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

Abstract

Prothymosin ␣ is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin ␣ 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 ␣ in vivo by pulse-labeling HeLa cells with [ 32 P]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 Prothymosin ␣ is a small, highly acidic (1, 2), nuclear (3-6) protein found in virtually all mammalian tissues (7-11). Under conditions of rapid growth, a cultured cell accumulates upwards of 17 million molecules, a number roughly equivalent to that of histone cores (12). When cells are disrupted with detergents, the protein readily leaks out of the nucleus, suggesting that stable interactions with nucleosomes or with the nuclear matrix are not an inherent part of its activity (2, 3). Its precise function is unknown. Nevertheless, a role in cell proliferation has been proposed based on the following observations: prothymosin ␣ mRNA is plentiful only in rapidly dividing cells (13)(14)(15); the level of the protein declines 10-fold in cells forced to subsist in stationary phase (12); the amount of prothymosin ␣ is directly proportional to the proliferative activity of the tissue from which it is isolated (13); and the uptake of antisense oligodeoxyribonucleotides directed toward various locations in prothymosin ␣ mRNA prevents synchronized human myeloma cells from entering mitosis (16). Hence, a deficiency in prothymosin ␣ is associated with failure to complete the cell cycle.Prothymosin ␣ has several unusual features. The human protein, which is almost identical to that of all other mammals (17), has 109 amino acids, nearly 50% of which are acidic (1, 2). A potent nuclear localization signal consisting of five basic amino acids has been identified near the carboxyl terminus (3, 6), while a second cluster of five basic residues near the amino terminus has no unambiguous role (3,18). The absence of all aromatic residues renders the protein transparent at 280 nm; it also lacks methionine, cysteine, and histidine. Based on biophysical data, it is believed to have an unfolded structure (19), a conclusion consistent with the presence of only seven widely dispersed hydrophobic residues in the human protein. Prothymosin ␣ does not bind SDS and stains anomalously with silver stain (2). The protein and the peptides derived from it exhibit poor immunogenicity. However, due to yet another aberrant property, the ability to partition quantitatively into the aqueous phase ...

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).…”

Prothymosin α in Vivo Contains Phosphorylated Glutamic Acid Residues

“…2 With so many features in common, it is tempting to speculate that prothymosin ␣, like nucleoplasmin, interacts with chromatin. Further evidence for a role involving chromatin comes from studies of the half-life of prothymosin ␣'s glutamyl phosphates in vivo (36). Our laboratory will show that these phosphates undergo a pronounced increase in stability upon the cessation of transcription, 3 suggesting the nucleosome as a likely target for the functional activity of prothymosin ␣.…”

“…6) Cells cannot phosphorylate Ser 1 of prothymosin ␣ (36). When specific peptides bearing N-terminal acetylserine or the amino-terminal sequence of prothymosin ␣ were added to the lysis buffer under a variety of conditions including those most conducive to phosphorylation by a traditional kinase ([␥-32 P]ATP and Mg 2ϩ ) or incubated with extracts of whole cells labeled with [ 32 P]inorganic phosphate in vivo, no phosphorylated specific peptides were recovered (36). These experiments argue against a cellular activity capable of phosphorylating prothymosin ␣ directly on Ser 1 in vivo.…”

Factors influencing the inhibition of protein kinases

“…Since the cellular concentration of ATP is close to 1 mM [26][27][28] , the potency (IC 50 ) of AEW541 and STAURO was measured in the biochemical assay in the presence Figure 2) and from these values, β and K I were calculated using Equation 4 (Materials and methods) ( Table 2). The presence of the bound peptide affects the binding of AEW541 (β 0.5 mM = 15; β 10 mM = 39) more than that of STAURO (β 0.5 mM = 0.9; β 10 mM = 5 ] f = 10 mM (543 nM; Table 2).…”