Identification of an ectokinase activity in cerebellar granule primary neuronal cultures

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.