742:plastin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011493:Protein Kinase C; D064546:Protein Kinase C beta