title: Vasodilator-stimulated phosphoprotein regulates inside-out signaling of beta2 integrins in neutrophils.
abstract: The monomeric GTPase Rap1 controls functional activation of beta2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 and inside-out signaling of beta2 integrins. We found that fMLP-induced activation of Rap1 in human polymorphonuclear leukocytes or neutrophils and differentiated PLB-985 cells was blocked by inhibitors of the NO/guanosine-3',5'-cyclic monophosphate-dependent protein kinase (cGKI) pathway [N-(3-(aminomethyl)benzyl)acetamidine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, DT-3 peptide, 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate, Rp-isomer triethylammonium salt-guanosine-3',5'-cyclic monophosphate], indicating that the downstream signaling events in Rap1 activation involve the production of NO and guanosine-3',5'-cyclic monophosphate, as well as the activation of cGKI. Silencing the expression of vasodilator-stimulated phosphoprotein (VASP), a substrate of cGKI, in resting PLB-985 cells or mice neutrophils led to constitutive activation of Rap1. In parallel, silencing VASP in differentiated PLB-985 cells led to recruitment of C3G, a guanine nucleotide exchange factor for Rap1, to the plasma membrane. Expression of murine GFP-tagged phosphodeficient VASP Ser235Ala mutant (murine serine 235 of VASP corresponds to human serine 239) in PLB-985 cells blunted fMLP-induced translocation of C3G to the membrane and activation of Rap1. Thus, bacterial fMLP triggers cGKI-dependent phosphorylation of human VASP on serine 239 and, thereby, controls membrane recruitment of C3G, which is required for activation of Rap1 and beta2 integrin-dependent antibacterial functions of neutrophils.
authors.affiliation: Centre for Infection and Immunity, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Deevi
authors.forename: Madhuri
authors.initials: M
authors.lastname: Koney-Dash
authors.forename: Adrien
authors.initials: A
authors.lastname: Kissenpfennig
authors.forename: James A
authors.initials: JA
authors.lastname: Johnston
authors.forename: Kai
authors.initials: K
authors.lastname: Schuh
authors.forename: Ulrich
authors.initials: U
authors.lastname: Walter
authors.forename: Karim
authors.initials: K
authors.lastname: Dib
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology; D015153:Blotting, Western; D018821:CD18 Antigens / Q000276:immunology* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000276:immunology* / Q000378:metabolism; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D010750:Phosphoproteins / Q000276:immunology* / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology*; D020827:rap1 GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D001426:Bacterial Proteins; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D020735:Guanine Nucleotide-Releasing Factor 2; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D020827:rap1 GTP-Binding Proteins