Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells

Assays of Raf activity on eEF1A isoforms purified from E. coli

On the basis of our previous results,²³ we analyzed the ability of Raf kinases to phosphorylate recombinant eEF1A1-His and eEF1A2-His purified from E. coli in vitro according to the procedures described in Supplementary Information. The kinase assays were performed as reported in Materials and Methods using recombinant B-Raf (wt B-Raf or constitutively active mutant B-Raf V600E)²⁵ as well as C-Raf DD (constitutively active C-Raf DD)²⁶ purified from baculovirus-infected Sf9 cells as described previously.²⁷ Inactive C-Raf K75D and B-Raf K75D were used as negative controls. The results, reported in Figure 1, showed the presence of a radioactive band with a size corresponding to that of eEF1A1-His (50.5 kDa), thus indicating that wt B-Raf and the constitutively active B-Raf V600E were able to phosphorylate both eEF1A1-His and eEF1A2-His in vitro (Figure 1a, lanes 1–4 and Figure 1d, lanes 1–2). To verify the reproducibility of the results, the kinase assay was performed using a different concentration of B-Raf obtaining similar results (Figure 1g, lanes 1–2 and Figure 1k, lanes 1–2, respectively). The correspondence of the ³²P-signal with eEF1A-His was confirmed by probing the membranes with anti-eEF1A antibody (Figures 1b, e and h). In addition, the radioactive band was also analyzed for trypsin digestion (Supplementary Information). C-Raf DD instead did not promote any phosphorylation on eEF1A1-His and eEF1A2-His (Figure 2a, lanes 2 and 3, respectively). Therefore, based on the hypothesis that the presence of both eEF1A isoforms could enhance C-Raf activity in vitro, equal amounts of eEF1A1-His and eEF1A2-His were mixed, preincubated at 37 °C in kinase buffer for 10 min and then, in case of a C-Raf DD-mediated phosphoryation, radioactively marked by ³²P-labelled ATP. A phosphorylation signal corresponding to the size of eEF1A-His was observed in the sample containing both proteins (Figure 2a, lane 4). This data could be confirmed by the fact that the relative densitometric evaluation of C-Raff DD autophosphorylation showed a significant reduction in samples containing both isoforms and in the control (Figure 2b, lanes 4 and 5). The correspondence of the ³²P-band and eEF1A-His was confirmed by probing the same membrane with anti-eEF1A antibody (Figure 2c).

Identification of phosphorylation sites on eEF1A1 and eEF1A2 mediated by Raf kinases in vitro

To identify the amino-acid residues of eEF1A1-His and eEF1A2-His phosphorylated by Raf, recombinant eEF1A-His isoforms expressed in E. coli were incubated with either B-Raf or C-Raf DD in the presence of unlabelled ATP and analyzed by mass spectrometry. As reported in Table 1, analysis of the phosphopeptides showed that B-Raf phosphorylated eEF1A1-His and eEF1A2-His, thus confirming the above reported results of radioactive kinase assays. C-Raf DD did not show any phosphorylation activity on eEF1A1-His, as also observed for radioactive kinase assays whereas an eEF1A2-His phosphopeptide containing phosphorylated S21 was detected. The latter result, apparently in contrast to the radioactive kinase assays (Figure 2) can be explained by a low C-Raf DD activity on eEF1A2-His that is not detectable using conventional methods (e.g., in vitro kinase assay). Thus, this finding is because of the specificity of mass spectrometry. Table 1 and Supplementary Information report the mass spectrometry identified phosphopeptides.

Identification of phosphorylation sites of eEF1A1 expressed in COS 7 cells

To assess if the identified eEF1A phosphorylation sites were present in functional and active proliferating cells, glutathione S-transferase (GST)-eEF1A1 and eEF1A2-HIS were expressed in COS 7 cells and, 24 h after transfection, the recombinant proteins were purified according to the procedure described in Supplementary Information. Mass spectrometry analysis of phosphopeptides obtained after tryptic digestion of the proteins extracted from the SDS-PAGE, identified two phosphorylation sites on GST-eEF1A1 (Table 2). One of these sites (T88) was identical to that identified on recombinant eEF1A1-His purified from E. Coli after in vitro kinase assay in the presence of B-Raf (Table 1). No phosphopeptides were instead identified on eEF1A2-HIS.

Expression and stability of eEF1A1 and eEF1A2 and their mutants in COS 7

To study the relevance of eEF1A phosphorylation sites found in vitro and in vivo, S21 and T88 on eEF1A1 and S21 on eEF1A2 were mutated using site-directed mutagenesis and expressed in COS 7 cells. To mimic the negative charge of the phosphate group, serine/threonine was replaced with aspartic acid (S21D and T88D), whereas the phosphorylation-deficient site was realized by substituting serine/threonine with alanine (S21A and T88A). To compare the expression levels of these proteins, wt GST-eEF1A1, wt eEF1A1-HIS, wt eEF1A2-HIS and their corresponding mutants were expressed for 48 h and analyzed by immunoblotting. As reported in Figure 3, western blot analysis with anti-His antibody or anti-GST antibody of cell lysates showed that compared with wt, the modified proteins were expressed at lower levels. In particular, compared with wt eEF1A1-HIS, the densitometric evaluation of the band intensities from cell lysates (Figure 3a) with respect to that of β-actin (Figure 3c) showed that the expression level of S21A and S21D mutants was lower than that of the T88A and T88D mutants (Figure 3b). Similar results were also obtained by analyzing the expression level of eEF1A1 and its mutant fused to GST. In fact, western blots of the cell lysates (Figure 3d) with anti-GST antibody confirmed the lower expression of the GST-eEF1A1 mutants with respect to GST-eEF1A1 wt (Figure 3e). Compared with wt eEF1A2-HIS, also the expression of its corresponding S21 mutants with anti-His antibody (Figure 3g) appeared to be reduced as shown by the densitometric evaluation of the band intensity (Figure 3h) from cell lysates with respect to that of β-actin (Figure 3i). These findings suggested that compared with wt GST-eEF1A1, eEF1A1-HIS and eEF1A2-HIS the respective mutant proteins appeared less stable in vivo.

S21 has a crucial role in the ubiquitination status and subsequent proteasomal degradation of eEF1A isoforms in vivo

As reported above, mutation of RAF-mediated phosphorylation sites on eEF1A interfere with eEF1A protein expression levels. This indicates that Raf might regulate the stability of both eEF1A isoforms in an ubiquitin dependent and thus proteasome-dependent manner. To investigate this possibility, COS 7 cells were co-transfected transiently with eEF1A1-HIS or eEF1A2-HIS, respectively, and HA-ubiquitin for 36 h. In the following 12 h, cells were treated with the proteasomal inhibitor MG-132. Overexpressed eEF1A-HIS isoforms were precipitated using Ni-nitrilotriacetic acid (NTA) and immunoblotted against HA-ubiquitin using an anti-HA antibody. As shown in Figure 4 ubiquitination levels of both isolated phosphorylation resistant mutants of eEF1A-HIS (S21A and T88A) were increased in comparison with the ubiquitination levels of isolated phosphorylation-mimicking mutants (S21D and T88D) in MG132 non-treated cells (Figure 4a). Remarkably, in MG132-treated cells ubiquitination levels of isolated eEF1A1-HIS wild-type and corresponding mutants were comparable (Figure 4b). Similar results were obtained when eEF1A2-HIS wt and corresponding mutants (S21A and S21D) were co-expressed together with HA-ubiquitin in COS 7 cells (Figure 4e). In detail, the ubiquitination level of eEF1A2-HIS S21A was increased in comparison with the ubiquitination level of eEF1A2-HIS S21D in the absence of MG132, whereas in the presence of MG132 ubiquitination levels of eEF1A2-HIS wt, eEF1A2-HIS S21A and eEF1A2-HIS S21D were quite similar (Figure 4f). Similar results were also obtained when in a similar independent experiment. Immunoblotting analysis with anti-His antibody of the cell extracts after His-pull-down showed that compared with intact eEF1A1 and eEF1A2, the polyubiquitination level was slightly higher for the mutants (Figures 4i and k, respectively).

S21 positively regulates apoptosis in H1355 cells

In order to assay the biological effects of the eEF1A isoforms and their mutants, H1355 cells were transfected with wt eEF1A-HIS and the corresponding S21A and S21D constructs. As reported in Figure 5, compared with wt, transfections of H1355 cells with eEF1A-HIS mutants showed in general an increase in the apoptosis evaluated after 72 h by FACS analysis following double labelling of the cells with annexin V and propidium iodide. In particular, eEF1A1-HIS S21A and S21D mutants induced mainly an increase of early apoptosis whereas eEF1A2-HIS S21A and S21D mutants induced an increase of late apoptosis. Details regarding the FACS analysis of the samples are reported in Supplementary Information.

Expression and purification of recombinant eEF1A1-His and eEF1A2-His from E. coli

eEF1A1 and eEF1A2 cDNAs were cloned in pET22b(+) expression vectors (Novagen, Darmstadt, Germany) in frame to the His-Tag sequence and overexpressed in E. coli strains (Rosetta, BL21C43) according to the procedure reported in Supplementary Information. Activity of the purified proteins was tested in vitro according to the procedure already described⁴² and summarized in Supplementary Information.

Expression and purification of recombinant eEF1A1 and eEF1A2 from COS 7 cells

eEF1A1 and eEF1A2 cDNAs were cloned in mammalian expression vectors (pEBG and pCDNA 3.1, respectively) and expressed in COS 7 cells. Because of the lack of an isoform-specific antibody raised against the two isoforms, eEF1A1 was fused to GST (GST–eEF1A1) whereas eEF1A2 was provided with a His-Tag (eEF1A2-HIS). Vectors were transfected with jet polyethylenimine solution (Polyplus tranfection) according to the manufacturer's protocol. The procedure for the purification of both proteins is reported in Supplementary Information.

In vitro kinase assay

The Raf kinase assay was performed in vitro on recombinant eEF1A-His purified from E. coli. Depending on the kinase and the substrate, recombinant proteins were mixed in a 1 : 3/1 : 5 or 2 : 3/2 : 5 ratio ensuring substrate excess. The reaction mixture contained 300 or 600 ng of purified kinase, purified substrate 600–1000 ng, 1 × kinase buffer (10 × kinase buffer: 25 mM glycerol-phosphate, 50 mM TrisHCl, pH 7.5, 2 mM DTT, 10 mM MgCl2 and 25 mM ATP) to a final volume of 30 μl. For the radioactive labelling assay 5–10 μCi [γ-³²P]ATP (Hartmann Analytics, Braunschweig, Germany), corresponding to 0.5–1.0 μl, were added. The mixture was incubated at 37 °C for 1 h on a thermomixer at 600 r.p.m. The reaction was stopped by addition of 4 × Laemmli loading buffer (2.4 ml 1 M Tris pH 6.8, 0.8 g SDS, 4 ml 100% glycerol, 0.01% bromophenol blue, 1 ml β-mercaptoethanol and 2.8 ml water),⁴³ and analyzed by immunoblotting with phosphospecific antibodies or, in case of radioactive labelling, the nitrocellulose membrane was exposed to an X-ray film at −80 °C overnight (ON). The protein concentration was determined according to Lowry.⁴⁴ Analysis of trypsin digestion of the radio-labelled band extracted from gels was performed according to van der Geer and Hunter.⁴⁵

Vector construction

Full-length eEF1A1 and eEF1A2 cDNAs were cloned in PET-22b(+) using pAN7⁴⁶ and eEF1A2 pcDNA 3.1. (gift of Charlotte R Knudsen, Aarhus)⁴⁷ vectors as templates. The following primers were used: EF1 (forward) 5′-CCCTAAAAGCCCATATGGGAAAGG-3′ and EF2 (reverse) 5′-CTTCCACCACGGATCCAGAGTGGG-3′. A2 (forward) 5′-TGGAATTCACTCATATGGGCAAG-3′ A2 (reverse) 5′-CTCGAGTCAGTGATGGTGATG-3′.

Full-length eEF1A1 cDNA was cloned in pcDNA 3.1 using the pAN7 vector as a template. The following primers were used: EFB (forward) 5′-GCCAAAATGGGATCCGAAAAGACT-3′ corresponding to the region containing M₁GKEKT and EFA (reverse) 3′-ATTCATTTACCGGTCTGAGCTTTCTG-5′ overlapping with the Q₄₅₆KAQKAK (stop codon) region of eEF1A.

For the pEBG vector containing full-lenght EF1A1 fused to GST the following primers were used: Pebg (forward) 5′ AAAAAAGGATCCATGGGAAAGGAAAAGACTCATATCAACATTGTC-3′ and PEBG (reverse) 5′-AAAAAATCTAGATCATTTAGCCTTCTGAGCTTTCTGGGCAGACTT-3′.

The eEF1A mutants were generated by site-direct mutagenesis using the corresponding vectors as templates. The following self-complementary oligonucleotides were used: eEF1A1 S21A (forward): 5′-GTA GAT TCG GGC AAG GCG ACC ACT ACT GGC CAT-3′, eEF1A1 S21A (reverse): 5′-ATG GCC AGT AGT GGT CGC CTT GCC CGA ATC TAC-3′, eEF1A1 S21D (forward): 5′-GTA GAT TCG GGC AAG GAT ACC ACT ACT GGC CAT-3′, eEF1A1 S21D (reverse): 5′-ATG GCC AGT AGT GGT ATC CTT GCC CGA ATC TAC-3′, eEF1A1 T88A (forward): 5′-AGC AAG TAC TAT GTG GCG ATC ATT GAT GCC CCA-3′, eEF1A1 T88A (reverse): 5′-TGG GGC ATC AAT GAT CGC CAC ATA GTA CTT GCT-3′, eEF1A1 T88D (forward): 5′-AGC AAG TAC TAT GTG GAT ATC ATT GAT GCC CCA-3′, eEF1A1 T88D (reverse): 5′-TGG GGC ATC AAT GAT ATC CAC ATA GTA CTT GCT-3′, eEF1A2 S21A (forward): 5′-GTG GAC TCC GGA AAG GCG ACC ACC ACG GGC CAC-3′, eEF1A2 S21A (reverse): 5′-GTG GCC CGT GGT GGT CGC CTT TCC GGA GTC CAC-3′, eEF1A2 S21D (forward): 5′-GTG GAC TCC GGA AAG GAT ACC ACC ACG GGC CAC-3′, eEF1A2 S21D (reverse): 5′-GTG GCC CGT GGT GGT ATC CTT TCC GGA GTC CAC-3′.

Following amplification, performed with Pfu polymerase (Stratagene, Santa Clara, CA, USA), the PCR products were treated with Dpn I endonuclease (Fermentas, Milan, Italy, 1 h at 37 °C plus inactivation for 30 min at 80 °C) specifically digesting the methylated parental DNA strand. The duration of the elongation cycle was calculated based on the activity of the Pfu polymerase (2 min/1000 bp).

Pull-down and western blot analysis

Pull-down of GST-eEF1A1 or eEF1A2-HIS was performed using GST-sepharose (Amersham, Milan, Italy) or Ni-NTA agarose (Qiagen, Milan, Italy), respectively. Cell extracts (500 μg) were incubated with equal amounts of pre-equilibrated resin (150 μl slurry/1 mg protein extract) for 2 h at room temperature (RT) or ON at 4 °C, respectively. After the incubation, the resin was washed two times (centrifugation for 2 min at 2000 r.p.m. and 4 °C) in 1 ml of 1 × phosphate buffered saline (PBS), in the case of GST-sepharose, or in 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, in the case of Ni-NTA to reduce nonspecific bound proteins, 0.05% Tween 20, pH 8.0, resuspended in 30 μl of 4 × Laemmli loading buffer, heated to 95 °C for15 min and subjected to western blot analysis. Blots were developed using enhanced chemiluminescence detection (SuperSignal West Pico, Pierce, Milan, Italy). All films were scanned using Adobe photoshop software (San Jose, CA, USA).

Antibodies used were as follows: mouse monoclonal antibody anti-EF1A (Upstate, Billerica, MA, USA), rabbit polyclonal antibody anti-GST (Santa Cruz, Santa Cruz, CA, USA), mouse monoclonal antibody anti-actin (Santa Cruz), mouse monoclonal antibody anti-His (Qiagen) and rabbit polyclonal anti-HA antibody (Abcam, Cambridge, MA, USA).

Mass spectrometry measurements

Mass spectrometry analysis of eEF1A-His samples overexpressed in E. coli was performed after separation of the recombinant proteins by SDS-PAGE (NuPAGE (Darmstadt, Germany) Novex 4–12% Bis-Tris gels; MOPS buffer system). Gels were subjected to silver staining, and the respective bands were excised and washed according to Shevchenko et al.⁴⁸

Mass spectrometry analysis of GST-eEF1A and eEF1A2-HIS overexpressed in COS 7 cells was performed after separation of the proteins on 4–12% BisTris SDS Gels (Invitrogen, Karlsruhe, Germany). Afterwards protein band was visualized according to Blum et al.⁴⁹ with slight modifications, excised, washed and in-gel digested as described previously.^{50, 51}

Detailed procedures for mass spectrometry measurements are reported in Supplementary Information.

Cell culture and transfection

COS 7 cells (African green monkey kidney) were grown in Dulbecco's modified Eagle medium (Gibco, Monza, Italy) with 10% fetal bovine serum (FBS), 2 mM -glutamine (Gibco) and antibiotics (penicillin–streptomycin, Gibco). The human lung epidermoid carcinoma cell line (H1355), obtained from American Type Tissue Collection (Rockville, MD, USA), was grown in an RPMI 1640 medium supplemented with heat-inactivated FBS, 100 U/ml penicillin, 100 μ/ml streptomycin and 1% -glutamine at 37 °C. The cells were grown in a humidified atmosphere of 95% air, 5% CO₂ at 37 °C.

Cells were detached from confluent 100-mm dishes. About 2 × 10⁵ cells were incubated in electroporation buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄ and 6 mM glucose) and 15 μg/μl of DNA construct in 20 mM HEPES. Cells were electroporated at 250 V and at 975 mF for 6 s and incubated at 37 °C for 24 h. Following incubation the cells were processed for western blotting analysis and apoptosis detection as described below.

Immunoprecipitation

H1355 cells were scraped, washed twice in cold PBS and resuspended in 20–40 μl of lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0,25% sodium deoxycholate, 150 mM NaCl, 1 μg/ml aprotinin, leupeptin, pepstatin, 1 mM Na₃VO₄ and 1 mM NaF) for 30 min on ice and centrifuged at 14 000 × g for 20 min at 4 °C. Total protein extracts (500 μg) were subjected to immunoprecipitation with 2 μg of anti-eEF-1A for 16 h at 4 °C. Immune complexes were bound to 20 μl of protein A-agarose for 2 h at 4 °C. The protein A-agarose/immune complex was washed four times with cold PBS and resuspended in 20 μl of SDS loading buffer, heated for 5 min at 95 °C and subjected to western blotting analysis. Cytosolic proteins (2 μg) were separated by SDS-PAGE, electrotransferred to nitrocellulose and probed with anti-eEF-1A and anti-His antibodies. Blots were developed using enhanced chemiluminescence detection reagents (SuperSignal West Pico, Pierce) and exposed to X-ray films. Bands were quantified by using Adobe photoshop software.

Ubiqutination assay

COS 7 cells were grown and co-transfected, as above mentioned, either with eEF1A1-HIS or with eEF1A2-HIS constructs and HA-ubiquitin for 36 h and subsequently incubated in the presence or absence of MG132 (Calbiochem, Milan, Italy) for 12 h. Immunoblotting analysis of Ni-NTA isolated proteins has been performed as above reported, probing the corresponding membrane with anti-HA antibody.

Flow cytometric analysis of apoptosis

Apoptotic cell death was analysed by Annexin-V–fluorescein isothiocyanate (FITC) staining. Annexin-V–FITC binds to phosphatidylserine residues, which are translocated from the inner to the outer leaflet of the plasma membrane during the early stages of apoptosis. Labelling of apoptotic cells was performed using an Annexin-V kit (MedSystems Diagnostics, Vienna, Austria). Briefly, after transfection, H1355 cells were incubated in complete RPMI 1640 for 72 h. Subsequently, all cells (flowing and attached) were incubated with Annexin-V–FITC in a binding buffer (provided by the manufacturer) for 10 min at RT, washed and resuspended in the same buffer as described by the manufacturer. Apoptosis was evaluated measuring the % of mean fluorescence intensity (% MFI) using FL1-H (Log scale) by the FACSCalibur software (Applied Biosystems, Monza, Italy). For each sample, 20 000 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.