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 specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.
Enkemann
S A
SA
Section on Genes and Gene Products, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Pavur
K S
KS
Ryazanov
A G
AG
Berger
S L
SL
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D054736:Elongation Factor 2 Kinase; D005459:Fluorides / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011498:Protein Precursors / Q000378:metabolism*; D013947:Thymosin / Q000031:analogs & derivatives* / Q000378:metabolism; D014162:Transfection
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011498:Protein Precursors; C040782:prothymosin alpha; D013947:Thymosin; C517689:EEF2K protein, human; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517690:Eef2k protein, mouse; D054736:Elongation Factor 2 Kinase; D005459:Fluorides