INTRODUCTION

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, mitogen-stimulated, and malignant lymphocytes (9, 10); and regenerating liver (10). There are also positive correlations among prothymosin  mRNA levels and those of growth-related molecules such as histone H3, proliferating nuclear antigen, and Myc (11-13).

The specific function of prothymosin  has eluded detection. Despite a report to the contrary, prothymosin  is not present in yeast, nor are there close relatives, with the exception of the X. laevis protein, nucleoplasmin. Both prothymosin  and nucleoplasmin share a presumptive histone binding sequence, localize to the nucleus, and achieve high concentrations (5, 6, 14-16). However, unlike nucleoplasmin, which disappears during embryogenesis (14), prothymosin  persists as an abundant protein in proliferating cells throughout the life of the organism. Other observations from which prothymosin 's function can be intimated include 1) binding to histones in vitro (17, 18), implicating a role in chromatin remodeling; 2) interacting with the Rev protein of human immunodeficiency virus in vitro (19), suggesting involvement in RNA export from the nucleus; 3) up-regulation in the presence of Myc in specialized cells (20, 21); and 4) phosphorylation (16, 22).

There are two views of prothymosin 's phosphates. According to Sburlati et al. (16), phosphorylation of the human and bovine protein occurs on the N-terminal acetylserine residue, and not on Ser at positions 8 and 9 or Thr at positions 7, 12, 13, 100, and 105 (see Fig. 1). Ser⁸³, which is an alanine in most other mammals, and Thr⁸⁵ were not rigorously excluded as possible sites in this study. The second study of phosphoprothymosin , a much less exhaustive analysis in mouse splenic lymphocytes, placed the labeled phosphate(s) on unspecified threonine residue(s) near the N terminus (22). Because prothymosin  sequences from different species are nearly identical, with ~95% sequence homology over the entire protein and 100% sequence homology within the amino-terminal 30 residues (23, 24), the discrepancy was unsettling.

Diagram of pBC12BIProG, the human prothymosin  gene from HindIII to ScaI cloned into an expression vector, pBC12BI, cleaved with HindIII and SmaI.

The dynamic aspects of prothymosin 's phosphates were equally puzzling. Prothymosin  is metabolically stable and becomes phosphorylated equivalently at all stages of the cell cycle (16). Additionally, only 2% of the bovine protein is phosphorylated at steady state (16). Based on these properties, prothymosin  seemed to be involved in the continuing activities performed by the cell and not, as previously postulated (25), in the regulation of an intermittent function of the cell cycle.

Here, both the location and the stability of prothymosin 's phosphates have been evaluated. We find that the initial sites of phosphorylation are glutamic acid residues, that the phosphates are extremely labile and readily hydrolyze during the earliest steps of the isolation procedure, and that phosphorylation on serine or threonine may occur solely in vitro when labile phosphates transfer to stable positions. Our data resolve the discrepancies between observations of Ser/Thr phosphorylation made in the human and mouse systems and explain the minute amount of phosphate found on the protein regardless of the organism studied. Based on an analysis of the peptides of human and monkey prothymosin , the phosphorylated residues have been localized to an extremely acidic region that is homologous with the histone binding site of nucleoplasmin. Since the free energy of hydrolysis of a glutamyl phosphate is higher than that of ATP (26) and since our evidence suggests that several of prothymosin 's glutamic acids bear phosphate simultaneously, we surmise that the protein is able to supply abundant energy for processes in the nucleus.

EXPERIMENTAL PROCEDURES

Mutant clones were obtained by making modifications in pBC12BIProG, the 5-kilobase pair human prothymosin  gene cloned into pBC12BI (5). A triple mutant in which Ser¹, Ser⁸³, and Thr⁸⁵ were replaced by Ala residues was prepared using the polymerase chain reaction and appropriate oligomers to generate the desired mutations. Construction involved three steps. A single mutant in which Ser¹ was replaced by Ala was generated first; in a second construct, Ser⁸³ and Thr⁸⁵ were mutated; and finally, the triple mutant was obtained by shuffling fragments derived from the two mutant genes using hapaxomers (27). A map of the gene illustrating the locations of important sites and regions is shown in Fig. 1. Similar methods were used to generate clones coding for prothymosin  wild type or mutant proteins bearing six carboxyl-terminal histidine residues.

Expression of human prothymosin  in Escherichia coli was achieved by cloning the cDNA into NdeI-BamHI-cut pET3a from Stratagene. Sites were introduced into the cDNA using the polymerase chain reaction. In a related clone, the codons for six C-terminal histidine residues were inserted by including them in the BamHI-containing primer. All polymerase chain reaction-generated DNA was sequenced to select error-free molecules for further study. Recombinant genes were expressed in E. coli BL21(DE3) from Stratagene, and protein was recovered by lysing bacteria in 8 M urea made 0.1 M in sodium phosphate and 0.01 M in Tris-HCl at pH 8.0. Prothymosin  was obtained by means of a phenol extraction (6) and purified to homogeneity on a Bioscale Q2 column run with a Biologics (Bio-Rad) medium pressure liquid chromatography system.

Plasmids derived from pCH110 containing the gene for -galactosidase fused downstream of fragments of prothymosin  cDNA were constructed and characterized by Manrow et al. (5). Similarly, codons for KKKRK were inserted into pCH110 using restriction sites and methods identical to those of Manrow et al. (5). The SV40 nuclear localization signal, VPKKKRKVP, in the engineered -galactosidase drove the protein into the nucleus as confirmed by staining with 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (data not shown).

HeLa cells and African green monkey kidney cells (COS-1) were grown in Dulbecco's modified Eagle's medium from Life Technologies, Inc. or BioFluids (Rockville, MD) containing 10% fetal bovine serum (Hyclone, Logan, UT, or Life Technologies, Inc.) and 2 mM glutamine, 500 units/ml penicillin, 2.5 µg/ml streptomycin, and 5000 units/ml amphotericin from Life Technologies, Inc. in an atmosphere of 5% CO₂ at 37 °C. Cells were harvested by washing with Puck's saline and treating them with 0.05% trypsin in Hanks' balanced salts containing 0.5 mM EDTA. Transient transfections of COS cells were carried out in 60-mm dishes in a total volume of 0.6 ml using the DEAE-dextran method (28) with DNA that was purified by chromatography in Qiagen columns (Studio City, CA). The cells were incubated for 48-60 h and labeled either with 100 µCi/ml [³²P]orthophosphoric acid for 4 h or with 200 µCi/ml of L-[G-³H]glutamic acid (TRK445, 49 Ci/mmol; Amersham Corp.) for 4 h in complete medium.

Characterization of Prothymosin , Phosphoprothymosin , and Fusion Proteins

To obtain prothymosin , washed cells from one dish were lysed with 1 ml of the standard buffer (10 mM Tris-HCl at pH 7.5, 5 mM EDTA, 12% sucrose, 1% Triton X-100, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride-HCl (AEBSF,¹ Sigma)) at 0 °C; the nuclei that lost their prothymosin  were removed by centrifugation at 14,000 × g in a Tomy Tech USA Inc. refrigerated microcentrifuge; and both prothymosin  and its histidine-tagged derivative were isolated by means of phenol extractions of the supernatant fluids, diluted with an equal volume of water. Protein was recovered by precipitation and, where necessary, purified further by digestion with ribonuclease in any convenient buffer. These methods were detailed by Sburlati et al. (6, 16). The wild type and tagged proteins were separated from each other and nonprotein contaminants in 18% polyacrylamide gels in the presence of SDS (Novex). ³²P-Protein in dried gels was imaged directly with X-AR x-ray film (Kodak). ³H-Protein was visualized by soaking fixed gels in Enlightning (NEN Life Science Products) according to the accompanying instructions, drying the gels, and exposing them to film. In both cases, gels were stained with Coomassie Brilliant Blue; quantitative evaluations of stained and autoradiographed proteins were obtained with a Molecular Dynamics Personal Laser Densitometer.

Fusion proteins containing -galactosidase attached to all or part of prothymosin  sequences were obtained from COS-1 cells transfected with pCH110 containing a variety of inserts. In the experiments shown, cells were labeled with [³²P]orthophosphate as described above. Lysates were prepared with the standard buffer enriched with 0.1% SDS. Immunoprecipitations made use of 100 µl of Cappel rabbit anti--galactosidase IgG conjugated to Sepharose 4B, 1 ml of cell lysate, and the methods recommended by the manufacturer. In short, binding reactions were carried out overnight at 4 °C on a platform rocker, and the beads were collected by low speed centrifugation, washed many times with calcium- and magnesium-free phosphate-buffered saline, and treated with 50 µl of 2 × SDS gel running buffer for 5 min at 100 °C to release bound molecules. -Galactosidase fusion proteins were analyzed in 10% polyacrylamide gels (Novex), which were stained, exposed to film, and quantified as noted above.

Mock transfected or transfected COS cells were labeled with [³²P]orthophosphate, recovered from 60-mm dishes, and lysed at 4 °C in 1 ml of 5 mM EDTA, 12% sucrose, 1% Triton X-100, and 1 mM AEBSF containing 20 mM MES at pH 3.2, 4.0, 5.2, 6.1, 7.0, 8.0, 9.0, or 10.2. Postnuclear supernatants were prepared immediately; approximately 20-30 min was required for handling samples and centrifuging them to remove nuclear debris. The supernatant fluids were then brought to pH 5.1 by the addition of 3 volumes of ACE buffer (10 mM sodium acetate at pH 5.1, 50 mM NaCl, 3 mM EDTA) and made 0.5% in SDS. Prothymosin  was purified with phenol and ribonuclease-treated as described above. Control samples, in which [³²P]prothymosin  was studied in crude lysates, were prepared by lysing labeled cells in buffer at pH 7 (5 mM MES at pH 7.2, 5 mM EDTA, 1% Triton X-100, 12% sucrose, and 1 mM AEBSF), incubating them for 20 min on ice, diluting the lysates to approximately twice the volume at the stated pH with water and 200 mM MES adjusted with either NaOH or HCl as needed, and incubating them for 30 min at the stated pH. Prothymosin  was recovered as noted. Alternatively, histidine-tagged prothymosin  was isolated from transfected, [³²P]orthophosphate-labeled COS cells by nickel-nitrilotriacetic acid column chromatography using 0.5 ml of matrix and instructions from Qiagen. The purified, tagged [³²P]prothymosin  was added to unlabeled cells that were lysed in MES. After 1 h at 0 °C at one of several pH values, prothymosin  was purified. When the fate of purified [³²P]prothymosin  was assessed in defined solutions, it was dissolved in MES at the stated pH, refrigerated overnight, and recovered using phenol. All samples were evaluated electrophoretically and quantified as described above. One to two dishes of cells were used for each assay.

Phosphorylation of Prothymosin  Molecules in Vitro

Extracts of 2.5 × 10⁶ COS cells in 100 µl of the standard buffer were supplemented with 30 µg of recombinant bacterial prothymosin  and 300 µCi/ml [-³²P]ATP (Amersham, 3000 Ci/mmol) either in the presence or absence of 10 mM unlabeled ATP. The solutions were incubated for 30 min at 37 °C to allow labeling of exogenous prothymosin  to occur. Prothymosin  was recovered by means of a phenol extraction.

To test for transfer of phosphate from one prothymosin  molecule to another, two 60-mm dishes of cells were mixed and lysed together. One dish contained untransfected cells labeled with 100 µCi/ml of [³²P]orthophosphate for 4 h, whereas the other dish contained cells expressing the transiently transfected gene coding for prothymosin  with six carboxyl-terminal histidine residues. The standard buffer was used for lysis. Prothymosin  proteins were recovered as described for phosphoprothymosin  above and analyzed electrophoretically.

Reductive cleavage of prothymosin  with NaBH₄ was carried out with 2 × 10⁷ COS cells, which were washed with phosphate-buffered saline, recovered by centrifugation, and lysed in 1 ml of 10 mM Tris-HCl at pH 7.5, 30 mM EDTA, and 1.0% Triton X-100 made 0.1 M in NaBH₄. Alternatively, lysis was achieved in 2 ml of Me₂SO containing 30 mM [³H]NaBH₄ (specific activity 1000 mCi/mmol; NEN Life Science Products) at a specific activity of 47 mCi/mmol (low specific activity method) or in 1 ml of 5-mCi [³H]NaBH₄ (222 mCi/mmol) in Me₂SO (high specific activity method). Reactions were carried out for 1-3 h. Aqueous samples were diluted with 4 volumes of 0.5% SDS, and aprotic samples composed of precipitated protein in addition to unwanted insoluble debris were either dissolved in 6 ml of ACE buffer made 0.5% in SDS or washed with Me₂SO several times and dissolved in 3 ml of the same ACE/SDS solution. Prothymosin  was recovered by means of several phenol extractions. In some experiments, cells were labeled with [³²P]orthophosphate for 4 h in order to incorporate radioactivity into prothymosin  before reducing with borohydride.

Purification and Endoproteolytic Digestion of Prothymosin

Prothymosin  was purified to homogeneity in a three-step process consisting of phenol extractions of postnuclear supernatants, DEAE column chromatography, and C-18 reverse phase column chromatography. For purifications involving DEAE, samples in 400 µl of deionized water were injected onto a TosoHaas TSK-gel DEAE-5PW, 7.5 mm inner diameter × 7.5 cm column and eluted at a flow rate of 1 ml/min with a gradient of 0-500 mM NaCl in 20 mM Tris-HCl at pH 7.5 using the Waters HPLC system described previously (16). Prothymosin  was recovered as a single peak at 330 mM NaCl. Further purification was carried out by C-18 reverse phase column chromatography (16). Prothymosin  eluted as a sharp peak at 30% acetonitrile; the protein was collected and dried under vacuum with a Speed Vac (Savant Instruments). When [³H]samples were purified, radioactivity was determined in fractions collected in Hydrofluor (National Diagnostics) and assayed in a Packard Tri-Carb model 1500 liquid scintillation analyzer.

Peptides were generated from ~60 µg of prothymosin  with Lys-C and purified by C-4 reverse phase column chromatography essentially as detailed by Sburlati et al. (16). The stated digestion buffer was replaced with 0.1 M Tris-HCl at pH 7.5 made 5% in acetonitrile. Further digestion of the peptides generated with Lys-C was performed with the proteolytic enzyme, Asp-N, in 50 mM sodium phosphate at pH 8 containing 5% acetonitrile for 2 h at 37 °C at an enzyme:peptide ratio of 1:100. The Asp-N peptides were purified using the same system employed for the Lys-C peptides. The enzymes were purchased from Boehringer Mannheim.

Amino acid analysis was performed using the Pico-Tag methods developed by Waters. The steps include vapor phase acid hydrolysis for 22-24 h at 107 °C, evacuation of the samples, redrying with twice the recommended volume of ethanol:water:triethylamine, and precolumn derivatization with phenylisothiocyanate. Injection volumes were 15 or 60 µl for unlabeled samples and up to 160 µl for samples labeled with [³H]NaBH₄ or for the standards used to evaluate them (see below). Fractions were collected every 23 s and counted in Hydrofluor twice for 10 min as described above. Additional analyses made use of a Beckman 6300 amino acid analyzer equipped with an SP-4270 integrator (6).

Homoserine and homoserine lactone, the products of borohydride reduction of aspartyl phosphate (29) were purchased from Sigma and subjected to acid hydrolysis as well as redrying and derivatization. Hydroxynorvaline, obtained similarly from glutamyl phosphate (29), was synthesized by deaminating polyornithine with NaNO₂ using reactions described by Malin et al. (30). After acid hydrolysis of the treated polyornithine, the initial products are hydroxynorvaline and ornithine. However, because incubation in acid converts hydroxynorvaline to chloronorvaline (30) and because chloronorvaline cyclizes to form proline in base (31), there are at least three amino acid products, depending on the pH. Using Pico-Tag chemistry and Waters chromatography equipment as noted above, three amino acids were obtained. Ornithine was identified by hydrolyzing polyornithine in acid and finding one peak eluting near lysine on the amino acid analysis column; proline was characterized both by its elution time and by mass spectrometry; the remaining peak was subjected to mass spectrometry and found to be hydroxynorvaline. With Pico-Tag chemistry, which includes the coupling of phenylisothiocyanate in base, there was no chloronorvaline.

RESULTS

Stable Phosphate in Native and Mutant Prothymosin

The introduction of stable phosphate was investigated in COS cells using the native COS prothymosin  protein and the products of four transfected human prothymosin  genes: the wild type gene, a tagged variant encoding six histidine residues at the carboxyl terminus, a triple mutant in which Ser¹, Ser⁸³, and Thr⁸⁵ were replaced with alanine codons, and a histidine-tagged triple mutant. A gene map and amino acid sequence are presented in Fig. 1. The substitution of alanine residues for a subset of Ser/Thr eliminated all of the proven and suspected sites of stable phosphate in human prothymosin  (16), whereas the histidine tag made it possible to distinguish the endogenous protein, with its greater mobility, from exogenous tagged protein, which is slightly retarded when analyzed electrophoretically in polyacrylamide gels. As shown in Fig. 2, A and B (lanes 2-5), all of the transfected genes gave rise to approximately 10-fold more prothymosin  than found in mock transfected cells (Fig. 2A, lane 1) regardless of whether the total protein (indicated by Coomassie Blue staining (Fig. 2A)) or the newly synthesized protein (labeled with [³H]glutamic acid (Fig. 2B)) was examined. Furthermore, the ratio of stable phosphate to protein was the same for the endogenous and wild type transfected prothymosin  proteins (Fig. 2C, lanes 1 and 2), slightly diminished for the tagged wild type prothymosin  (lane 3), and greatly reduced for the mutant prothymosin  (lane 4) and for the tagged mutant protein (lane 5). Quantitatively, the tagged mutant prothymosin  contained 15% of the phosphate found in the tagged wild type molecule. From these observations, one can infer that the histidine tag has little effect on the amount of stable phosphate and that Ser¹ is important but not the sole site of phosphate incorporation. Among the possible explanations for the unexpected incorporation of phosphate in the absence of previously identified locations are 1) low level phosphorylation at heretofore unsuspected sites or 2) phosphorylation at primary sites common to both wild type and mutant prothymosin  proteins followed by the transfer of phosphate to a hierarchy of stable positions.

Phosphorylation of endogenous, wild type, and mutant prothymosin s in mock transfected and transfected COS cells.

The pH Stability of Prothymosin 's Phosphates

At the moment of cell lysis, prothymosin  should contain only those phosphates acquired in vivo. If phosphate attaches initially at Ser and Thr residues and persists, the phosphate should be resistant to changes in pH under gentle conditions regardless of their locations. Alternatively, if phosphorylation in vivo occurs on any other type of amino acid found in prothymosin , it might be possible to infer the nature of the phosphoamino acid bond from its stability as a function of pH. When prothymosin  in transfected COS cells was exposed to a range of lysis buffers at different pH values and subsequently purified using the standard methods, the specific activity of the [³²P]ectopic wild type protein or the ³²P-tagged triple mutant exhibited bell-shaped pH stability curves (Fig. 3A). Endogenous prothymosin  in untransfected cells and the tagged wild type protein behaved similarly (data not shown). Such behavior is typical of an acyl phosphate such as acetyl phosphate (32), aspartyl phosphate (33), or the phosphorylated residue of acetate kinase (see Ref. 34 and references therein). It is not characteristic of phosphoramidates (35). These data show that 1) the amount of stable phosphate obtained depends profoundly on the conditions of lysis; 2) at least 90% of the phosphate is labile as indicated by its susceptibility to hydrolysis at the extremes of pH; and 3) the shape of the pH stability curve does not substantively change in the presence or absence of introduced mutations that replace serine and threonine residues. Taken together, the data argue against Ser or Thr as the primary site of phosphorylation but, instead, support the idea of unstable phosphates whose transient survival influences the production of phosphoserine or phosphothreonine as a secondary event. Clearly, labile phosphate will either hydrolyze and remain undetected or transfer to more stable positions, where it can be analyzed.

The pH stability of phosphoprothymosin  immediately upon cell lysis, after incubation in crude cell lysates, and after purification to homogeneity.

Controls that support these conclusions make use of the data in Fig. 3B. Here, [³²P]prothymosin , which was purified to homogeneity and incubated in buffers from pH 3 to 11, was evaluated and found to be completely resistant to strong acid and base (solid line). Furthermore, purified [³²P]histidine-tagged prothymosin , when added to cells before lysis, incubated briefly in cell extracts prepared as in Fig. 3A, and subsequently purified by nickel chelate chromatography, also remained stable (dotted line). These controls show that at mild temperatures the stable phosphate of prothymosin  is unaffected by defined solutions at different pH values and by any of the components of complete cell extracts prepared over a broad range of pH. In the third curve (dashed line in Fig. 3B), the fate of [³²P]prothymosin , which was labeled in vivo, was determined after a brief delay at pH 7; lysates at neutrality were incubated 20 min on ice, diluted with each of a range of buffers to generate extracts comparable with those in Fig. 3A, and used for analysis of phosphoprothymosin . Again, the specific activity of prothymosin  was independent of pH. Labile phosphate in vivo, after a brief sojourn in extracts at pH 7, gave rise to phosphate that was stable in vitro throughout the pH range studied. These results emphasize the fact that phosphate on freshly isolated prothymosin  undergoes change and that the static conditions encountered when phosphoprothymosin  is purified to homogeneity do not reflect the properties immediately apparent upon cell disruption. It is worth reiterating that even at pH 7, where phosphate recovered on prothymosin  is maximal, only 2% of the total purified prothymosin  contains phosphate (16) and the amount of phosphate initially incorporated and then lost is unknown.

Phosphatases as the cause of the disappearance of prothymosin 's phosphates were also investigated. As illustrated in Table I, the specific activity of [³²P]prothymosin  labeled in vivo and purified remained unchanged regardless of whether a phosphatase inhibitor such as sodium fluoride, okadaic acid, or calyculin A was included in the lysis buffer. From these data, one suspects that the loss of labile phosphate occurs independently of phosphatases and, therefore, stems from an inherent property of the phosphorylated residue.

Table I. Effect of phosphatase inhibitors on the recovery of [32P]prothymosin  labeled in vivo Prothymosin  was isolated from cells labeled with [32P]orthophosphate in vivo and lysed in standard buffer in the presence of 1 mM okadaic acid, 0.5 mM calyculin A, or 50 mM sodium fluoride, where noted. Each row represents an experiment in which Coomassie Blue-stained protein and radioactivity in the same gel were quantified. [32P]Prothymosin /Prothymosin   Control Okadaic acid Calyculin A NaF 9.4 9.9 10.5 8.7 10.5 8.4 9.2 8.4

To tally prothymosin 's phosphates, one must consider phosphorylation in vivo as well as the acquisition or loss of phosphate occurring as artifacts during the purification procedure. Phosphorylation of prothymosin  was examined during isolation in the standard EDTA-containing buffer at pH 7. Extracts of COS cells supplemented with [-³²P]ATP either in the presence or absence of unlabeled ATP were unable to label prothymosin  (data not shown). EDTA appears to sequester the Mg²⁺ necessary for kinases utilizing ATP. In a slightly different format, ³²P-labeled endogenous prothymosin  labeled in vivo with [³²P]orthophosphate was tested as a phosphate donor; when the labeled COS cells were mixed and lysed together with unlabeled COS cells, which were transfected with the tagged prothymosin  gene, ³²P-labeled molecules including labeled prothymosin  were unable to transfer phosphate to an excess of tagged prothymosin  molecules (data not shown). Furthermore, in the accompanying paper (36), we searched for and failed to find kinases capable of phosphorylating serine 1 in cell extracts composed of whole cells or the isolated cytosol under a wide variety of conditions. These data are consistent with the idea that the phosphate on Ser¹ of prothymosin  is not acquired intermolecularly from cell extracts, that cells lack the capability of direct phosphorylation of serine 1, and that phosphate on serine or threonine is captured as a result of an intramolecular transfer from unstable to stable positions.

Identification of the Region of Prothymosin  Responsible for the Transfer of Phosphate

If labile phosphate is indeed transferred from a region of prothymosin  bearing acyl phosphates to its N-terminal portion, it should be possible to identify the acidic region and use it to distribute phosphate to stable positions in an irrelevant recipient. The experiment was performed by creating proteins composed of -galactosidase with all or part of the prothymosin  coding sequence (minus its own initiator codon) fused in frame upstream of the -gal gene but downstream of the AUG codon in pCH110 (5). A construct in which the SV40 nuclear localization sequence was inserted into the same position in the -galactosidase gene in the same vector was also made. When these genes were transfected into COS cells, it was evident that -galactosidase targeted to the nucleus by means of the SV40 nuclear targeting signal and -galactosidase bearing 51 amino acids from prothymosin 's amino terminus were poorly phosphorylated or unphosphorylated on stable positions (Table II). In contrast, the addition of either prothymosin  or the C-terminal portion of prothymosin  to the bacterial protein resulted in a substantial increase in stable phosphate. It is important to note that the C-terminal fragment of prothymosin , PTMA-(30-109), unlike the protein in its entirety, does not include the sites of stable phosphorylation found within the first 14 amino acids of prothymosin  but does include the nuclear localization signal. These data suggest that the acyl phosphates are located between amino acid 30 and the C terminus of prothymosin  (or, more restrictively, downstream of residue 51) and that the presence of this region is capable of conferring stable phosphate on an irrelevant protein in very close proximity.

Table II. Phosphorylation of -galactosidase fusion proteins -Gal was fused either to the SV40 nuclear localization signal (NLS) or to parts of the PTMA amino acid sequence as follows: PTMA-(1-51), the amino-terminal 51 amino acids of prothymosin , which include the preferred sites of stable phosphorylation located within the first 14 amino acids; PTMA-(30-109), the carboxyl terminus of prothymosin , which bears the nuclear localization signal and an extended acidic stretch; PTMA-(1-109), the entire prothymosin  amino acid sequence. Genes for the fusion proteins were transfected into COS-1 cells. -Galactosidase fusion proteins were isolated with the aid of specific antibodies, gel-purified, and quantified. The numbers, in arbitrary units, represent the amounts of stable phosphate incorporated into the fusion protein divided by the total amount of the fusion protein. Fused PTMA--gal genes were constructed from pCH110 and the prothymosin  cDNA (5), but, due to an unusual alternative splice site that adds one glutamic acid codon between codons 38 and 39 in 10% of prothymosin  transcripts (37), there are two numbering systems for amino acids. Here the extra codon is present, but unkeyed, in order to maintain consistency of the amino acid numbering system.  -Gal fusion partner Specific activity (32P-labeled fusion/total fusion) Exp. I Exp. II SV40 NLS <8.6a PTMA residues 1-51 0 <5.2a PTMA residues 30-109 7 22.4 PTMA residues 1-109 20 13.1 a Values recorded are overestimates because the optical density on the film was diffuse, unlike the sharp discrete bands exhibited by all fusion proteins on the stained gel and on the autoradiograms for -gal-PTMA-(1-109) and -gal-PTMA-(30-109).

Reductive Cleavage of Covalently Bound Phosphate on Prothymosin  by Sodium Borohydride

Aspartyl phosphate and glutamyl phosphate, but not phosphoserine, phosphothreonine, or phosphotyrosine, react with sodium borohydride to give altered amino acids that no longer contain phosphate (29). Such reactions can be carried out in an aqueous medium or in aprotic solvents such as Me₂SO. Since the phosphate on [³²P]prothymosin  rapidly disappears upon rupturing cells, it was not possible to purify a suitably phosphorylated protein before initiating treatment with sodium borohydride. Accordingly, [³²P]prothymosin  phosphorylated in vivo was studied in cells lysed either in aqueous solution or in Me₂SO, both with and without NaBH₄. Fig. 4 shows an electropherogram of Coomassie Blue-stained prothymosin  and the accompanying autoradiogram obtained under aqueous conditions. The presence of the borohydride had little effect on the recovery of prothymosin , compared with a sample isolated in the absence of the reagent (Fig. 4, left), but had a major effect on the retention of phosphate on prothymosin (Fig. 4, right). In quantitative terms, the specific activity ([³²P]prothymosin /total prothymosin ) was ~8-fold higher in the absence of borohydride than in its presence. Similar results were obtained in Me₂SO except that the untreated [³²P]prothymosin  had a specific activity 5-fold higher than the borohydride-reacted sample (data not shown). The results are consistent with the presence of acyl phosphates in prothymosin , which are displaced by borohydride.

Reductive cleavage of phosphoprothymosin  with sodium borohydride.

Estimation of the Number of Phosphorylated Acidic Residues in Prothymosin

The reaction of prothymosin  with borohydride yields informative products. 1) reductive cleavage of acyl phosphates by borohydride treatment generates the corresponding alcohols; 2) the alcohols and their acid hydrolysis products allow identification of the type of amino acid bearing the activated carbonyl group; and 3) tritium donated by [³H]NaBH₄ becomes incorporated into the protein at the location of the acyl groups (29). Cells containing unlabeled prothymosin  were lysed in the presence or absence of very high concentrations of sodium borohydride in aqueous solutions or necessarily lower concentrations in Me₂SO, and the products were scrutinized. After reduction with borohydride, prothymosin  was purified to homogeneity using phenol to remove other proteins, and two successive column chromatography steps (see "Experimental Procedures") to remove nucleic acids and other contaminants. The purified protein was hydrolyzed and subjected to amino acid analysis. Table III displays the results from different approaches. Using the Pico-Tag method to visualize amino acids, the number of acidic residues found in the absence of borohydride was always greater than that in its presence. There were 4-8 missing acidic residues in borohydride-reduced samples. The large number of acidic amino acids in prothymosin  and the proximity of the peaks for derivatized glutamate and aspartate made it difficult to determine which type(s) was missing. It was clear from many analyses that the Pico-Tag methods almost always underestimated the number of acidic residues in prothymosin  but that borohydride treatment always caused a reduction in acidic residues. The correct number of residues was obtained for all other amino acids, in particular lysine and arginine.

Table III. Effect of sodium borohydride reduction on the amino acid composition of prothymosin   COS cells were lysed either in an aqueous buffer or in Me2SO, each in the presence and absence of sodium borohydride. The numbers in the table represent the average number of residues found in replicate samples. Numbers in parentheses indicate the number of acidic residues missing after treatment with sodium borohydride. Samples were normalized using glycine; valine is shown as an example of an amino acid that should be unaffected by reduction. Analysis Solvent NaBH4 Amino acid residues after acid hydrolysis Glu/Gln + Asp/Asn Gly Val Pico-Tag H2O   57 9.0 4.0 Pico-Tag H2O + 49 (8)a 9.0 4.7 Pico-Tag Me2SO   64 9.0 4.7 Pico-Tag Me2SO + 60 (4)b 9.0 4.2 Ninhydrin H2O   60 9.0 3.7 Ninhydrin H2O + 56 (4)c 9.0 3.6 Theoretical 61 or 62 9 5 a Six replicates. b Four replicates. c Two replicates.

As a control, purified prothymosin  was also treated with borohydride. Prothymosin  was first purified to homogeneity (a process during which the labile phosphates disappear), reacted with NaBH₄, and repurified to homogeneity using the methods noted above. Amino acid analysis of these samples revealed no deficit in acidic residues relative to controls (data not shown). Hence the methods themselves are not responsible for the results. To corroborate these findings, we repeated the analyses of borohydride-treated and -untreated aqueous prothymosin  samples using the ninhydrin method to visualize amino acids. Here too, reduced prothymosin  was missing an average of four acidic residues in a reaction that did not underestimate the acidic residues in untreated protein. Taken together, the data suggest that there are a few to many phosphorylated acidic residues/molecule of prothymosin  and that other phosphorylated amino acids are not present at detectable levels. Because the borohydride must compete with water in the reaction, our methods can only underestimate the number of mixed anhydrides.

Treatment of Prothymosin  with [³H]NaBH₄

The reaction of prothymosin  in crude cell lysates was repeated using [³H]NaBH₄ in Me₂SO. When labeled borohydride was included in the cell lysis solution, tritium became incorporated into prothymosin  as well as into most other macromolecules. Tritium may be incorporated as a consequence of reduction, but in the vast majority of cases, radioactive protons generated during the reaction merely exchange with their nonradioactive counterparts. Thus, purification of prothymosin  was required not only to obtain pure protein but also to eliminate large amounts of exchangeable tritium from the products. With this goal, we designed the procedure shown in Fig. 5. Here phenol-extracted prothymosin  was partially purified on a DEAE-methacrylate copolymer column and visualized at 214 nm (Fig. 5A). Although the pattern appears to be complex, prothymosin  was easily recognized as the peak at 214 nm that did not have absorbance at either 260 (data not shown) or 280 nm due to the absence of aromatic residues. The chromatogram displays entities not usually seen in gels (see Fig. 2, A and B), because RNA, as well as salts and solvents, absorbs at 214 nm and acquires exchangeable tritium but does not stain with Coomassie Blue or label with [³H]glutamic acid. Prothymosin , indicated by the arrow, was purified to homogeneity using a C18 reverse phase column developed in acetonitrile (which also absorbs at 214 nm) (Fig. 5B). Although a large amount of tritium eluted at the front, radioactivity remained with prothymosin , an observation consistent with the reduction of acyl phosphates with [³H]borohydride.

Purification of [³H]NaBH₄-treated prothymosin  by sequential DEAE and C18 column chromatography using HPLC.

The reduced, tritiated prothymosin  described above was subjected to acid hydrolysis and amino acid analysis to characterize the labeled products. Toward this end, appropriate markers were obtained: homoserine and homoserine lactone, the expected reduction products of aspartyl phosphate, and hydroxynorvaline, the expected product of glutamyl phosphate. When subjected to the same reactions used for the analysis of proteins, homoserine and its lactone persist as products (Fig. 6A, dashed line), but hydroxynorvaline is converted to proline under the basic conditions of Pico-Tag derivitization (31) (Fig. 6A, thin line). Ornithine (Orn) (Fig. 6A, thin line) appears as a result of the incomplete deamination of polyornithine, the starting material for hydroxynorvaline synthesis (30). To verify that the peak at 6.5 min was indeed proline and that the peak at 5.75 min was hydroxynorvaline, identity was established by mass spectrometry (data not shown). A peak eluting at 10.7 min, diphenylthiourea, is produced by the reaction of phenylisothiocyanate with ammonia.

Amino acid analyses of [³H]NaBH₄-treated prothymosin  and standards using Pico-Tag technology.

Fig. 6A also displays the behavior of the radioactive products obtained from prothymosin  that was reduced with [³H]NaBH₄ in Me₂SO immediately as cell lysis occurred, purified to homogeneity, and hydrolyzed (thick solid line). There are two major peaks of radioactivity: a peak eluting at 8 min, which is composed in part of Tris and NH₃ but does not contain any known amino acid or any of the derivatives, and a smaller peak of radioactivity, which coelutes with proline. The presence of labeled proline following the reductive cleavage of prothymosin  with tritiated sodium borohydride strongly suggests that prothymosin  initially contained -glutamyl phosphate.

To demonstrate that the radioactivity associated with proline was incorporated specifically, rather than by nonspecific exchange of [³H]H₂O with ionizable hydrogen ions in the protein, a sample of purified prothymosin  was used as a control. Purified prothymosin  does not contain acyl phosphates, based on the pH stability studies shown in Fig. 3, and should not generate [³H]proline upon treatment with [³H]borohydride. Cells were lysed in Me₂SO/[³H]borohydride, and in the same experiment the homogeneous protein was suspended in a Me₂SO/H₂O/[³H]borohydride mix to duplicate the conditions encountered with crude samples. The amino acid analysis of the reduced, purified protein (Fig. 6B, dashed line) gave rise to a pattern almost identical to that of the crude prothymosin  (solid line) with one exception; radioactivity in proline was absent from hydrolyzed prothymosin  that had been purified to homogeneity first and only later treated with the reducing agent. Hence, radioactivity associated with proline was specifically incorporated into prothymosin  when the reaction with borohydride occurred immediately upon cell lysis. Our data suggest that -glutamyl phosphate is present on prothymosin  in vivo but disappears during the subsequent scheme of purification.

Peptides derived from [³H]borohydride-reduced fresh prothymosin  were prepared by treating the isolated protein with endopeptidases, purifying the products, and subjecting the peptides to amino acid analysis. As Table IV illustrates, all of the radioactivity was found in a single Lys-C peptide composed of residues 21-87; other Lys-C peptides were not radioactive. However, of the 34 glutamic acid residues in prothymosin , Glu¹⁸ and Glu¹⁰⁷ were not included in peptides of sufficient size to be retained by the HPLC column and were not pursued. Upon further digestion of Lys-C-(21-87) with Asp-N, four major products, Asp-N-(31-87), Asp-N-(21-30), Asp-N-(31-47), and Asp-N-(48-87), were obtained, purified, and identified by amino acid analysis (Table IV). Further characterization of these peptides indicated that residues 21-30 contained an occasional glutamyl phosphate, which, because of poor labeling, was difficult to quantify; residues 31-47 did not include a phosphorylation site; and residues 48-87 harbored virtually all of the radioactivity incorporated specifically into prothymosin  (Table IV). This region conforms precisely to that tentatively identified by the experiments with -galactosidase fusion proteins in Table II, i.e. it is an acidic region located downstream of amino acid 51 of prothymosin .

Table IV. Location of glutamyl phosphates Prothymosin  was treated with [3H]borohydride in dimethylsulfoxide at the moment of cell lysis. Peptides were purified and analyzed by acid hydrolysis and amino acid analysis. All radioactivity incorporated was found in the peptide Lys-C-(21-87) shown below. Lys-C peptides in which no radioactivity was found are included only to emphasize the absence of label. Lys-C-(21-87) was further digested with Asp-N. Major peptides were purified to homogeneity and analyzed. Values represent the dpm in tritium, with background subtracted, found in proline in the stated peptide upon amino acid analysis. Sp. Act. refers to the specific activity/mol of peptide. Carrier prothymosin  present only in experiments I and II was not included in specific activity calculations. BKG, background; NA, not applicable. , the peptide was produced in the experiment but not analyzed. Peptide Disintegrations/min in proline Sp. Act. Exp. I Exp. II Exp. III Exp. I Exp. II Exp. III dpm mCi/mol Lys-C-(21-87) 310 1120 50 80 Asp-N-(31-87) 330 300 160 50 Asp-N-(21-30) 20   2   Asp-N-(31-47) BKG   NA   Asp-N-(48-87) 170a 560 120a 80 Lys-C-(1-14)b BKG   NA   Lys-C-(88-101) BKG   NA   a Corrected value based on analysis of approximately one-third of the total quantity of peptide. b Value corrected for contamination by Lys-C-(21-87) as indicated by the presence of substoichiometric amounts of arginine in the hydrolysate.

The chromatogram of the amino acid analyses of four representative peptides is illustrated in Fig. 7. Tritiated Lys-C-(21-87), Asp-N-(31-87), and Asp-N-(48-87) each contain [³H]proline as the sole radioactive amino acid, whereas the fourth peptide in Fig. 7, Lys-C-(88-101), was devoid of [³H]proline. As the peptides underwent purification, radioactivity in the unidentified peaks at the beginning and end of the chromatogram become minor, and the peak at 8 min, attributed primarily to contaminating Tris, also became less pronounced relative to proline. (Compare the amino acid analysis of the intact protein in Fig. 6A with the patterns in Fig. 7.) It is worth reiterating that this peak does not overlap with any known amino acid or derivative and cannot be assigned to spurious products generated from putative phosphoramidates, because arginine and lysine are not found in two of the peptides analyzed in Fig. 7. Because experiment III (Table IV) did not include carrier, it was used to compare the specific activities of radioactivity incorporated per mole of peptide; the specific activity of the large Lys-C peptide from 21 to 87 was nearly identical to that of the smaller Asp-N-(48-87) peptide. Therefore, it is unlikely that significant radioactivity occurred elsewhere in the protein. The presence of glutamyl phosphate in this highly acidic region of prothymosin  is notable because the presumed histone binding region of nucleoplasmin, a chromatin remodeling protein found in the germinal vesicle of X. laevis, is almost identical in sequence.

Amino acid analyses of peptides of [³H]NaBH₄-treated prothymosin  using Pico-Tag technology.

DISCUSSION

Prothymosin  contains acyl phosphates based on the following five observations. 1) Mutant human prothymosin  molecules lacking Ser¹, the sole confirmed repository of phosphate, continued to become phosphorylated. Because prothymosin  in solution is unfolded (38), the data cannot readily be understood by invoking cryptic sites of phosphorylation in mutants. Rather, our data fit a model in which both mutant and wild type prothymosin  molecules initially acquire phosphate at the same site and subsequently transfer it to a stable location. 2) The pH stability curves of fresh [³²P]prothymosin  and the histidine-tagged mutant were similar to the bell-shaped curve exhibited by acetyl phosphate (32). Neither phosphoserine and phosphothreonine, which are resistant to base at mild temperatures and stable in acid, nor the phosphoramidates (lysyl and arginyl phosphate) demonstrate such behavior (35). 3) A short incubation of crude solutions at pH 7 changed the pH stability curve of the phosphate in [³²P]prothymosin  from the pattern expected for an acyl phosphate, with rapid hydrolysis at the extremes of pH, to that displayed by serine or threonine phosphate. Loss of phosphate was unaffected by the presence of a series of phosphatase inhibitors, suggesting that phosphate instability is an inherent property. From these experiments, it can be argued that the amount of stable phosphate found in prothymosin  depends heavily on the lysis conditions and that larger amounts of stable phosphate are recovered under conditions that favor the survival of unstable phosphate. 4) Reductive cleavage of prothymosin  with sodium borohydride diminished the amount of radioactive phosphate attached to prothymosin  by 5-8-fold and reduced the number of acidic residues observed upon acid hydrolysis by 4-8. Since borohydride is unreactive toward phosphate esters, the data rule out phosphoserine or phosphothreonine as significant components of in vivo phosphorylation events. Instead, the disappearance of phosphate and the deficit in acidic residues after hydrolysis point to the presence of activated carbonyl groups bearing phosphate as an integral part of prothymosin . 5) When [³