Does Prothymosin α Affect the Phosphorylation of Elongation Factor 2?

Abstract

Prothymosin ␣ 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 ␣ 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 ␣, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin ␣ 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 ␣. 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 ␣ and the phosphorylation of eEF-2.The detailed function of prothymosin ␣ remains cryptic and controversial. The protein is not, as its name intimates, a precursor for much smaller, processed, thymic hormones (1-5). Rather, prothymosin ␣ is exclusively mammalian (see Refs. 6 and 7, and references therein), extremely abundant (8, 9), probably unfolded (10), and highly acidic throughout its length of 109 -110 amino acids (1,11,12). It localizes to the nucleus in intact form in all tissues (13-15). An overview of the prothymosin ␣ literature strongly suggests that the protein plays an essential, but nevertheless undefined, role in the growth and survival of virtually all cells (1, 16 -19). Several laboratories have noted (i) that an increase of greater than 10-fold in the mRNA and the protein occurs when quiescent cells proliferate, (ii) that the amount of prothymosin ␣ in a tissue mirrors its inherent growth rate, and (iii) that prothymosin ␣ is expressed in a chronologically contemporaneous manner with other growth related molecules such as Myc, histone H3, and proliferating cell nuclear antigen (1, 3, 8, 20 -24). However, the most compelling evidence comes from synchronized human myeloma cells, which, when exposed to antisense oligodeoxyribonucleotides directed toward prothymosin ␣ mRNA at several locations, undergo growth arrest (25).The particulars of prothymosin ␣'s function are contested. The amino acid sequence, which should provide clues, is essentially unique. Except for a "classical" bipartite nuclear localization signal included in many otherwise unrelated nuclear proteins, the only shared featured is a reiterated region of glutamic acid residues found also in the Xenopus chromatin remo...

Prothymosin Alpha Selectively Enhances Estrogen Receptor Transcriptional Activity by Interacting with a Repressor of Estrogen Receptor Activity

“…In addition to preferentially binding histone H1, PT␣ also displays high affinity for histones H3 and H4 (6). It has also been suggested to stimulate the phosphorylation of transcription factors such as E2F, although this is controversial (7). PT␣ is a small, 109-amino-acid protein that is rich in acidic (aspartic acid and glutamic acid) residues such as are also found in the yeast transcription-regulating proteins GAL4 and GCN4 (13), the viral protein VP16, and the N terminus of the GR (11,12).…”

Prothymosin α Is a Nonspecific Facilitator of Nuclear Processes: Studies of Run-on Transcription

“…Native prothymosin ␣ from HeLa cells and prothymosin ␣ bearing the FLAG epitope (25) expressed in COS-1 cells were isolated with phenol. All samples were chromatographically purified further using Q2, a quaternary amine anion exchange column from BioRad and C-18, a reverse phase column from Vydac (25). Prothymosin ␣ was stored frozen in water at Ϫ20°C.…”

“…Prothymosin ␣ to which six C-terminal histidine residues had been attached (19) was expressed either in bacteria or in COS-1 cells and purified either by extraction with phenol (4,19,20) or by chromatography on nickel nitrilotriacetate resin (Qiagen) (25). Native prothymosin ␣ from HeLa cells and prothymosin ␣ bearing the FLAG epitope (25) expressed in COS-1 cells were isolated with phenol.…”

“…A vague role in the post-transcriptional regulation of RNA maturation has also been proposed (23). Furthermore, there is a report claiming that prothymosin ␣ inhibits translation by promoting the phosphorylation of eukaryotic elongation factor 2 during mitosis (24), an idea that has been vigorously challenged (25).…”

Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells

“…Calmodulin mediates many of the intracellular effects of Ca 2+ (Park, 2001), and the eEF-2 kinase is entirely dependent on Ca 2+ /CaM for activity in mammalian tissues. Increased phosphorylation of eEF-2 occurs in several types of cells in response to stimuli, which elevates cellular Ca 2+ levels or stimulates cellular proliferation (Enkemann et al, 1999). Decreased kinase activity is seen in the presence of drugs that increase the concentration of cAMP (Hovland et al, 1999).…”