title: Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms.
abstract: The targets of the p38 MAPK pathway responsible for regulation of neutrophil chemotaxis and exocytosis are unknown. One target of this pathway is the actin-binding protein, heat shock protein 27 (Hsp27). Therefore, we tested the hypothesis that Hsp27 mediates p38 MAPK-dependent chemotaxis and exocytosis in human neutrophils through regulation of actin reorganization. Sequestration of Hsp27 by introduction of anti-Hsp27 Ab, but not an isotype Ab, inhibited fMLP-stimulated chemotaxis, increased cortical F-actin in the absence of fMLP stimulation, and inhibited fMLP-stimulated exocytosis. Pretreatment with latrunculin A prevented actin reorganization and the changes in fMLP-stimulated exocytosis induced by Hsp27 sequestration. To determine the role of Hsp27 phosphorylation, wild-type, phosphorylation-resistant, or phosphorylation-mimicking recombinant Hsp27 was introduced into neutrophils by electroporation. The phosphorylation-resistant mutant significantly reduced migration toward fMLP, whereas none of the Hsp27 proteins affected fMLP-stimulated or TNF-alpha-stimulated exocytosis or actin polymerization. Endogenous Hsp27 colocalized with F-actin in unstimulated and fMLP-stimulated neutrophils, whereas phosphorylated Hsp27 showed cytosolic localization in addition to colocalization with F-actin. Our results suggest that Hsp27 regulates neutrophil chemotaxis and exocytosis in an actin-dependent, phosphorylation-independent manner. Phosphorylation of Hsp27 regulates chemotaxis, but not exocytosis, independent of regulation of actin reorganization.
authors.affiliation: Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY 40202, USA.
authors.forename: Neelakshi R
authors.initials: NR
authors.lastname: Jog
authors.forename: Venkatakrishna R
authors.initials: VR
authors.lastname: Jala
authors.forename: Richard A
authors.initials: RA
authors.lastname: Ward
authors.forename: Madhavi J
authors.initials: MJ
authors.lastname: Rane
authors.forename: Bodduluri
authors.initials: B
authors.lastname: Haribabu
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: McLeish
mesh_terms: D000199:Actins / Q000276:immunology / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000276:immunology*; D005089:Exocytosis / Q000187:drug effects / Q000276:immunology*; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000276:immunology*; D018832:Molecular Chaperones; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000276:immunology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000276:immunology; D053778:Thiazolidines / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000276:immunology / Q000378:metabolism
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D053778:Thiazolidines; D009240:N-Formylmethionine Leucyl-Phenylalanine; D048051:p38 Mitogen-Activated Protein Kinases; C037067:latrunculin A