THROMBIN PRODUCES PHOSPHORYLATION OF CYTOSOLIC PHOSPHOLIPASE A(2) BY A MITOGEN-ACTIVATED PROTEIN-KINASE KINASE-INDEPENDENT MECHANISM IN THEHUMAN ASTROCYTOMA CELL-LINE 1321N1

The release of [H-3]arachidonic acid was studied in the 1321N1 astrocy toma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simulta neously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A(2) (cPLA(2)) takes part in thrombin-ind uced arachidonate release was provided by the finding that thrombin in duced retardation of the mobility of cPLA(2) in SDS/polyacrylamide gel s, which is a feature of the activation of cPLA(2) by mitogen-activate d protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPL A(2), namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA(2) more clearly t han the activation of p42-MAK kinase did. Both cPLA(2) and c-Jun kinas e activation were not affected by PD-98059, a specific inhibitor of MA P kinase kinases, which indeed completely blocked p42-MAP kinase shift . Heat shock, a well-known activator of c-fun kinase, also phosphoryla ted cPLA(2) but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin rec eptor stimulation that activates a kinase cascade acting on the Pro-Le u-Ser-Pro consensus primary sequence, activates cPLA(2), and associate s the release of arachidonate with nuclear signalling pathways.