Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils.
The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.
Lominadze
George
G
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, KY 40202, USA.
Rane
Madhavi J
MJ
Merchant
Michael
M
Cai
Jian
J
Ward
Richard A
RA
McLeish
Kenneth R
KR
D000595:Amino Acid Sequence; D040502:Calgranulin B / Q000378:metabolism* / Q000502:physiology; D002469:Cell Separation; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism* / Q000502:physiology
D040502:Calgranulin B; D011994:Recombinant Proteins; D013912:Threonine; D048051:p38 Mitogen-Activated Protein Kinases