Thrombin activates mitogen‐activated protein kinase in primary astrocyte cultures

Abstract

Thrombin is known to evoke numerous inflammatory and proliferative responses in a wide variety of its target cells. Recent studies have demonstrated morphoregulatory and mitogenic effects of thrombin on astroglial cells (astrocytes). The present study deals with thrombin‐induced activation of mitogen‐activated protein (MAP) kinase in primary cultures of rat astrocytes. Treatment of serum‐starved astrocytes with thrombin resulted in a rapid activation of tyrosine (Tyr) phosphorylation of a set of proteins including a prominent one with a molecular mass of 42 kDa (p42). The identity of p42 with MAP kinase was confirmed by MAP kinase‐immunoreactivity of isolated [i.e., immunoprecipitated with anti‐phosphotyrosine (PY) antibodies] p42 and by increased myelin basic protein (MBP) kinase activity present in MAP kinase immunoprecipitates of thrombin‐treated cultures. Pertussis toxin (PTX) pretreatment failed to inhibit thrombin stimulation of p42 phosphorylation, indicating the lack of involvement of PTX sensitive G proteins in the mechanism of activation of MAP kinase by thrombin. Chronic exposure of cultures to phorbol 12‐myristate 13‐acetate to down‐regulate PKC resulted in an attenuation of thrombin‐induced p42 Tyr phosphorylation, although H‐7, a known PKC inhibitor, failed to block thrombin effect. However, staurosporine, a nonspecific protein kinase inhibitor, prevented the activation of p42 phosphorylation. It is concluded that thrombin induces MAP kinase activation in astrocytes by a mechanism involving a staurosporine‐sensitive pathway.