Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.
Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils.
Tudan
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C
Department of Medicine, University of British Columbia, Biomedical Research Centre, Kinetek Biotechnology Corporation, Suite 500, 520 West 6th Avenue, Vancouver, BC, V5Z 1A1, Canada.
Jackson
J K
JK
Charlton
L
L
Pelech
S L
SL
Sahl
B
B
Burt
H M
HM
D000730:Androstadienes / Q000494:pharmacology; D002131:Calcium Pyrophosphate / Q000494:pharmacology*; D003460:Crystallization; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases / Q000097:blood*; D015398:Signal Transduction; D000077191:Wortmannin
D000730:Androstadienes; D002131:Calcium Pyrophosphate; D004791:Enzyme Inhibitors; D007211:Indoles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D019893:Ribosomal Protein S6 Kinases; D000077191:Wortmannin; C064758:Ro 31-8220