Thrombin produces phosphorylation of cytosolic phospholipase A(2) by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1

The release of [H-3]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A(2) (cPLA(2)) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA(2) in SDS/polyacrylamide gels, which is a feature of the activation of cPLA(2) by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA(2), namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA(2) more clearly than the activation of p42-MAK kinase did. Both cPLA(2) and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-fun kinase, also phosphorylated cPLA(2) but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA(2), and associates the release of arachidonate with nuclear signalling pathways.