Prothymosin α Stimulates Ca2+-dependent Phosphorylation of Elongation Factor 2 in Cellular Extracts*

Prothymosin α (PTA) stimulates in a dose-dependent manner the phosphorylation of a 105-kDa protein (p105) in cell extracts from different cell types. Protein sequencing and immunological analysis indicated that this protein is elongation factor 2 (EF-2). We propose that calcium/calmodulin-dependent protein kinase III is responsible for the PTA-dependent EF-2 phosphorylation based on the following lines of evidence: (a) Ca2+ is required for the effect; (b) calmodulin enhances the reaction, and calmodulin inhibitors block the phosphorylation; and (c) no phosphorylation is seen in cell extracts depleted of calmodulin-binding proteins. To obtain a strong phosphorylated EF-2 band, we found it necessary to add PTA to cytosolic extracts from synchronized dividing cells in various phases of the cell cycle except in mitosis. Since PTA is a nuclear protein everywhere in the cell cycle except in mitosis, when it is found in the cytoplasm, we hypothesize that, if PTA activates EF-2 phosphorylation in vivo, as present data suggest, its presence in the cytoplasm during mitosis could explain why EF-2 phosphorylation is mainly restricted to that phase of the cell cycle. Moreover, other bands in addition to EF-2 were phosphorylated in a calmodulin- and PTA-dependent manner, and several of them (in a range between 50 and 60 kDa) have similar M r to those that conform to the holoenzyme calcium/calmodulin dependent protein kinase II, suggesting that PTA could have a more general function modulating the activity of various Ca2+/CaM-dependent enzymes along the cell cycle.

cal studies have also shown that PTA is expressed in proliferating but not quiescent cells in all tissues studied so far (7)(8)(9)(10)(11)(12); (c) PTA mRNA antisense oligomers were shown to inhibit cell division in myeloma cells (13); (d) we and others have found that PTA levels are increased in various human malignant tumors (7, 14 -16).
PTA physiological function remains unknown. It is a nuclear protein (17,18) present throughout the cell cycle (4). PTA has been found phosphorylated, although the physiological relevance of this finding is not known (19,20). On the other hand, the intracellular signaling pathways governing PTA expression are poorly known. PTA expression seems to be under the direct control of MYC (21)(22)(23)(24); however, Mol et al. (25) did not confirm this finding. Recently, Vareli et al. (26) have reported that the transcription factor E2F activates a reporter gene under the PTA promoter, indicating that E2F could directly control PTA expression.
Calcium, an intracellular second messenger, is known to regulate various cellular responses. Many aspects of calcium action are mediated by calcium/calmodulin-dependent protein kinases (CaM-kinases). There are different CaM-kinases controlling a wide range of physiological processes including cell proliferation (27). In the present report, we assessed phosphorylation changes in response to PTA presence in cell extracts and found that the phosphorylation state of several proteins was affected. One of them was identified as the eukaryotic elongation factor 2 (EF-2), suggesting that PTA activates CaMkinase III, thus providing a new link between calcium and other mitogenic signaling pathways.

EXPERIMENTAL PROCEDURES
Materials-PTA from bovine thymus was obtained from Peninsula Laboratories Europe. Calmodulin from bovine brain and calmodulinagarose were from Sigma. Calmodulin inhibitors trifluoperazine and W-7 were purchased from Calbiochem. [␥-32 P]ATP was from Amersham Pharmacia Biotech.
Human Recombinant Prothymosin ␣ Expression and Purification-A polymerase chain reaction-modified human PTA cDNA (28) was subcloned in the NdeI-EcoRI sites of a T7-7 bacterial expression vector (29), and the recombinant plasmid pEP was introduced into BL21(DE3)/pLysS cells (Novagen). Bacterial cultures were grown to A 600 ϳ 1.0 and induced with 1 mM isopropyl ␤-D-thiogalactopyranoside for 1 h. Cells harvested by centrifugation were resuspended in 5% perchloric acid, sonicated, and cleared by centrifugation, and the supernatant (perchloric soluble fraction) was stored at Ϫ80°C. Perchloric soluble fraction was neutralized with NaOH and precipitated with 2.5 volumes of ethanol at Ϫ20°C overnight. Precipitated proteins were resuspended in phosphate-buffered saline and subjected to phenol extraction (30) using 1 volume of phenol saturated in 0.3 M sodium acetate, pH 5.2, 150 mM NaCl at 65°C. After vigorous mixing and centrifugation, purified PTA was recovered in the aqueous phase, desalted through a Sephadex G-50 minicolumn, and precipitated with ethanol as above. Pellets were now resuspended in water and stored at Ϫ80°C for no longer than 1 month. Protein purity and concentration was determined by SDS-PAGE.
Cell Culture-NIH 3T3 mouse fibroblasts were grown at 37°C and * This work was supported in part by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social and Consellería de Sanidade e Servicios Sociais, Dirección Xeral de Saú de Pú blica, Programa de Screening de Cancer de Mama (to F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ These two authors equally contributed to this work. ʈ To whom correspondence should be addressed: Dept. de Fisiología, Facultad de Medicina, Universidad de Santiago, 15705 Santiago, Spain. Tel.: 34 81 582658; Fax: 34 81 574145; E-mail: fsfedopu@usc.es. 1 The abbreviations used are: PTA, prothymosin ␣; CaM-kinase, calcium/calmodulin-dependent protein kinase; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; EF-2, eukaryotic elongation factor 2; W-7, N(6-aminohexyl)-5-chloro-1-naphtalene-sulfonamide. 5% CO 2 in a humid atmosphere, in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine. Cells were synchronized as described by Zalvide et al. (4).
Human promyelocytic leukemia tumor cell line HL-60 was grown in RPMI 1640 medium with 2 mM glutamine, 10% fetal calf serum. Fisher rat thyroid cells, FRTL-5, were cultured in Coon's modified Ham's F-12 medium in the presence of a 5H hormone mixture as described previously (31).
Cell Extracts-Cells were harvested by trypsinization (monolayer cultures) or centrifugation (suspension cultures), washed twice with ice-cold phosphate-buffered saline, resuspended in homogenization buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, 250 mM sucrose, 5 g/ml soybean trypsin inhibitor, 2 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) and sonicated. After centrifugation at 100,000 ϫ g for 30 min, the supernatant was purified by gel filtration through Sephadex G-50 and eluted in sucrose-free homogenization buffer. Protein concentration in the supernatant was quantified with the Bio-Rad dye-binding assay.
To deplete extracts of CaM-binding proteins, 2 mg of the cell extract were made 2 mM Ca 2ϩ by the addition of CaCl 2 and diluted 1:1 in washing buffer (40 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM CaCl 2 , 1 mM phenylmethylsulfonyl fluoride). The diluted extract was then incubated with 400 l of CaM-agarose beads during 30 min at 4°C. After incubation, the mixture was centrifuged, and the supernatant was collected and stored.
Phosphorylation Assay-Standard phosphorylation assays (50 l) were performed with 5-20 g of cell extract in 40 mM Hepes, pH 7.0, 3 mM MgCl 2 , 50 mM NaF, and 0.1 mM CaCl 2 in the presence or absence of the indicated amounts of PTA, EGTA, and CaM. Phosphorylation was started by the addition of 10 Ci of [␥-32 P]ATP (3000 Ci/mmol). Reactions were incubated at 37°C for 5 min and then stopped by the addition of electrophoresis sample buffer. Phosphorylated products were analyzed on 7.5% SDS-PAGE gels under reducing conditions. Gels were stained with Coomassie Blue, dried, and subjected to autoradiography.
Protein Sequencing-Samples for sequence analysis were reduced with dithiothreitol and pyridylethylated with 4-vinylpyridine in the sample buffer prior to running the gel electrophoresis. After Coomassie staining, the 105-kDa band was excised from the gel and subjected to "in gel" digestion with trypsin essentially as described by Hellman et al. (32). Resulting internal tryptic fragments were isolated by narrow bore reversed phase liquid chromatography on a RPC C2/C18 SC 2.1/10 column, operated in SMART System (Amersham Pharmacia Biotech). Amino acid sequence analysis of selected fragments was performed on an ABI model 470A gas phase sequenator, following the manufacturer's instruction.
Western Blot Analysis-Cell extracts phosphorylated in the phosphorylation assay were subjected to SDS-PAGE and transferred onto nitrocellulose filters. These were incubated overnight with a 1:2000 dilution of anti-EF-2 antiserum (33). Detection was performed with the Western Light Immunoblotting System (Tropix).

FIG. 1. PTA induces the phosphorylation of a 105-kDa protein.
A, ten micrograms of extract from asynchronously proliferating NIH3T3 cells were phosphorylated as described under "Experimental Procedures" in the absence (Ϫ) or presence (ϩ) of 8 M PTA and subjected to SDS-PAGE. The left part (CBB) shows a dried Coomassie Blue-stained gel where reactions were resolved, and the right part ( 32 P) shows the corresponding autoradiogram. B, to show the dose-response effect of PTA on p105 phosphorylation, 20 g of NIH3T3 cell extract were employed for phosphorylation assays in the presence of the indicated amounts of PTA. The reactions were resolved in SDS-PAGE and electrotransferred onto nitrocellulose, and the filter was exposed to obtain the autoradiography that is shown. C, the indicated amounts of HL-60 cells extract (right part) or 20 g of FRTL-5 cells extract (left part) were subjected to phosphorylation assays in the absence (Ϫ) or presence (ϩ) of 8 M PTA to show that PTA enhanced the phosphorylation of p105 in extracts from different cell types. Samples were processed as described under "Experimental Procedures," and an autoradiogram is shown. In each panel, the positions of the molecular mass markers are indicated. The arrows mark the position of p105.

RESULTS
When cell extracts obtained from exponentially growing NIH3T3 cells were incubated in the presence of radioactive ATP, several phosphorylated bands were observed. The addition of PTA to the reaction affected this pattern, stimulating the phosphorylation of some of these proteins. Initially, we focused on one of them, with an apparent molecular mass of 105 kDa (p105), that was very weakly or not at all phosphorylated in the absence of PTA (Fig. 1A), and its phosphorylation was induced by PTA in a dose-dependent manner (Fig. 1B). Both bovine PTA and human recombinant PTA were active in the phosphorylation assay (data not shown); thus, we used human PTA to obtain the data reported in the present work. As seen in Fig. 1C, PTA also induced the phosphorylation of a 105-kDa protein in extracts prepared from FRTL-5 and HL-60 cell lines, a normal rat and a human tumor cell line, respectively, indicating that the PTA-induced phopshorylation of the 105-kDa band might be a common phenomenon in mammalian cell lines. When a large amount (20 g) of cell extract was used, the phosphorylation of p105 was observed even in absence of exogenous PTA. Decreasing the ratio of cell extract to added PTA produced a concomitant reduction of the phosphorylated p105 band, pointing out that the stimulation of phosphorylation is dependent on the amount of the extract added to the sample.
To gain an insight into the conditions needed for the phosphorylation of p105, we explored its Ca 2ϩ requirements. The addition of EGTA abolished the PTA-induced p105 phosphorylation, while the addition of Ca 2ϩ , at concentrations as low as 0.1 M, restored that effect, showing that the presence of Ca 2ϩ is required for the phosphorylation effect (Fig. 2).
The p105 band was isolated from HL-60 extracts, and its internal peptide fragments were obtained by the in gel digestion procedure. Two fragments were characterized with the following sequences: FSVSPVV and FDVHDVTLHADAI. These sequences corresponded with the fragments 499 -505 and 702-714 of EF-2. Further immunological analysis by Western blot, using a polyclonal antibody against EF-2 (a generous gift from Dr. Angus C. Nairn), showed that indeed p105 and EF-2 comigrate in SDS-PAGE (Fig. 3). Moreover, we found that PTA modified the phosphorylation of EF-2 without affecting the amount of this protein present in the cell extract. To our knowledge, CaM-kinase III is the only known kinase that phosphorylates EF-2, and EF-2 is the only known substrate for CaM-kinase III, at least in vitro (34). So we studied whether the PTA-induced phosphorylation of EF-2 might be mediated by CaM-kinase III. To do this, we exploited the fact FIG. 3. Immunological identity of p105 and EF-2. Proteins from standard phosphorylation reactions were run in SDS-PAGE and electrotransferred onto a nitrocellulose filter. The membrane was exposed to obtain an autoradiogram (upper panel). After the decay of the radioactive signal, the same filter was subjected to a Western blot (WB) with an antiserum against EF-2. The chemiluminiscent register is shown in the lower panel. that CaM-kinase III binds CaM with high affinity and depleted the cell extracts of CaM-binding proteins by means of CaM affinity chromatography. As Fig. 4 shows, EF-2 phosphorylation was not detectable in the presence of PTA when the extracts were depleted from CaM-binding proteins despite the fact that EF-2 was still present (albeit at half the amount present in total extracts (data not shown)), strongly suggesting that CaM-kinase III, a CaM-binding protein, mediates the effect of PTA.
Since calmodulin is the main activator of CaM-kinases, we tested the effect of the simultaneous addition of PTA and CaM to cell extracts. Fig. 5A shows that the simultaneous addition of PTA and CaM caused a reinforcement of phosphorylated EF-2 band in a synergistic way. Other bands in a range between 50 and 60 kDa were phosphorylated in a CaMand PTA-dependent manner (Fig. 5A, open arrow). Next, to determine if PTA-stimulated phosphorylation of EF-2 depended on CaM activity, two widely used CaM inhibitors were employed in the reactions. W-7 inhibited the stimulation due to PTA alone or to the addition of PTA plus CaM (Fig. 5B), and trifluoperazine inhibited the PTA effect in a dose-dependent manner (Fig. 5C).
We further investigated the PTA-induced phosphorylation of EF-2 in different stages of the cell cycle. Such phosphorylation was not detectable when reactions were performed with extracts from serum-starved quiescent NIH3T3 cells even in the presence of exogenous PTA, pointing out that some essential factor is not present, at least in sufficient amounts, in quiescent cells; this essential factor is not EF-2 that was present in the quiescent extracts as seen by Western blot (data not shown). Eight hours after serum refeeding, PTA induced the phosphorylation of EF-2 (Fig. 6A). PTA-dependent phosphorylation of EF-2 was detectable in extracts from cells synchronized at different points between S and M phases of the cell cycle. The basal phosphorylation of EF-2, without exogenous PTA, was also seen albeit at a lower amount than when PTA was added (Fig. 6B). Interestingly, in nocodazole-arrested cells, the addition of PTA was not necessary to see a strong band of phosphorylated EF-2 (Fig. 6B). In all samples, the band of phosphorylated EF-2 reached similar intensity in the presence of PTA, independently of the basal phosphorylation observed, suggesting that the stimulated pathway is the same in both cases (with or without exogenous PTA). Since PTA is a nuclear protein everywhere in the cell cycle except in mitosis, when it is found in the cytoplasm (data not shown), nocodazole-arrested cells (mitotic cells) represent a situation when PTA-induced EF-2 phosphorylation might have a physiological significance, since PTA, CaM-kinase III, and EF-2 are found together in the cytoplasm. DISCUSSION Here we report that in cell extracts PTA stimulates the phosphorylation of several proteins. Initially, we focused on one of them, with an apparent molecular mass of 105 kDa, that was very weakly or not at all phosphorylated in the absence of PTA, and its phosphorylation was induced by PTA in a dose-dependent manner. Two fragments of this band were characterized with sequences that were identical to the eukaryotic elongation factor 2, an essential factor for the extension of the polypeptide chain in the ribosome. Further immunological analysis by Western blot, using a polyclonal antibody against EF-2, confirmed this identity, showing that indeed p105 and EF-2 comigrated in SDS-PAGE. Moreover, we found that PTA modified the EF-2 phosphorylation without affecting the amount of EF-2 present in the cell extract. To our knowledge, CaM-kinase III is the only known kinase that phosphorylates EF-2 (34). We think that CaM-kinase III is responsible for the PTA-stimulated EF-2 phosphorylation based on the following evidence: (a) Ca 2ϩ is required; (b) calmodulin enhances the reaction, and calmodulin inhibitors block the phosphorylation; (c) no phosphorylation is seen in extracts depleted of CaM-binding proteins. Moreover, we found PTA-induced phosphorylation of EF-2 in various cell lines in agreement with the finding that CaM-kinase III has a widespread tissue distribution (33). On the other hand, quiescent, nonproliferating cells did not show kinase activity despite the addition of PTA to the reaction, indicating that some other factor was lacking in the extracts from nonproliferating cells. This finding could be explained by the fact that CaM-kinase III is present at low levels in nondividing cells (35,36).
The mechanism by which PTA stimulates EF-2 phosphorylation is not clear now. Several scenarios can be proposed: (a) PTA may directly act on CaM-kinase III; (a) PTA may displace CaM from binding sites on proteins present in the cell extracts, increasing its availability to bind CaM-kinase III; or (c) PTA may bind to EF-2 and conformationally alter the latter, resulting in exposure of new sites of phosphorylation. These alternate scenarios may be distinguished by future experiments.
When eukaryotic cells undergo mitosis, the rate of protein synthesis declines by up to 35-40%. This decline is due to phosphorylation of EF-2 (37), since this phosphorylation affects the rate of translation (38,39). To obtain a strong phosphorylated EF-2 band, we found it necessary to add exogenous PTA to cytosolic extracts prepared from synchronized dividing cells in the various phases of the cell cycle except in mitosis. In mitotic cell extracts, the addition of exogenous PTA did not further enhance EF-2 phosphorylation, as if the reaction was already saturated with endogenous PTA. Interestingly, PTA is a nuclear protein throughout the cell cycle except in mitosis, when it is found in the cytoplasm; therefore, if PTA modulates EF-2 phosphorylation, as present data suggest, then it could be explained why EF-2 phosphorylation is mainly restricted to mitosis despite the presence of both CaM-kinase III and EF-2 in the cytoplasm throughout the cell cycle.
Moreover, other bands in addition to EF-2 were phosphorylated in a CaM-and PTA-dependent manner (Fig. 5A); the apparent molecular masses of these bands (in a range between 50 and 60 kDa) are similar to those reported for the different subunits that conform the holoenzyme CaM-kinase II. Initial evidence obtained by Western blot using an anti-CaM-kinase II antibody points to the fact that these bands are in fact CaMkinase II, 2 suggesting that PTA could have a more general function modulating the activity of various Ca 2ϩ /CaM-dependent enzymes throughout the cell cycle.
MYC has been reported to induce PTA expression (21)(22)(23)(24). More recently, E2F has also been implicated in promoting PTA expression (26). Here we present evidence that PTA modulates the activity of CaM-kinases. Therefore, if the present findings are confirmed in vivo, PTA could represent a novel link between various intracellular signaling mitogenic pathways such as Ca 2ϩ , MYC, and E2F. Studies to assess this issue are currently under way.