ROLE OF EXTRACELLULAR SIGNAL-REGULATED PROTEIN-KINASE-1 AND PROTEIN-KINASE-2 IN OLIGODENDROGLIAL PROCESS EXTENSION

The relationship between extracellular signal-regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendro cytes (OLGs) was investigated, Process extension was induced through t he exposure of cultured OLGs to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), for various intervals. During the isolation of these OLGs from bovine brain, the original processes were lost, Therefore, any reinitiation of process extension via PMA s timulation was easily discernible through morphological monitoring, It was found that exposure of OLGs to PMA for 10 min was enough to induc e OLG process extension 24-72 h later, Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indi cating that OLGs do not need continuous PKC activation to sustain proc ess extension. Control and PMA-stimulated OLGs were also subjected to immunocytochemistry using an anti-ERK antibody selective for the mitog en-activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isofo rms. ERK immunoreactivity in the nucleus was evident after PMA stimula tion of OLGs but not in control OLGs. In parallel experiments, the con trol and PMA-stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [gamma-P-32]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate bas ed on the Thr(97) phosphorylation site in MBP. These assays indicated that in PMA-treated OLGs, ERK activation was at least 12-fold higher t han in control OLGs. Anti-ERK and anti-phosphotyrosine western blots o f the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA-treated fractions relative to control fractions. When OLG s were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 09 8059 before PMA stimulation, they exhibited a 67% decrease in ERK acti vation as compared with cells treated with PMA alone. Furthermore, the se MEK inhibitor-pretreated cells were still viable but showed no proc ess extensions up to 1 week later. Therefore, we propose that a thresh old level of ERK activity is required for the initiation of OLG proces s extension.