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. In addition, both rat brain white matter and glioma cells contained CaM kinase IV activity and immunoreactive protein, an enzyme previously found only in neuronal tissues which phosphorylates Rap-1b, a member of the Ras superfamily of CTP-binding proteins. The subcellular distribution of the CaM kinases differed between the normal and malignant tissues. The activity of CaM kinases was inhibited by calmodulin inhibitors such as trifluoperazine. Alterations in subcellular distribution and endogenous substrates for CaM kinases in glioblastoma may contribute to the altered signal transduction seen in this disease, and inhibition by drugs such as trifluoperazine may explain the cytotoxic actions of these drugs.