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.
Highlights
From the Departments of ‡Biochemistry and Molecular Biology and §Medicine, University of Louisville, and ¶Robley Rex VA Medical Center, Louisville, KY
Use of pharmacologic inhibitors and knockout mice has identified several signal transduction pathways that are involved in neutrophil granule exocytosis
Pathways containing p38 mitogen activated protein kinase (MAPK) and the nonreceptor Src tyrosine kinases regulate neutrophil specific and azurophilic granule exocytosis, and those kinases are activated by several stimuli that induce exocytosis (14 –18)
Summary
From the Departments of ‡Biochemistry and Molecular Biology and §Medicine, University of Louisville, and ¶Robley Rex VA Medical Center, Louisville, KY. Molecules necessary for bacterial killing, processing of cytokines and chemokines, induction of increased vascular permeability, and monocyte recruitment are released into phagosomes or extracellularly [3,4,5,6] The contents of these granules contribute to tissue damage in autoimmune and inflammatory diseases such as rheumatoid arthritis, ANCA-associated vasculitis, and acute coronary syndromes [7,8,9]. The goal of the present study was to identify granule phosphoproteins and their respective phosphorylation sites as an approach to elucidate the mechanisms of kinase-regulated neutrophil exocytosis. To accomplish this goal, proteomic techniques allowing large-scale identification of phosphopeptides were combined with a novel reagent developed in our laboratory that inhibits granule exocytosis. The expression and phosphorylation of one of the identified proteins, G protein-coupled receptor kinase 5 (GRK5), was validated
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