title: Identification of phosphoproteins associated with human neutrophil granules following chemotactic peptide stimulation.
abstract: Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100±10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40202, USA.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Luerman
authors.forename: David W
authors.initials: DW
authors.lastname: Powell
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Uriarte
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Cummins
authors.forename: Michael L
authors.initials: ML
authors.lastname: Merchant
authors.forename: Richard A
authors.initials: RA
authors.lastname: Ward
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: McLeish
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002633:Chemotaxis / Q000187:drug effects*; D019295:Computational Biology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism; D015696:Gene Products, tat / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000145:classification / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D050766:Qb-SNARE Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D050600:SNARE Proteins / Q000494:pharmacology*; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
chemical_list: D015696:Gene Products, tat; D010748:Phosphopeptides; D010750:Phosphoproteins; D050766:Qb-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C582513:TAT-SNAP-23 fusion protein; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019061:src-Family Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D048051:p38 Mitogen-Activated Protein Kinases