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.

Role of calmodulin-dependent phosphorylation of elongation factor 2 in the proliferation of rat glial cells.

Calmodulin (CaM) is believed to play an important role in the regulation of cellular proliferation. The mechanism of regulation, although unknown, may involve CaM-binding proteins, particularly CaM-dependent protein kinases. Previously, we have shown that CaM-dependent protein kinase III phosphorylates elongation factor 2 (EF-2) in proliferating, C6 glioma cells but not in normal white matter, a tissue rich in nonproliferating glia. To determine whether CaM-dependent phosphorylation of EF-2 is linked, in general, to cellular division, we studied the phosphorylation of EF-2 in proliferating and growth-arrested C6 cells and in proliferating, primary cultures of normal glia. Phosphorylation of EF-2 was not detectable in C6 cells arrested in their growth by serum deprivation. When serum-deprived cells were stimulated to proliferate by the re-addition of serum, the amount of phosphorylated EF-2 correlated with levels of [3H]thymidine incorporation into DNA. Primary cultures of dividing, normal glia, obtained from neonatal rats, also demonstrated phosphorylation of EF-2. Therefore, the CaM-dependent phosphorylation of EF-2 appears to be associated with cellular proliferation in normal and malignant glia in the rat.

Calmodulin-dependent protein kinases in rat glioblastoma.

The mitogenic activity of several growth factors is mediated by calcium-dependent signal transduction. Calmodulin (CaM) binding proteins such as CaM-dependent protein kinases are important components of this pathway and may be altered in diseases characterized by abnormal cell growth. CaM kinase II is believed to regulate the phosphorylation of microtubular-associated proteins and control the initiation of DNA synthesis. Furthermore, drugs that inhibit CaM-mediated signal transduction also inhibit cellular proliferation and are cytotoxic to numerous malignant cell lines, including those established from malignant gliomas. Yet, little is known about CaM-dependent protein kinases in these tumors. Therefore, we have investigated the activity and distribution of CaM-dependent protein kinase II in normal and malignant glial tissues, a kinase believed to play a critical role in cell cycle regulation. C6 and 9L cells contained kinase activities that were activated by Ca2+/CaM and inhibited by trifluoperazine. Tissue extracts from these cell lines and from rat brain white matter phosphorylated exogenous synapsin I in a pattern consistent with the presence of CaM kinase II activity as determined by phosphopeptide mapping. CaM kinase II activity was confirmed using a specific peptide substrate and inhibitor. An unexpected finding was that glioma lines, but not rat brain white matter, also contained a CaM-dependent protein kinase detected by the phosphorylation of a M(r) 100,000 protein, subsequently identified as elongation factor 2, the only known substrate for CaM kinase III.(ABSTRACT TRUNCATED AT 250 WORDS)