Prothymosin α in Vivo Contains Phosphorylated Glutamic Acid Residues

Abstract

2) Immediately upon cell lysis, the pH stability curves of metabolically labeled native [ 32 P]prothymosin ␣ or a [ 32 P]histidine-tagged variant resembled the pH stability curve of acetyl phosphate. 3) After a brief incubation at pH 7, these curves changed from a pattern diagnostic for an acyl phosphate to that characteristic of a serine or threonine phosphate, an observation consistent with transfer of phosphate in vitro. Our data indicate that most of prothymosin ␣'s phosphates are subject instantaneously to hydrolysis, based on the observation that greater than 90% of the phosphate initially found at pH 7 disappeared at the extremes of pH. Rapid loss of phosphate was not affected by the presence of phosphatase inhibitors including 50 mM sodium fluoride, 1 mM okadaic acid, and 0.5 mM calyculin A. The amount of phosphate missing could not be ascertained, but the trifling amount recovered on Ser or Thr depended heavily on conditions favoring the transient survival of labile phosphate. Further analysis using COS cells lysed in the presence of sodium borohydride showed that: 1) phosphate recovered on prothymosin ␣ decreased 8-fold when lysates were treated with borohydride; 2) the reagent caused 4 -8 glutamic acid residues/molecule to vanish; 3) using [ 3 H]NaBH 4 , label was introduced into proline, a product derived from reductive cleavage of phosphoglutamate; and 4) [ 3 H]proline was localized almost exclusively to a peptide with pronounced homology to the histone binding site of nucleoplasmin, a chromatin remodeling protein found in Xenopus laevis. Our data demonstrate that prothymosin ␣ is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.Prothymosin ␣ is a highly unusual protein with an unfortunate name. The protein is neither a precursor for processed polypeptides nor specifically associated with the thymus nor a member of a family with ␤ or ␥ homologues (1-3). Instead, it is probably the most acidic naturally occurring polypeptide in the eukaryotic world, with 54 carboxyl groups in 109 amino acids, resulting in an isoelectric point at or below pH 3.5 (1, 2, 4). The mRNA for prothymosin ␣ is distributed ubiquitously among mammalian nucleated cells and tissues (1). The protein possesses a potent nuclear localization signal and is present in amounts equivalent to those of histone H1 (5, 6). Because the amount of prothymosin ␣ mRNA (and presumably protein) found in a cell is directly proportional to cell growth, the protein is believed to play a role in cell proliferation (1). This idea was reinforced by the observation that synchronized human myeloma cells, in the presence of antisense oligodeoxyribonucleotides directed at prothymosin ␣ mRNA, were unable to divide while detectable amounts of the antisense oligonucleotides remained inside the cell (7). There are now many examples of a link between prothymosin ␣ and growth in systems as diverse as developing mouse embryos (8); normal, mitogenstimulated, and malignant lymphocytes (9, 10); and regenerating liver (10). There are also positive ...

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

“…Its level is also higher in malignant breast tumors than in benign breast lesions or in adjacent normal breast tissue (4,36), and it may be a prognostic factor in breast cancer, as there is an association between PT␣ level and increased risk of death from breast cancer. There is also evidence that PT␣ binds to tRNAs (20), that PT␣ contains phosphorylated residues, including acylphosphates (phosphoglutamic acid), and that the activity of PT␣ may involve turnover of its acylphosphates (34,39) and may fuel an energy-requiring step in the production or processing of RNA (32).…”

Substitution of Aspartate and Glutamate for Active Center Histidines in the Escherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Maintain Phosphotransfer Potential

“…The creation of two mutant proteins, which are able to form a phosphoglutamyl residue is significant due to the rarity of these linkages in nature. There are few reported examples of phosphoglutamates in the literature; acetate kinase (15) and prothymosin alpha (36). The infrequent observation of phosphoglutamyl residues may in part be due to the instability of these linkages.…”

Does Prothymosin α Affect the Phosphorylation of Elongation Factor 2?

“…The Berger group has shown that prothymosin ␣ is phosphorylated in vivo on glutamic acid residues in the region homologous with nucleoplasmin (33). These acyl phosphates are unstable in vivo and energy rich (33,34).…”

“…The Berger group has shown that prothymosin ␣ is phosphorylated in vivo on glutamic acid residues in the region homologous with nucleoplasmin (33). These acyl phosphates are unstable in vivo and energy rich (33,34). Upon cell lysis, most hydrolyze almost instantaneously, while a tiny fraction migrate to more stable positions on nearby serine or threonine residues (33,35).…”

“…These acyl phosphates are unstable in vivo and energy rich (33,34). Upon cell lysis, most hydrolyze almost instantaneously, while a tiny fraction migrate to more stable positions on nearby serine or threonine residues (33,35). By observing the changes in stability of prothymosin ␣'s phosphates in vivo in cells overexpressing prothymosin ␣, in synchronized cells at different stages of the cell cycle, and in cells treated with metabolic inhibitors, the group concluded that prothymosin ␣'s phosphates become stabilized and inactive during mitosis and in transcriptionally compromised cells (36).…”