PROTEIN-KINASE-C ISOFORMS IN HUMAN GLIOBLASTOMA CELLS

Protein kinase C (PKC), an enzyme involved in signal transduction, responds to diacyl glycerol and also to phorbol ester, a ligand analogous to diacyl glycerol. We have studied the expression of the major isoforms (alpha, betaI, betaII, and gamma) in eight human glioblastoma cell lines. In all eight lines, PKC-alpha mRNA and protein were expressed. In none of the eight did a probe for PKC-betaI and betaII mRNA give positive results nor were Western blots for PKC-betaII positive. The half-life for PKC a mRNA was approximately 16 h and levels of the mRNA were increased slightly following addition of phorbol myristate acetate (PMA) or transforming growth factor-beta (TGFbeta). PKC-gamma was present in most of the glioblastomas. In cell line A172, 82% of the PKC-alpha was present in the cytosol with the remainder evenly divided between plasma membrane and nucleus. Thirty minutes after addition of PMA, 33% of the total original protein was in the plasma membrane and 48% in the nuclear fraction. By 21 h, no PKC-alpha was recovered from any fraction. PKC-gamma was also down-regulated in the presence of PMA, but there was no evidence for translocation to the plasma membrane or nuclear fraction. In a more detailed study, translocation of PKC-ot in the presence of PMA was complete by 10 min, and a major decrease in the PKC translocated to the plasma-membrane fraction occurred some time between 2 and 4 h after PMA addition, while a major decrease in the translocated nuclear fraction occurred some time after 6 h. cAMP alone had no effect on the PKC alpha protein level or distribution, nor did it alter the translocation and down-regulation due to PMA exposure. In these studies the level of PKC-alpha mRNA in tumors was similar to that in normal glial cells.