Phosphorylation of specific, distinct proteins in synaptosomes and axons from squid nervous system

Synaptosomes and axons from squid were incubated with [γ-32 P]ATP or [32 P]orthophosphate and specific, distinct proteins were found to be labeled in each preparation. In axoplasm, only the major 200,000M r neurofilament protein and a specific protein of ≈400,000M r were labeled, as reported previously [Pant, H. C., Shecket, G., Gainer, H. & Lasek, R. J. (1978)J. Cell Biol. 78, R23-R27]. These results were independent of whether the cosubstrates were32 PO4 2- or [γ-32 P]ATP. However, synaptosomes lacked the 200,000M r neurofilament protein and several lower molecular weight proteins were labeled instead, the most prominent being a 47,000M r species. [γ-32 P]ATP was much more effective in labeling the 47,000M r species than32 PO4 2- . Synaptosomes also contained a distinct 250,000M r protein species which, however, was not labeled.The protein kinase activity in synaptosomes was sensitive to various pharmacological agents, depending on whether the labeled phosphate came directly from ATP or orthophosphate. Carbonyl cyanidep -trifluoromethoxyphenyl hydrazone, a mitochondrial H+ uncoupler, almost completely inhibited incorporation of32 P into protein with32 PO4 2- as cosubstrate, as expected, but produced only 32% inhibition with [γ-32 P]ATP as cosubstrate. The activity could be augmented by incubating synaptosomes in a calcium-free medium and could be suppressed by increasing intrasynaptosomal Ca2+ with A23187, a Ca2+ ionophore. The latter effect was more prominent with32 PO4 2- than with [γ-32 P]ATP as cosubstrate. Depolarizing agents such as veratridine and high K+ also suppressed activity, and the veratridine effect was completely reversed by tetrodotoxin or by omission of Ca2+ when [γ-32 P]ATP was used, and partially reversed when32 PO4 2- was used. We conclude that the morphological transformation of an axon into a terminal is accompanied by significant changes in protein and phospho-protein composition that may be related to synaptic transmission.