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 also been proposed to be an ecto-protein kinase capable of phosphorylating itself as well as exogenous proteins. We have investigated the phosphorylation 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-32P]ATP, SDS/PAGE of whole cell lysates showed a single band which was PC-1; this band was absent in cells not expressing PC-1. Immunoprecipitates of soluble PC-1 isolated from culture supernatants of cells expressing PC-1 were also capable of autophosphorylation, and the size of the labeled protein was the same as previously reported for soluble PC-1. PC-1 was also labeled with [alpha-32P]ATP and [35S]dATP[alpha S]. We found no evidence that PC-1 was capable of phosphorylating proteins other than itself, and conclude that it is not a true kinase, and that the observed labeling with [gamma-32P]ATP, [alpha-32P]ATP and [35S]dATP[alpha S] reflect transient covalent adducts that are part of the catalytic cycle of phosphodiesterase/pyrophosphatase activity rather than intrinsic kinase activity. Mutation of the active-site threonine to tyrosine, serine or 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.
Belli
S I
SI
Department of Pathology and Immunology, Monash Medical School, Alfred Hospital, Victoria, Australia.
Mercuri
F A
FA
Sali
A
A
Goding
J W
JW
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D051379:Mice; D008969:Molecular Sequence Data; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism*; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase