Does prothymosin alpha affect the phosphorylation of elongation factor 2?

Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specif ically increase the phosphorylation of eukaryotic elongation factor 2 (eEF- 2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernst edt, C., and Dominguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using s imilar lysates prepared by four methods (detergent lysis, Dounce homogeniza tion, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native p rotein, and a histidine-tagged recombinant prothymosin alpha expressed eith er in bacteria or in COS cells), we failed to find a response. A reconstitu ted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and cal cium was also unaffected by prothymosin alpha. However, unlike our optimize d buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, w hich when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effe ctive fluoride concentration will relieve the inhibition and appear to acti vate. Our data do not support a direct relationship between the function of prothymosin a and the phosphorylation of eEF-2.