MAP KINASE CASCADE IN ASTROCYTES

We have studied in cultured rat astroglial cells MAP kinases, known fo r their role in intracellular signal transduction. The MAP kinase acti vity was stimulated by growth factors (FGFb, FGFa, EGF, PDGF, and IGF1 ), by a phorbol ester (TPA) activating-protein kinase C (PKC), by a ne uropeptide (endothelin-1), and by a neuromediator (carbachol). Astrocy tes pretreated for 18 h with TPA were still stimulated by growth facto rs and endothelin, suggesting that down-regulated isoforms of PKC are not involved in MAP kinase activation. In contrast, the small effect o f carbachol was suppressed by TPA pretreatment. Astrocytes contained t wo proteins (p41 and p44) recognized by MAP kinase antibody. These pro teins were phosphorylated on tyrosine residues in the cytosols of stim ulated astrocytes. The kinetics of MAP kinase activation by FGFb and I GF1 were very different. FGFb promoted a rapid activation of MAP kinas e (about 10 min) plus a prolonged phase that lasted at least 12 h. IGF 1 produced only a rapid transient peak of activation at about 20 min. Hence, extracellular signals might generate different effects in astro cytes by differentially modulating the MAP kinase cascade. On a Mono a nd column the growth factor-stimulated MAP kinase activity was separat ed into two peaks containing p41 and p44. Stimulation of astrocytes al tered the elution pattern of p44 as a result of its phosphorylation. A n ATP-dependent MAP kinase activator (MW = 40-45 kDa) was found in fra ctions of FGFb-stimulated cells which were not retained on Mono Q colu mn, indicating the existence of a MAP kinase kinase (MEK) in astrocyte s. C-Raf, identified in other cells as a MAP kinase kinase kinase, was also present in astrocytes. Thus astrocytes contain many components o f the MAP kinase cascade activated by growth factors that may also be implicated in the action of neuropeptides and neuromediators. (C) 1994 Wiley-Liss, Inc.