Dr. R. L. Stariha and Dr. S. Kikuchi are co-first authors.
Role of Extracellular Signal-Regulated Protein Kinases 1 and 2 in Oligodendroglial Process Extension
Article first published online: 18 NOV 2002
Journal of Neurochemistry
Volume 68, Issue 3, pages 945–953, March 1997
How to Cite
Stariha, R. L., Kikuchi, S., Siow, Y. L., Pelech, S. L., Kim, M. and Kim, S. U. (1997), Role of Extracellular Signal-Regulated Protein Kinases 1 and 2 in Oligodendroglial Process Extension. Journal of Neurochemistry, 68: 945–953. doi: 10.1046/j.1471-4159.1997.68030945.x
- Issue published online: 18 NOV 2002
- Article first published online: 18 NOV 2002
- Received June 25, 1996; revised manuscript received October 8, 1996; accepted October 21, 1996.
- Extracellular signal-regulated protein kinase;
- Extracellular signal-regulated protein kinase isoforms;
- Mitogen-activated protein kinases;
- Process extension;
- Protein kinase C activation
Abstract: The relationship between extracellular signal-regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendrocytes (OLGs) was investigated. Process extension was induced through the 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 stimulation was easily discernible through morphological monitoring. It was found that exposure of OLGs to PMA for 10 min was enough to induce OLG process extension 24–72 h later. Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indicating that OLGs do not need continuous PKC activation to sustain process extension. Control and PMA-stimulated OLGs were also subjected to immunocytochemistry using an anti-ERK antibody selective for the mitogen-activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isoforms. ERK immunoreactivity in the nucleus was evident after PMA stimulation of OLGs but not in control OLGs. In parallel experiments, the control and PMA-stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [γ-32P]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate based on the Thr97 phosphorylation site in MBP. These assays indicated that in PMA-treated OLGs, ERK activation was at least 12-fold higher than in control OLGs. Anti-ERK and anti-phosphotyrosine western blots of the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA-treated fractions relative to control fractions. When OLGs were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 098059 before PMA stimulation, they exhibited a 67% decrease in ERK activation as compared with cells treated with PMA alone. Furthermore, these MEK inhibitor-pretreated cells were still viable but showed no process extensions up to 1 week later. Therefore, we propose that a threshold level of ERK activity is required for the initiation of OLG process extension.
Abbreviations used: ERK, extracellular signal-regulated protein kinase; GalC, galactocerebroside; MAP2, microtubule-associated protein 2; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MEK, extracellular signal-regulated protein kinase kinase; MS, multiple sclerosis; OLG, oligodendrocyte; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBS+, phosphate-buffered saline containing 5% normal goat serum and 0.2% Triton X-100; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SDS, sodium dodecyl sulfate.