Phosphorylation of elongation factor 2 in normal and malignant rat glial cells.
Certain calmodulin (CaM)-dependent protein kinases phosphorylate substrates have been implicated in regulating cellular proliferation. In this study, CaM-dependent phosphorylation has been examined in normal and tumor tissue from rat brain to determine whether differences exist. Using in vitro phosphorylation reactions, we compared endogenous substrates for Ca2+/CaM-dependent protein kinases in rat brain white matter (RBWM), a tissue rich in normal glia, to those of C6 rat glioma cells. A major phosphoprotein having a M(r) of 100,000 was observed in proliferating C6 cells that was not present in RBWM or in nonproliferating cells. Phosphorylation was stimulated by Ca2+ and CaM and inhibited by trifluoperazine. An antibody to elongation factor 2 (EF-2) immunoprecipitated the M(r) 100,000 protein from C6 cells. EF-2 was present in RBWM but was not phosphorylated. Homogenates of RBWM did not phosphorylate exogenous EF-2, which suggested the absence of CaM kinase III activity in normal glial tissue. Furthermore, the addition of purified, exogenous CaM kinase III to homogenates of RBWM resulted in EF-2 phosphorylation. These data demonstrate that a basal level of EF-2 phosphorylation exists in proliferating glioma cells that is markedly diminished or absent in normal glial tissue and is due to the activity of CaM kinase III.
Bagaglio
D M
DM
Department of Pharmacology and Internal Medicine, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5638.
Cheng
E H
EH
Gorelick
F S
FS
Mitsui
K
K
Nairn
A C
AC
Hait
W N
WN
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D054736:Elongation Factor 2 Kinase; D005910:Glioma / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D009457:Neuroglia / Q000201:enzymology / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014268:Trifluoperazine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D014268:Trifluoperazine; D011494:Protein Kinases; C517691:Eef2k protein, rat; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D002118:Calcium