A 180-kDa Protein Kinase Seems to Be Responsible for the Phosphorylation of Prothymosin alpha  Observed in Proliferating Cells*

(Received for publication, October 11, 1996)

Antonio Pérez-Estévez , Cristina Díaz-Jullien , Guillermo Covelo , M. Teresa Salgueiro and Manuel Freire Dagger

From the Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Santiago, Santiago de Compostela, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Prothymosin alpha  (ProTalpha ) is an acidic protein involved in cell proliferation. Its phosphorylation status is correlated with proliferative activity. Here we report the isolation and characterization of a ProTalpha -phosphorylating kinase (ProTalpha K) from mouse splenocytes that seems to be responsible for the in vivo phosphorylation of ProTalpha and that differs from other protein kinases reported to date. This enzyme, mainly located in the cytosol, has an molecular mass of 180 kDa and appears to be made up of two proteins of 64 and 60 kDa. Its activity was markedly enhanced by mitogenic activation of cells. The ProTalpha residues phosphorylated by the enzyme in vitro are a Thr at position 7 and another Thr at positions 12 or 13, both located within casein kinase 2 (CK-2) consensus motifs; these are the same residues as are phosphorylated in vivo. The new enzyme shows a number of clear structural and catalytic differences from CK-2. It phosphorylates histones H2B and H3, although with weaker activity than ProTalpha . An enzyme with the same characteristics was also found in other murine tissues and cell lines.


INTRODUCTION

Prothymosin alpha  (ProTalpha )1 is a polypeptide comprising 109-111 amino acid residues, according to protein (1), cDNA (2, 3), and genome (4) analysis. Its sequence is highly conserved (5) and includes an extensive central acidic region (residues 41-85) comprised of Glu and Asp residues. Under physiological conditions, ProTalpha behaves as a monomeric protein with a random coil conformation (6, 7). It is widely distributed in mammalian tissues and particularly abundant in lymphocytes (8-10).

The changes over time in the levels of ProTalpha (11) and ProTalpha mRNA (12-14) observed in dividing cells suggest that its function is related to cell proliferation. This is in agreement with the efficiency of antisense oligonucleotides complementary to ProTalpha mRNA for paralyzing cell growth (15). Furthermore, the proposed implication of the c-Myc protein in the transcriptional regulation of the ProTalpha gene (16-18), although controversial (19), would support this hypothesis.

Although the precise function of ProTalpha remains unclear (for review, see Ref. 20), there is strong evidence to suggest a nuclear role: its C terminus region bears a karyophilic signal (21) and ProTalpha migrates to the nucleus in proliferating cells (22, 23). Recent work in our laboratory has shown that ProTalpha binds histones and cooperates in nucleosome assembly in vitro (24), suggesting that the putative nuclear function may be related to chromatin remodeling; this possibility is consistent with the structural similarities between ProTalpha and various nuclear proteins known to be involved in chromatin activity (25, 26).

Phosphorylation of ProTalpha has recently been reported by us (27) and corroborated by other authors (28). This post-translational modification constitutes an additional clue to its biological role. The available data indicate that the extent of phosphorylation of ProTalpha is positively correlated with cell proliferation activity (27, 28). The phosphorylation sites are included in the first 14 amino acids of the sequence: however, there is controversy as to whether the residues which undergo phosphorylation are Thr residues (our findings; Ref. 27), or Ser residues (Sburlati et al. (28)).

Regardless of whether the residues phosphorylated are Thr or Ser, the N-terminal sequence of ProTalpha (AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys) suggests that the in vivo phosphorylation sites are casein kinase 2 (CK-2) consensus sites (29). This is in accordance with our previous results which show that CK-2 is able to phosphorylate the 14-residue N-terminal fragment of ProTalpha in vitro (30). However, phosphoamino acid analysis showed that in vitro phosphorylation with CK-2 leads to phosphorylation of both Thr and Ser residues, in approximately equal proportion.

In the work reported here, we aimed to characterize the enzyme responsible for the phosphorylation of ProTalpha in proliferating cells. At the same time, we set out to resolve the controversy as to which amino acid residues undergo phosphorylation. Fractionation of cell extracts by affinity chromatography on a ProTalpha -Sepharose column and then by ion-exchange HPLC yielded a protein kinase, different from CK-2, which phosphorylated the N-terminal 14-mer of ProTalpha . Analysis of the sites phosphorylated both in vivo and by the purified enzyme in vitro confirmed that the residues phosphorylated in the N-terminal peptide of ProTalpha are Thr residues, and indicate that the purified enzyme is probably responsible for the phosphorylation of ProTalpha observed in vivo.


EXPERIMENTAL PROCEDURES

Materials

The triethylammonium salts of adenosine 5'-[gamma -32P]triphosphate ([gamma -32P]ATP, 3000 Ci/mmol) and guanosine 5'-[gamma -32P]triphosphate ([gamma -32P]GTP, 5000 Ci/mmol) were purchased from Amersham International. [32P]Orthophosphate (1 Ci/mmol) was from DuPont NEN. TPCK-treated trypsin, alkaline phosphatase from bovine intestinal mucosa, poly-L-lysine hydrobromide, dephosphorylated beta -casein from bovine milk, protamine (grade IV) from salmon, and concanavalin A were from Sigma. Thin-layer cellulose plates (0.1 mm) were from Merck. Interleukin-2, DNase I, RNase I, histones, the artificial casein kinase II substrate RRRDDDSDDD and protease V8 (sequencing grade) from Staphylococus aureus were obtained from Boehringer Mannheim. Antibodies to human casein kinase II were obtained from Upstate Biotechnology Inc. and casein kinase II from Promega. ProTalpha was purified from calf thymocytes as described (10). All other reagents and materials were of analytical grade.

Methods

Cell Culture and Subcellular Fractionation

Splenocytes, thymocytes, and hepatocytes were obtained from 45-day-old female BALB/c mice. Cells were isolated by pressing a suspension of the freshly excised and minced tissue in RPMI 1640 medium through a 65-µm mesh stainless steel screen. In all cases the resulting suspension was treated with erythrocyte lysis buffer (0.75% NH4Cl, 0.21% Tris-HCl, pH 7.2) and washed twice in RPMI 1640 before use. Splenocytes and thymocytes (5 × 106 cells/ml) were grown for various times in RPMI 1640 containing 10% fetal calf serum, 100 units ml-1 of penicillin, and 100 µg ml-1 of streptomycin, in the presence or absence of concanavalin A (2.5 µg ml-1) and interleukin-2 (10 units ml-1), in a humidified 5% CO2 atmosphere at 37 °C. Cell viability was determined by trypan blue exclusion.

HeLa and NC37 cells were grown in minimum Eagle's medium or RPMI 1640, respectively, at 37 °C in a humidified CO2 atmosphere. In both cases the medium contained 10% fetal calf serum, 100 units ml-1 of penicillin, and 100 µg ml-1 of streptomycin.

Subcellular fractionation of the different cell types was as follows. Cells were collected from culture, washed twice, suspended (about 5 × 107 cells/ml) in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, 0.5 mM dithiothreitol, 5 mM EGTA, 1 mM EDTA, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 10 µg/ml leupeptin), incubated on ice for 10 min, homogenized (10 strokes) in a Potter Teflon glass blender, then centrifuged (2000 × g for 10 min) to yield pellet I (nuclei). The supernatant was ultracentrifuged (110,000 × g for 1 h at 4 °C) to yield pellet II (microsomes) and a supernatant which was dialyzed against buffer A (50 mM Tris-HCl, pH 7.5, containing 5% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) to yield the cytosol fraction. Pellet II was suspended in buffer A containing 0.5 M NaCl and 0.5% sodium deoxycholate, incubated at 0 °C for 10 min, and centrifuged at 20,000 × g for 15 min: the supernatant was then dialyzed against buffer A to yield the microsomal fraction. Nuclei were suspended (about 2 × 108 nuclei/ml) in buffer B (10 mM Tris-HCl, pH 7.4, containing 0.1 mM MgCl2, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) supplemented with 80 µg/ml DNase I and 10 µg/ml RNase I, incubated at 0 °C for 1 h, and then centrifuged at 20,000 × g for 15 min at 4 °C. The supernatant was dialyzed against buffer A to yield the nucleoplasm fraction. The pellet was suspended in buffer B containing 0.5 M NaCl, incubated for 5 min at 0 °C, and centrifuged at 20,000 × g for 15 min; the supernatant was then dialyzed against buffer A to yield the nuclear envelope fraction. All fractions were either used immediately or stored at -70 °C until use.

Isolation and Purification of the ProTalpha Protein Kinase

The various subcellular fractions were chromatographed on ProTalpha -Sepharose columns as described previously (24). Briefly, the fraction under study, in buffer A, was first passed through a bovine serum albumin-Sepharose column (1 mg of bovine serum albumin/ml of Sepharose) at a ratio of about 10 mg of protein per ml of bovine serum albumin-Sepharose matrix. The flow-through fractions were then slowly loaded onto a 1-ml column of ProTalpha -Sepharose (0.8 mg of ProTalpha per ml of Sepharose). The column was then sequentially eluted with 0.3 M NaCl and with 1 M NaCl in buffer A. The flow-through fraction and the two eluate fractions were dialyzed against buffer A and concentrated to final volumes of 0.5 ml. ProTalpha kinase activities of the different concentrates were then assayed as described below. All operations were carried out at 4 °C.

To further purify components in the affinity chromatography fractions with ProTalpha kinase activity, these fractions were loaded onto a Spherogel-TSK DEAE-SPW column (7.5 × 75 mm) previously equilibrated with buffer A, in a Beckman Gold HPLC system. The column was developed for 10 min with buffer A and then eluted with 0-0.8 M NaCl (linear gradient) in the same buffer. Flow rate was 0.5 ml/min and fractions were collected every minute. ProTalpha kinase activity was assayed in aliquots of each fraction as described below. Fractions with the highest ProTalpha kinase activity were collected and concentrated at final volumes of 0.5 ml in a Centricon 10 and stored at 4 °C or -70 °C. Protein concentrations were determined by the Bradford assay (31).

Isolation and Purification of 32P-ProTalpha from Metabolically Labeled Cells

32P-Labeled ProTalpha was obtained from proliferating cells following modification of the procedure of Haritos et al. (32), as we have described previously (27). Briefly, splenic lymphocytes activated with concanavalin A and interleukin-2 were incubated with [32P]orthophosphate (50 µCi/ml), then frozen in liquid nitrogen, powdered, and suspended in boiling 0.15 M NaCl. After cooling, the suspension was centrifuged and the supernatant brought to pH 2.5, clarified by centrifugation, made up to 50 mM Tris-HCl (pH 8) and 70 mM NaCl, and applied to a DEAE-cellulose column, which was eluted with 3 volumes of loading buffer (50 mM Tris-HCl, pH 8) containing 0.2 M NaCl, followed by 6 volumes containing 0.5 M NaCl. The components eluted with the latter buffer were purified by reverse-phase HPLC. Phosphorylated ProTalpha , which co-purified with calf ProTalpha , was characterized by SDS-PAGE and isoelectric focusing, and on the basis of immunoreactivity with an antibody raised against the first 28 amino acids of ProTalpha (27). This procedure has previously been shown by us (10) and by others (5, 9) to yield 43-130 µg of ProTalpha /g of cell, depending on tissue. In the present study, the yields of radioactive ProTalpha after HPLC purification were about 4 × 105 cpm/108 mitogen-activated splenic lymphocytes.

Peptide Mapping and Phosphoamino Acid Analysis

Peptide mapping on the basis of tryptic digestion of 32P-labeled phosphorylated ProTalpha , and phosphoamino acid analysis of the ProTalpha fragments were as described (27). Briefly, 32P-ProTalpha , either purified from metabolically labeled cells or from kinase-assay reaction mixtures, was digested with TPCK-trypsin for 10 h at 37 °C. Tryptic peptides were then separated by reverse-phase HPLC. Aliquots of the radioactive peak coeluting with the 14-residue N-terminal fragment of calf ProTalpha were then subjected to phosphoamino acid analysis, by hydrolysis with 6 N HCl, mixing with nonradioactive phosphoamino acid standards, thin-layer electrophoresis, and autoradiography. Alternatively, aliquots of the radioactive peak (of the HPLC-separated tryptic digests, whether from metabolically labeled cells or from kinase-assay reaction mixtures) were further digested with endopeptidase V8 (Boehringer Manheim) (protease:peptide ratio 1:10) in 25 mM ammonium carbonate (pH 7.8). The resulting peptides were separated by reverse-phase HPLC in an Ultrasphere-ODS column (5 µm, 4.6 × 250 mm), and phosphoamino acid analysis was performed as above. The amino acid compositions of the peptides derived from V8 digestion of the N-terminal fragment were determined after acid hydrolysis in a Biochrom 20 analyzer (Pharmacia).

Kinase Activity Assays

Assays of the ProTalpha phosphorylating activity of cell extracts or rat liver CK-2 were performed in 25-µl reaction mixtures containing 50 mM Tris-HCl (pH 7.4), 150 mM KCl, 26 mM MgCl2, 1.6 mM EGTA, 1 mM EDTA, 3.3 mM dithiothreitol, 80 ng/ml protamine, 83 mM beta -glycerol phosphate, and 100 µM [32P]ATP. Dephosphorylated calf thymus ProTalpha (5 µg), or that indicated under "Results," was used as substrate. After 30 min at 37 °C, the reaction was stopped with 5 µl of 100 mM ATP. Activity was quantified (a) by separating the components of the reaction mixture by SDS-PAGE (33), with autoradiography to estimate 32P-ProTalpha concentration and kinase activity subsequently expressed in arbitrary units, or (b) by separating the components by reverse-phase HPLC (as described above), then quantifying the radioactivity co-eluting with ProTalpha , with activity expressed as amount of 32P incorporated into the substrate. Assays of immunoreactivity with antibodies raised against the first 28 amino acids of ProTalpha (see above) were also used to characterize 32P-ProTalpha in the various reaction mixtures.

Immunodepletion assays were carried out in 50-µl aliquots of reaction mixture buffer, without ATP, containing 30 µl of the purified kinase and different concentrations of antibody to the alpha  subunit of CK-2. The mixture was incubated overnight at 4 °C with gentle shaking. Forty µl of Sepharose-bound protein A in reaction mixture buffer was then added, and the mixture gently shaken for 2 h at 4 °C. Immunocomplexes were collected by centrifugation and kinase activity was assayed in the supernatant after adding 100 µM [32P]ATP.


RESULTS

ProTalpha Phosphorylating Activity in Splenic Lymphocytes

Phosphorylation of ProTalpha is particularly marked in mitogen-activated splenic lymphocytes, as compared with non-activated lymphocytes and other proliferating cell types (27, 28). We thus searched for the ProTalpha kinase (ProTalpha K) in extracts of these cells. The first step was to isolate cellular components which show affinity for ProTalpha by using a ProTalpha -Sepharose column that we have previously shown to be effective for this purpose (24). ProTalpha phosphorylation activity in the various affinity chromatography fractions was assayed under conditions equivalent to those used for assaying CK-2 activity, since the in vivo phosphorylation sites of ProTalpha are similar to those of this enzyme (27, 28). Subcellular fractions (cytosol, nucleoplasm, microsome, and nuclear envelope) from mitogen-activated splenic lymphocytes were chromatographed on ProTalpha -Sepharose columns and ProTalpha phosphorylation activity was assayed in the different eluates. As shown in Fig. 1, components with high affinity for ProTalpha -Sepharose (lanes 4) in the cytosol (Fig. 1A), nuclear envelope (Fig. 1B), and nucleoplasm (Fig. 1C) fractions all showed ProTalpha phosphorylation activity, as judged by the radioactivity migrating with ProTalpha in the SDS-PAGE analysis, whereas no such activity was observed when ProTalpha was omitted from the reaction mixtures (Fig. 1, lanes 3). Components obtained from the microsome fraction showed no detectable ProTalpha phosphorylation activity (data not shown). Affinity chromatography appears to be efficient for isolating the ProTalpha phosphorylating activity, since no phosphorylated product migrating with ProTalpha was detected in the flow-through fractions (Fig. 1, lane 1), and since the phosphorylated product was absent (Fig. 1, B and C, lanes 2) or present in only low concentration (Fig. 1A, lane 2) in the moderate-affinity fractions. ProTalpha phosphorylation activity was imperceptible in the various crude subcellular fractions (not shown).


Fig. 1. ProTalpha phosphorylation activity in subcellular fractionates of mitogen-activated splenic lymphocytes. Subcellular fractionates of splenic lymphocytes (2 × 108 cells), previously activated with concanavalin A plus interleukin-2, were chromatographed on ProTalpha -Sepharose columns, the different eluate fractions concentrated to 0.5 ml and aliquots (10 µl) of these fractions assayed for kinase activity in the presence or absence of ProTalpha (5 µg) as described under "Methods." The figure shows autoradiographs of the gels on which reaction mixture components were separated by SDS-PAGE. Reaction mixtures contained the flow-through fraction (lanes 1), the fraction eluted with 0.3 M NaCl (moderate affinity; lanes 2), or the fraction eluted with 1 M NaCl (high affinity; lanes 4). Lanes 3 were loaded with reaction mixtures made up in the same way as lanes 4, except that ProTalpha was omitted. Arrows mark the position of dephosphorylated ProTalpha . Similar patterns were observed in similar fractions from four different subcellular extracts.
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Similar results were obtained when an HPLC-based assay (see "Methods"; results not shown) was used for determination of ProTalpha phosphorylation activity in the different fractions. That the radioactive peaks co-eluting with calf ProTalpha in HPLC were indeed radiolabeled ProTalpha was confirmed by immunoprecipitation with the anti-28-mer antibody (see "Methods"; results not shown).

Densitometric scanning of the gels shown in Fig. 1 indicates that ProTalpha phosphorylation activity is highest in the high-affinity fraction (HAF, lane 4) of the cytosol extract. Activities in the HAFs of the nuclear envelope and nucleoplasm extracts were about 25 and 5.9%, respectively, of that in the HAF of the cytosol extract. Activity in the moderate-affinity fraction of the cytosol extract was about 4.8% of that in the HAF of the cytosol extract. Total protein concentrations were 15, 9, and 4 µg/108 cells in the HAFs of cytosol, nucleoplasm, and nuclear envelope extracts, respectively.

ProTalpha phosphorylation activity in the HAF of the cytosol extract from non-activated splenic lymphocytes was about 14% of that in the HAF of the cytosol extract from mitogen-activated splenic lymphocytes (Fig. 2). As is also shown in Fig. 2, activity in the HAF of the nuclear envelope extract from non-activated cells was about 35% of that in this fraction from activated cells, whereas activity in the HAF of the nucleoplasm extract from non-activated cells was scarcely detectable.


Fig. 2. Effect of mitogenic activation of splenic lymphocytes on ProTalpha phosphorylation activity. ProTalpha phosphorylation activity was assayed in 10-µl aliquots of the concentrated (0.5 ml) high-affinity fraction obtained in ProTalpha -Sepharose chromatography from subcellular extracts of 3 × 108 splenic lymphocytes previously stimulated with concanavalin A plus interleukin-2 or unstimulated (see "Methods"). Bars show kinase activities in arbitrary units, as deduced by densitometric scanning of the radioactive bands comigrating with ProTalpha (lower panel) in autoradiographs of SDS-PAGE gels in which the corresponding reaction mixtures had been run. Each value is the average of activities determined in three different assays; for each fraction, replicate values differed by less than 15%.
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Taken together, these results indicate that the ProTalpha K activity in splenic lymphocytes is influenced by proliferation status and is largely contained in the cytosol. Some activity is contained in nuclear envelopes, while activity in the nucleoplasm is negligible. To characterize this activity, we further purified components of the cytosol, nuclear envelope, and nucleoplasm extracts of activated splenic lymphocytes with affinity for ProTalpha -Sepharose by ion-exchange HPLC. ProTalpha phosphorylation activity in the HAFs of the cytosol and nuclear envelope fractions showed different elution patterns, but was in both cases concentrated in single clearly defined peaks (Fig. 3A). Kinase activity eluting at the same position as that in the HAFs from cytosol was scarcely detectable either in the eluate fractions obtained by ion-exchange HPLC of the HAFs of the nucleoplasm fraction or in eluate fractions obtained by ion-exchange HPLC of moderate-affinity fractions (see Fig. 1A, lane 2) from cytosol (result not shown). The specific activity of the HPLC-purified ProTalpha K from cytosol extract was about 300-fold higher than in the extract prior to HPLC. No such assessment of the effectiveness of the second purification step is possible for the nuclear envelope extract, since protein content in the peak-activity HPLC fraction was below detection limits. Gel filtration (Fig. 3B) indicated that the HPLC-purified kinase from the cytosol extract has a molecular mass of about 180 kDa, while the molecular mass of the kinase from the nuclear envelope extract was about 130 kDa.


Fig. 3. Chromatographic separation of ProTalpha binding components in subcellular fractionates of splenic lymphocytes. Components in the subcellular fractionates showing high affinity for Sepharose-ProTalpha were further separated first by ion-exchange HPLC and then by gel filtration. Panel A shows the elution pattern of the ProTalpha kinase activity (PK) separated by ion-exchange HPLC from the high-affinity fractions (0.4 ml) of the indicated subcellular fractionates from 4 × 108 mitogen-stimulated splenic lymphocytes, in the conditions described under "Methods." ProTalpha phosphorylation activities were assayed in 20-µl aliquots of each HPLC eluate fraction, and are shown in the figures as arbitrary units (determined as indicated in the legend to Fig. 2). The two fractions with the highest activity were collected and concentrated to 0.3 ml. Panel B shows the results of separation by gel filtration on a Sephacryl S-200 column (40 × 1.2 cm) of 0.1-ml aliquots of the maximum activity fractions from ion-exchange HPLC. The column was equilibrated and eluted with 50 mM Tris-HCl (pH 7.6), containing 5% glycerol at a flow rate of 12 ml/h. ProTalpha phosphorylation activity was assayed in 20-µl aliquots of each 1-ml fraction collected, and is represented as in panel A. Positions of protein markers are indicated.
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Identification of Sites of Phosphorylation

To investigate whether the purified kinases are responsible for the phosphorylation of ProTalpha observed in vivo, we performed experiments aimed at more detailed characterization. In various animal cells ProTalpha has been shown to be phosphorylated in its 14-residue N-terminal region. However, controversy remains as to whether the residues phosphorylated are Thr, as we have found (27), or Ser, as proposed by Sburlati et al. (28). We thus set out to identify sites phosphorylated in vivo and sites phosphorylated in vitro by the kinases purified from cytosol and nuclear envelopes. To this end, 32P-ProTalpha purified by reverse-phase HPLC (see "Methods") from metabolically labeled mouse splenocytes, or from kinase activity assay reaction mixtures, was trypsin-digested, and the resulting peptides were separated by reverse-phase HPLC. The elution pattern of the peptides derived from the tryptic digestion of 32P-ProTalpha phosphorylated in vitro by the cytosol-fraction ProTalpha K is shown in Fig. 4A. The observed pattern is identical to those obtained with ProTalpha phosphorylated in vitro by the nuclear envelope fraction ProTalpha K or with ProTalpha phosphorylated in vivo (results not shown). As can be seen from Fig. 4A, all the radioactivity co-eluted with the 14-residue N-terminal fragment of ProTalpha . Phosphoamino acid analysis of the radioactive tryptic peptides derived from ProTalpha phosphorylated in vivo or in vitro by the purified enzymes are shown in Fig. 4B. This result indicates that only Thr residues were phosphorylated by the cytosolic enzyme and in the ProTalpha phosphorylated in vivo, whereas both Ser and Thr residues, in similar proportion, were phosphorylated by the kinase from nuclear envelopes. This result confirms our previous findings as regards the phosphorylation of ProTalpha in vivo (27), and at the same time indicates that the cytosolic ProTalpha K, which phosphorylates only Thr residues, is probably that responsible for the phosphorylation observed in vivo. To investigate this hypothesis, we next performed a structural study of the radioactive tryptic fragments of ProTalpha phosphorylated in vivo and in vitro which co-purified with the N-terminal fragment.


Fig. 4. Identification of the sites phosphorylated in ProTalpha by the purified kinases in vitro and in vivo in proliferating splenic lymphocytes. Peptide mapping and phosphoamino acid analyses were performed as described under "Methods." 32P-ProTalpha (aliquots of about of 150,000 cpm) was obtained either (i) from kinase activity assay reaction mixtures (250 µl) containing 100 µl of the ion-exchange HPLC-purified ProTalpha -kinase (PK) activity indicated, or (ii) from 4 × 107 metabolically labeled splenic lymphocytes (phosphorylation in vivo). Aliquots were then digested with TPCK-trypsin in the presence of 50 µg of dephosphorylated calf thymus ProTalpha . Panel A shows the reverse-phase HPLC elution profile of the tryptic peptides derived from ProTalpha phosphorylated by the cytosolic kinase. Radioactivity was determined by counting in 1/10 aliquots of the different fractions (0.5 ml). The peak corresponding to the 14-residue N-terminal fragment of ProTalpha is indicated with an arrow. Panel B shows autoradiographs of thin-layer electrophoresis plates loaded with acid hydrolysates of the radioactive peaks (65,000 cpm) co-eluting with the 14-residue N-terminal fragment of ProTalpha (as shown in panel A) derived from ProTalpha phosphorylated by ProTalpha kinase activity from the indicated subcellular fractions and from metabolic labeling (in vivo). S and T indicate the positions corresponding to Ser and Thr markers, respectively. Panels C, D, and E show the reverse-phase HPLC elution profiles of the peptides resulting from hydrolysis with endopeptidase V8 (3 µg) of the radioactive tryptic peptide (about 85,000 cpm) co-eluting with the N-terminal fragment of ProTalpha (about 20 µg equivalents to that shown in panel A). Radioactivity was determined by counting in 1/10 aliquots of the different fractions (0.5 ml). Aliquots of the radioactive peptides from panel D (about 35,000 cpm) co-eluting with the 14-residue N-terminal fragment were subjected to phosphoamino acid analysis, and aliquots (about 4-6 µg) of the peptides in panel C were subjected to amino acid composition analysis.
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The sequence of the first 14 amino acid residues of ProTalpha (see Fig. 4) forms part of the most conserved region of the protein and is identical in all mammals studied to date (5). Protease V8 (in ammonium carbonate buffer, pH 7.8) specifically cleaves this fragment between residues Glu-10 and Ile-11, giving rise to a decapeptide (AcSDAAVDTSSE) and a tetrapeptide (ITTK). Since only one of these peptides contains Ser residues, V8 digestion is a useful tool for identification of the sites phosphorylated by the purified enzymes. V8 treatment of the N-terminal peptide from ProTalpha phosphorylated by the cytosolic kinase (Fig. 4C), the nuclear envelope kinase (Fig. 4D), or from ProTalpha phosphorylated in vivo (Fig. 4E), in all cases efficiently cleaved the N-terminal fragment to yield a decapeptide and a tetrapeptide, the sequences of which were confirmed by amino acid analysis (results not shown). The coincidence of the radioactive peaks in Fig. 4, C-E, with those derived from V8 cleavage of the N-terminal fragment of ProTalpha (in the same figures) provides further confirmation that both the in vitro (Fig. 4, C and D) and in vivo (Fig. 4E) phosphorylation sites are contained within the N-terminal fragment. Moreover, these elution patterns again indicate that the cytosolic enzyme phosphorylates the same sites as in vivo. Specifically, the Thr at position 7 in the fragment AcSDAAVDTSSE and, to judge from the amount of radioactivity, the Thr in the fragment ITTK at positions 12 or 13 (or both partially) are phosphorylated both by the cytosolic enzyme (Fig. 4C) and in vivo (Fig. 4E). By contrast, the nuclear envelope kinase (Fig. 4D) phosphorylates Thr residues at positions 12 or 13 (or both) in the tetrapeptide, as well as the Thr at position 7, and either one or two Ser residues in the decapeptide; the latter conclusion is inferred from the fact that the results of phosphoamino acid analysis of the decapeptide phosphorylated by the nuclear envelope kinase (not shown) are similar to those of analysis of the whole 14-mer phosphorylated by this enzyme (Fig. 4B). These data support our contention that the 180-kDa cytosolic ProTalpha K is that responsible for the phosphorylation of ProTalpha in vivo.

Characterization of the Purified Kinases

We next carried out experiments aimed at characterizing the cytosolic ProTalpha K and also the kinase from the nuclear envelope fraction which, although apparently not responsible for the phosphorylation of ProTalpha in vivo, shows similar specificity to that of the cytosolic enzyme. Since the specificities of both enzymes implied a consensus phosphorylation site identical to that of CK-2 (29), the first step was to investigate the behavior of the two enzymes with CK-2 substrates and effectors. The results of kinase activity assays performed with dephosphorylated casein and with the synthetic peptide RRRDDDSDDD, an artificial CK-2 substrate, are shown in Fig. 5A. The two kinases clearly differ in substrate specificity, since the nuclear envelope kinase phosphorylated both substrates while the cytosolic ProTalpha K did not phosphorylate either. Experiments involving immunodepletion with antibodies to the alpha  subunit of CK-2 (Fig. 5B) confirmed the difference between the two kinases, since only the activity of the nuclear envelope kinase was blocked. Moreover, the behavior of the nuclear envelope enzyme in these experiments, together with the molecular size data, suggest that this enzyme is in fact CK-2. Phosphopeptide mapping and phosphoamino acid analysis of ProTalpha phosphorylated by CK-2 from rat liver (data not shown) gave identical results to those obtained with the nuclear envelope kinase (see Fig. 4), which corroborates this conclusion. The antibody to the alpha  subunit of CK-2 was likewise ineffective for depleting the minor ProTalpha phosphorylation activity present in the HAF of nucleoplasm and in the fraction of the cytosolic extract with moderate affinity for ProTalpha -Sepharose (see Fig. 1, nucleoplasm panel, lane 4, and cytosol panel, lane 2); as indicated above, these activities were undetectable after ion-exchange HPLC. This suggests that this minor kinase activity may correspond to the main 180-kDa ProTalpha kinase.


Fig. 5. Comparative study of the behavior of the cytosolic and nuclear envelope kinases. The phosphorylation activities of the cytosolic (CPK) and nuclear envelope kinases (NEPK), in both cases after the ion-exchange HPLC purification step (20 µl), were assayed (see "Methods") with dephosphorylated casein, the synthetic peptide RRRDDDSDDD or dephosphorylated calf thymus ProTalpha as substrates (5 µg in each case), in the latter case in the presence or absence of an antibody to the alpha  subunit of CK-2. In all cases kinase activities shown are the means of values obtained in three replicate assays; interassay variation in no case exceeded 15%. Panel A shows the activities of the two enzymes with casein and the synthetic peptide as substrates. Activity with casein as substrate was estimated by densitometric scanning of radioactive phosphocasein in autoradiographs of SDS-PAGE gels in which the corresponding activity-assay reaction mixture had been run. Activity with the synthetic peptide as substrate was determined by quantifying the amount of [32P]orthophosphate incorporated into the peptide after its purification by reverse-phase HPLC in the same conditions as described in the legend to Fig. 4. Panel B shows the effect of antibody to the alpha  subunit of CK-2 on the activity of the two enzymes. Kinase activities in the presence of the indicated concentration of antibody are shown as percentages of activity in the absence of antibody. Activities were evaluated as described in the legend to Fig. 2.
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The response of the cytosolic ProTalpha K to CK-2 effectors was, however, quite similar to that of CK-2; it was markedly inhibited by heparin (at 0.2-0.4 µg/ml) and activated up to 4-fold by protamine or polylysine at 0.04-0.08 µg/ml. Kinetic study indicated that the cytosolic ProTalpha K has a Km for ProTalpha of 50 µM and a Vmax of 235 pmol/min/mg. The enzyme uses ATP (with a Km of 55 µM) as phosphate donor; GTP is an inefficient donor.

Determination of cytosolic ProTalpha K activity in extracts obtained at different periods (0-24 h) after the start of mitogenic activation of non-synchronized splenic lymphocytes indicated a linear increase of up to 10-fold over the first 16 h of activation, activity subsequently remaining roughly constant over the next 8 h (results not shown). SDS-PAGE analysis of the HPLC-purified enzyme (results not shown) indicate that the kinase activity seems to reside in two proteins of 64 and 60 kDa; taken together with the gel filtration data (see Fig. 3B), this suggests that the enzyme has an oligomeric structure in vivo.

We have recently reported that ProTalpha interacts with core histones in vitro (24). It was thus of interest to investigate whether the cytosolic ProTalpha K phosphorylates histones. Kinase activity assays using histone H1 and core histones from calf thymus as substrates (Fig. 6) indicated that histones H2B and H3 are phosphorylated by the ProTalpha K. On the basis of densitometric scanning of the autoradiograph, H2B and H3 phosphorylation activities were, respectively, 2 and 4.5 times lower than ProTalpha phosphorylation activity. The cytosolic ProTalpha K is quite stable in the fractions from affinity chromatography (about 2 months at 4 °C, 7 months at -70 °C), but shorter-lived after purification by ion-exchange HPLC (about 8 days at 4 °C, 3 weeks at -70 °C).


Fig. 6. Phosphorylation of histones by the cytosolic kinase. Kinase activity assays of ion-exchange HPLC-purified cytosolic ProTalpha K (10 µl) were performed in the conditions described under "Methods," using H1 or one of the core histones (5 µg in each case) as substrate. The figure shows the autoradiograph of the SDS-PAGE gel in which the different reaction mixtures were separated. Similar results were obtained in two replicate experiments. The activity of the cytosolic kinase with ProTalpha (5 µg) as substrate, assayed in the same conditions, is also shown.
[View Larger Version of this Image (75K GIF file)]


ProTalpha Kinase Activity in Different Cell Types

The ProTalpha kinase was isolated from other cell types following the same procedures as for mouse splenocytes. Subcellular fractionates obtained from hepatocytes and mitogen-activated thymocytes (both from mouse), semiconfluent HeLa cell cultures, and NC37 cells were subjected to ProTalpha -Sepharose affinity chromatography, and ProTalpha phosphorylation activity was assayed in the resulting eluate fractions. Kinase activity, only detected in components with high affinity for ProTalpha , was found to be appreciable in cytosolic fractions from all the cell types tested. ProTalpha kinase activity was also detected in nuclear envelope extracts of NC37 cells (results not shown), although substrate specificity and immunodepletion with anti-CK-2 indicated that the enzyme was CK-2, as in nuclear envelope extracts of splenocytes. ProTalpha kinase activities in cytosolic fractions from the different cell types are shown in Fig. 7. As can be seen from this figure, activities appear to be dependent on the cell's proliferation activity. Cells with moderate proliferation activity (hepatocytes, mitogen activated thymocytes, and semiconfluent HeLa cells) showed activity which was about 26-40% of that observed in mitogen-activated splenocytes, while activity in extracts from NC37 cells was in the range of that in mitogen-activated splenocytes. Fractions from unstimulated thymocytes and confluent HeLa cells showed negligible ProTalpha K activity (results not shown). Ion-exchange HPLC separation of the cytosolic components with high affinity for ProTalpha from NC37, semiconfluent HeLa, and mitogen-activated mouse thymocytes gave kinase activity elution patterns similar to those observed with cytosolic components from mouse splenocytes. Peptide mapping and phosphoamino acid analysis of the HPLC-purified enzymes, following the same procedure as for cytosolic ProTalpha K from splenocytes, indicated that the cytosolic kinases from NC37, semiconfluent HeLa, and mitogen-activated thymocytes show the same specificity for ProTalpha as the splenocyte enzyme.


Fig. 7. ProTalpha kinase activity in different cell types. Cytosolic fractionates from about 0.3 g of the different cell types were chromatographed on ProTalpha -Sepharose columns, and the ProTalpha phosphorylation activity in aliquots (10 µl) of the diverse eluates was assayed as described under "Methods." The figure shows kinase activity in arbitrary units (upper panel) of the cytosolic ProTalpha kinase obtained from the different cell types, deduced on the basis of densitometric estimation of the amount of radioactive product migrating with ProTalpha in the SDS-PAGE gels, as indicated in the legend to Fig. 2 (lower panel). Activities shown are the means of values obtained in four (thymocyte and NC37) or three (hepatocyte and HeLa) replicate experiments; interassay variation was generally less than 18%.
[View Larger Version of this Image (39K GIF file)]



DISCUSSION

We have isolated and purified a 180-kDa protein kinase whose specificity for ProTalpha suggests that it is responsible for the phosphorylation of this protein observed in vivo in mouse splenic lymphocytes and other cells (27, 28). The data presented in this study, particularly the affinity of the enzyme for Sepharose-bound ProTalpha , and the results of peptide mapping and phosphoamino acid analysis of ProTalpha phosphorylated in vivo and in vitro by the purified enzyme, support this hypothesis.

Affinity chromatography on a ProTalpha -Sepharose column has been shown to be effective for isolating components which can interact with ProTalpha , including core histones (24). The results presented here corroborate the efficiency of this chromatographic system for isolating possible cellular targets of ProTalpha . Thus, although ProTalpha is similarly phosphorylated by the 180-kDa protein kinase and by CK-2, only the former (Fig. 3A, upper panel) was separated by ProTalpha -Sepharose affinity chromatography from cytosolic extracts of the various cell types studied, in which both enzymes are present with similar levels of activity. However, in nuclear envelope extracts, which lack the 180-kDa kinase activity, the enzyme present (characterized as CK-2) was bound to ProTalpha -Sepharose (Fig. 3A, lower panel). We checked these points by evaluating the activity in cytosol and nuclear envelope extracts of the respective kinases, after purification by conventional chromatography on DEAE-cellulose and phosphocellulose (data not shown).

That the protein kinase isolated from the cytosol of the various mammalian cells is that responsible for the phosphorylation of ProTalpha in vivo is supported by our comparative analysis of the pattern of incorporation of [32P]orthophosphate into ProTalpha in vivo or by the purified enzyme in vitro (Fig. 4). In both cases (i.e. phosphorylated in vivo or in vitro), 32P-ProTalpha was identified by SDS-PAGE, isoelectric focusing, reverse-phase HPLC, and immunoreactivity. Furthermore, the phosphoamino acid analysis and peptide mapping were performed by two different procedures to guarantee the identification of the phosphopeptides. These comparative analysis not only indicate that the cytosolic kinase is that responsible for phosphorylation of ProTalpha in vivo, but also indicate that the kinase from nuclear envelopes, apparently identical to CK-2, phosphorylates ProTalpha in vitro in such a way as to rule out any role in the phosphorylation of this protein observed in vivo.

Phosphorylation sites of the 180-kDa ProTalpha K match consensus motifs for CK-2 (29). Furthermore, the ProTalpha kinase is, like CK-2, highly sensitive to inhibition by heparin and to activation by polyamines. However, the purified enzyme cannot be included in the CK-2 family in view of its structure (molecular size and subunit composition), and given that it does not phosphorylate substrates of CK-2, is not inhibited by anti-CK-2 antibodies (Fig. 5) and is unable to use GTP as phosphate donor. There are also differences as regards subcellular location (34-36). On the other hand, the ability of CK-2 to phosphorylate acidic nuclear proteins structurally related to ProTalpha , such as HMG-14 (37), protein P1 (38), and nucleolin (39), suggests that this enzyme is related to ProTalpha K.

Interestingly, the ProTalpha K phosphorylates histone H2B and, to a lesser extent, histone H3 (Fig. 6). Although no biological significance can at present be inferred from this result, it should be noted that these proteins are not substrates of CK-2 (29) and that the pattern of phosphorylation of ProTalpha by the ProTalpha K does not bear any resemblance to that reported for kinases which phosphorylate H2B (40, 41). In addition, the kinases that phosphorylate this histone are cAMP or cGMP-dependent (40); by contrast, signal transduction experiments performed in our laboratory2 indicate that ProTalpha is not directly phosphorylated by protein kinase A or C, and its phosphorylation in proliferating splenic lymphocytes seems to be dependent on protein kinase C activity. The present data thus suggest that the purified 180-kDa ProTalpha K does not fall into any of the protein kinase categories described to date (41). That this is a new enzyme should be confirmed by further investigation of its structure and substrate specificity. It should be noted that the widespread distribution of ProTalpha in mammalian cells, together with its high concentration (0.03-0.15 pg/per cell), imply a central role in the cell proliferation; it is thus quite reasonable to hypothesize that there is a specific kinase for this protein.

An important question is whether the phosphorylation of ProTalpha is biologically significant. In view of the present data, we would argue that it is, at least in proliferating cells, for two main reasons: first, the existence of the protein kinase reported herein, which appears to be highly specific for ProTalpha ; second, the high concentration of phosphorylated ProTalpha in proliferating cells. The amount of [32P]orthophosphate incorporated into ProTalpha after 20 h of metabolic labeling of mitogen-stimulated mouse splenocytes (in the conditions indicated in "Methods") was about 0.3 nmol/108 cells. As ProTalpha concentration in splenic lymphocytes was about 0.8 nmol/108 cells (in accordance with estimates reported by other authors; Refs. 5 and 9), and since peptide mapping suggests that only two Thr residues are phosphorylated in ProTalpha , the above finding suggests that about 18% of ProTalpha molecules are phosphorylated in mitogen-stimulated mouse splenocytes (assuming no phosphorylation before labeling and a 100% yield in the recovery of 32P-ProTalpha from labeled cells). Finally, the direct relationship between the activity of the ProTalpha K and the concentration of phospho-ProTalpha in proliferating cells (present results; and Ref. 27) argues for a significant biological role.

Phosphorylation makes ProTalpha more similar to other structurally related acidic nuclear proteins which likewise undergo phosphorylation in vivo, such as nucleoplasmin (42), nucleolin (39), P1 (37), and HMGs (37). However, the putative effects of phosphorylation on the behavior of ProTalpha are unknown. Interestingly, the recently demonstrated capacities of ProTalpha to interact with histones and to enable nucleosome assembly activity in vitro (24) do not appear to be affected by phosphorylation. Although this does not rule out the possibility that phosphorylation affects in vivo interactions between ProTalpha and histones or other molecules in the cell, it seems probable that phosphorylation is involved in some other aspect of ProTalpha function, such as degradation to yield thymosins alpha  (10) or nuclear import. In this connection, it should be noted that the rate of nuclear import of some proteins is regulated by phosphorylation at putative CK-2 sites (43). In this sense, ProTalpha could be included in the group of proteins (nucleoplasmin, lamins, etc.) whose nuclear activity is regulated by phosphorylation of CK-2 consensus site motifs (44). Clearly, both structural and biochemical characterization of the 180-kDa ProTalpha K, together with elucidation of the effects of phosphorylation on the behavior of ProTalpha may help to shed light on the function of this protein.


FOOTNOTES

*   This work was supported by Dirección General de Investigación Científica y Técnica Grant PB93/0518 and Consellería de Ordenación Universitaria Grants XUGA 20005B93 and XUGA 2007B95 from the Spanish Ministerio de Educación y Ciencia and Xunta de Galicia, respectively.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dpto. Bioquímica y Biología Molecular, Facultad de Biología, 15706 Santiago de Compostela, Spain. Tel.: 81-563100 (ext. 13316); Fax: 81-596904.
1   The abbreviations used are: ProTalpha , prothymosin alpha ; CK-2, casein kinase 2; HPLC, high performance liquid chromatography; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; HAF, high-affinity fraction.
2   A. Pérez-Estévez, C. Díaz-Jullien, G. Covelo, M. T. Salgueiro, and M. Freire, unpublished results.

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