Turnover of the Acyl Phosphates of Human and Murine Prothymosin α in Vivo

Abstract

Prothymosin alpha is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin alpha contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin alpha in vivo by pulse-labeling HeLa cells with [32P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. Regardless of the method, the phosphate turned over with a half-life of 75-90 min. The ability of cells to phosphorylate old prothymosin alpha molecules was established by demonstrating equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30-35 and 70 min, respectively. Our data suggest that the "activity" of prothymosin alpha involves the turnover of its acyl phosphates and that it participates in a function common to all nucleated mammalian cells regardless of whether they are quiescent or undergoing rapid proliferation. This is the first measurement of the stability of protein-bound acyl phosphates in vivo.