AUTOPHOSPHORYLATION OF PC-1 (ALKALINE PHOSPHODIESTERASE-I NUCLEOTIDE PYROPHOSPHATASE) AND ANALYSIS OF THE ACTIVE-SITE

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I (EC 3.1 .4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It has a lso been proposed to be an ecto-protein kinase capable of phosphorylat ing itself as well as exogenous proteins. We have investigated the pho sphorylation capability of PC-1 and have developed a novel method for its detection and characterization based on autophosphorylation, which allows detection without the use of antibodies. When cells expressing membrane PC-1 were held on ice with [gamma-P-32]ATP, SDS/PAGE of whol e cell lysates showed a single band which was PC-1; this band was abse nt in cells not expressing PC-1. Immunoprecipitates of soluble PC-1 is olated from culture supernatants of cells expressing PC-1 were also ca pable of autophosphorylation, and the size of the labeled protein was the same as previously reported for soluble PC-1. PC-1 was also labele d with [alpha-P-32]ATP and [S-35]dATP[alpha S]. We found no evidence t hat PC-1 was capable of phosphorylating proteins other than itself, an d conclude that it is not a true kinase, and that the observed labelin g with [gamma-P-32]ATP, [alpha-P-32]ATP and [S-35]dATP[alpha S] reflec t transient covalent adducts that are part of the catalytic cycle of p hosphodiesterase/pyrophosphatase activity rather than intrinsic kinase activity. Mutation of the active-site threonine to tyrosine, serine o r alanine reduced the 5'-nucleotide phosphodiesterase activity of PC-1 and its ability to autophosphorylate to undetectable levels. Together , these data suggest that both activities depend on the same site.