The involvement of ecto-ATPase activity in the phosphorylation of intracellular proteins by the addition of extracellular [P]ATP in PC12 cells

We have investigated the possibility that ecto-phosphorylation by extracellular ATP may play a role in the development of PC12 cells. To test this model and to identify putative target membrane proteins, intact PC12 cells were radiolabeled by the addition of 20 μM γ-³²P]ATP. An analysis of the labeled proteins revealed that a 57 kDa protein was the most abundant phosphorylated protein even within time periods as short as 3 min and continued to be labeled over and above the level of other proteins. This protein was identified as tyrosine hydroxylase by immunoprecipitation with antiserum to tyrosine hydroxylase. When intact cells were incubated with either γ-³²P]ATP or ³²P_i of comparable specific radioactivity, the overall protein labeling pattern and the degree of phosphorylation of tyrosine hydroxylase were similar. There were no discrete proteins that were labeled by γ-³²P]ATP and not by ³²P_i that would provide evidence for ecto-kinase activity in PC12 cells. Also, the addition of nonradioactive P_i reduced the incorporation of radioactivity into the protein from extracellular γ-³²P]ATP. These results suggested that the phosphorylation of tyrosine hydroxylase by extracellular γ-³²P]ATP required the initial hydrolysis of ATP and the subsequent incorporation of the ³²P_i into the intracellular ATP pool. To support this interpretation, we have demonstrated directly the presence of ecto-ATPase activity in intact PC12 cells by measuring the hydrolysis of extracellular γ-³²P]ATP. Nearly 50% of the total ATP added (20 μM) was hydrolyzed within 10 min under conditions identical to those used to demonstrate intracellular protein phosphorylation. PC12 cells express both a Ca²⁺-dependent ecto-ATPase activity and a Mg²⁺-dependent ecto-ATPase activity. In addition, extracellular ATP is degraded enzymatically not only to ADP, but sequentially to adenosine. Our results also point out the difficulties inherent in attempts to identify ecto-kinase activity in cells that also contain ecto-ATPase activities.