ACTIVATION OF S6 KINASE IN HUMAN NEUTROPHILS BY CALCIUM PYROPHOSPHATEDIHYDRATE 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 (p70(S6K)) has been shown to be de pendent on PI 3-kinase activation in mammalian cells, and since the fo rmer is a key enzyme in the transmission of signals to the cell nucleu s, activation of p70(S6K) was investigated in crystal-stimulated neutr ophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD )-crystal-activated neutrophils were separated by Mono Q chromatograph y and analysed for phosphotransferase activity using a range of substr ates and probed by Western analysis using antibodies to p70(S6K) and m itogen-activated protein kinase (MBP 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 fist report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p7 0(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor ( Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD cry stals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive)and protein kinase B (wortman nin sensitive) in neutrophils. These data suggest that activation of p 70(S6K) may proceed through a PI 3-kinase- and protein kinase B-indepe ndent but PKC-dependent pathway in crystal-activated neutrophils.