ANTIGEN ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE IN MAST-CELLS THROUGH PROTEIN-KINASE C-DEPENDENT AND INDEPENDENT PATHWAYS

We demonstrate discrete pathways for activation of mitogen-activated p rotein (MAP) kinase in cultured RBL-2H3 mast cells through protein kin ase C (PKC), cytosolic calcium, and a third pathway that provides sust ained signals for activation in Ag-stimulated cells. Thus, p42 MAP kin ase was activated by increasing intracellular free Ca2+ with thapsigar gin or by stimulating PKC with PMA. The latter stimulation was selecti vely blocked by the protein kinase C inhibitor, Ro31-7549. Stimulation of p42 MAP kinase by Ag resulted in relatively sustained activation o f MAP kinase which was only partially suppressed by Ro31-7549. Kinetic studies revealed two components of the MAP kinase response to Ag: a r apid but transient component that was Ro31-7549 sensitive and presumab ly PKC dependent; and a more sustained component that was Ro31-7549 re sistant and presumably PKC independent. Similarly, Ro31-7549 inhibited the early but not late release of arachidonic acid, a finding that wa s consistent with the known regulation of phospholipase A(2) by MAP ki nase. Early tyrosine phosphorylation events which were thought to be e ssential for Ag-induced activation of p42 MAP kinase and release of ar achidonic acid were unaffected by Ro31-7549. The findings suggested th at release of arachidonic acid was regulated primarily through MAP kin ase but that PKC may transiently influence this release, either direct ly or indirectly through MAP kinase.