THREONINE AUTOPHOSPHORYLATION AND NUCLEOTIDYLATION OF THE HEPATIC MEMBRANE-PROTEIN PC-1

The membrane protein plasma-cell-differentiation antigen 1 (PC-1) has been described as a phosphodiesterase-I/nucleotide pyrophosphatase and as an autophosphorylating protein kinase. It has been suggested, howe ver, that PC-1 is not a real protein kinase and that the autophosphory lated enzyme represents a nucleotidylated derivative, which is formed on Thr238 (murine PC-1) as a catalytic intermediate during ATP hydroly sis [Belli, S. I., Mercuri, F. A., Sali, A. & Goding, J. W. (1995) Eur . J. Biochem. 228, 669-676]. We have investigated the proposed multifu nctional role of PC-1 and show here that ATP hydrolysis and autophosph orylation represent two distinct catalytic reactions. The enzyme was r adiolabeled when various concentrations (1-260 mu M) of [alpha-P-32]AT P or [alpha-P-32]ADP, but not [gamma-P-32]ATP, were used as substrates for the formation of tile pyrophosphatase catalytic intermediate, esp ecially in the presence of imidazole, which interferes with the hydrol ysis oi the nucleotidylated enzyme. In contrast, autoradiography revea led autophosphorylation only with [gamma-P-32]ATP as the phosphoryl do nor, and autophosphorylation has been shown to occur only at ATP conce ntrations below 5 mu M. Autophosphorylation could also be differentiat ed from nucleotidylation by its higher resistance to alkaline treatmen t and its more basic pH optimum. An intestinal nucleotide pyrophosphat ase with a structurally related catalytic site could not be autophosph orylated, which shows that autophosphorylation is not an intrinsic pro perty of the nucleotide pyrophosphatase reaction. Autophosphorylation of PC-1 was associated with inactivation of its phosphodiesterase-I/nu cleotide-pyrophosphatase activity. We propose that autophosphorylation of PC-1 on Thr238 at low ATP concentrations serves as an autoregulato ry mechanism that makes Thr-238 unavailable for participation in the h ydrolysis of extracellular nucleotides when they become scarce.