eEF1A Phosphorylation in the Nucleus of Insulin-stimulated C2C12 Myoblasts

Recent data indicate that some PKC isoforms are translocated to the nucleus, in response to certain stimuli, where they play an important role in nuclear signaling events. To identify novel interacting proteins of conventional PKC (cPKC) at the nuclear level during myogenesis and to find new PKC isozyme-specific phosphosubstrates, we performed a proteomics analysis of immunoprecipitated nuclear samples from mouse myoblast C2C12 cells following insulin administration. Using a phospho(Ser)-PKC substrate antibody, specific interacting proteins were identified by LC-MS/MS spectrometry. A total of 16 proteins with the exact and complete motif recognized by the phospho-cPKC substrate antibody were identified; among these, particular interest was given to eukaryotic elongation factor 1α (eEF1A). Nuclear eEF1A was focalized in the nucleoli, and its expression was observed to increase following insulin treatment. Of the cPKC isoforms, only PKCβI was demonstrated to be expressed in the nucleus of C2C12 myocytes and to co-immunoprecipitate with eEF1A. In-depth analysis using site-directed mutagenesis revealed that PKCβI could phosphorylate Ser53 of the eEF1A2 isoform and that the association between eEF1A2 and PKCβI was dependent on the phosphorylation status of eEF1A2.

Mass spectrometry identification of protein binding partners is an important strategy to understand the role of multifunctional proteins. Protein kinase C (PKC) isoforms are involved in the transduction of a number of signals important for the regulation of cell growth, differentiation, apoptosis, and other cellular functions. Recent evidence has indicated that some PKC isoforms are translocated to the nucleus, in response to certain stimuli, where they have an important role in nuclear signaling (1)(2)(3). Insulin provokes rapid changes in phospholipid metabolism and, thereby, generates biologically active lipids that serve as intracellular signaling factors. Conventional protein kinase Cs (cPKCs), 1 novel PKCs, and atypical PKCs that are activated by this insulin signaling cascade have key roles in the regulation of the metabolic effects of insulin (4). Deregulated protein phosphorylation is associated with many disease states, including cancer and diabetes. Because phosphospecific kinase substrate antibodies have aided in the identification and characterization of proteins involved in cell signaling and functional proteomics is a very powerful approach for the discovery of kinase targets (5), we decided to combine these two approaches to identify new phosphosubstrates of nuclearly localized cPKC in C2C12 cells early on during insulin-stimulated differentiation to myocytes.
Skeletal muscle development is due to transmission of a large number of relatively unknown intracellular signals (6). In these cells, upon a differentiation stimulus, muscle-specific transcription factors, such as myogenin, are induced as are the muscle genes they regulate. Subsequently, cells become elongated and fused to each other to form multinucleated myotubes. Another critical event during the differentiation process is a decrease in DNA synthesis and cell cycle arrest.
Recently it has been shown that nuclear PLC␤ 1 is required for C2C12 differentiation, and it might play a crucial role in the initiation of the genetic program responsible for muscle differentiation (7). In particular, investigations on skeletal muscle development strengthen the contention that nuclear PLC␤ 1 signaling is required for the activation of the cyclin D3 promoter in C2C12 cells (8,9). PLC␤ 1 is a key player in the regulation of nuclear inositol lipid signaling and of a wide range of cellular functions, such as proliferation and differentiation (10 -13). Because PLC activation results in the production of diacylglycerol (DAG), it is plausible that DAG-sensitive members, such as conventional isoforms ␣, ␤⌱, ␤⌱⌱, and ␥ of the PKC family, may be involved in insulin-stimulated nuclear signaling pathways.
Here we describe a proteomics approach to identify potential effectors of nuclear PLC␤ 1 -dependent signaling during insulin-stimulated myogenic differentiation. Skeletal muscle is one of the major insulin-responsive tissues that express PKC isoforms from each of the categories, and a number of the isoforms have been shown to be activated by insulin or conditions important for effective insulin stimulation (14). The specific role of each individual PKC isoform in insulin signaling and the molecular mechanism causing PKC activation by insulin are still unclear (15). This lack of information prompted us to undertake a search of substrates of cPKC in nuclei of insulin-stimulated C2C12 cells. When activated, cPKC isozymes phosphorylate substrates containing serine or threonine with arginine or lysine at the Ϫ3, Ϫ2, and ϩ2 positions and a hydrophobic amino acid at position ϩ1 (16). To identify new potential phosphosubstrates of cPKC, mouse C2C12 myoblasts were induced with insulin. Immunoprecipitation experiments on nuclei of C2C12 myoblasts were performed with a phospho(Ser)-PKC substrate antibody, which detects endogenous levels of many cellular proteins only when phosphorylated on serine residues surrounded by Arg or Lys at the Ϫ2 and ϩ2 positions and a hydrophobic residue at the ϩ1 position. This technology relies on detection of cPKC activation via its most direct and important physiologic readout, substrate phosphorylation. Our data demonstrate that eukaryotic elongation factor 1␣ (eEF1A) was phosphorylated by PKC␤I on Ser 53 and that these two molecules localized in nuclei of C2C12 cells under conditions of insulin stimulation.
Vectors, Site-directed Mutagenesis, and Transfection-pGEX-4T-2 vectors coding for human eEF1A1 and eEF1A2 were a kind gift from Dr. Lee (18). The mutation of EF1A2 S53A was performed on this plasmid using the QuikChange XL II site-directed mutagenesis kit from Stratagene (La Jolla, CA) by using the following primer pair: 5Ј-GAGATGGGGAAGGGAGCCTTCAAGTATGCCTG-3Ј and 5Ј-CAG-GCATACTTGAAGGCTCCCTTCCCCATCTC-3Ј. The mutation was verified by DNA sequencing. eEF1A1 and wild type and mutated eEF1A2 DNA sequences were cloned into pCMV-TAG 4 vector (Stratagene) to obtain their fusion proteins with FLAG tag. The sequences were amplified by PCR using primers containing an EcoRI restriction site. Following the digestion with the restriction enzyme, the DNA fragments were ligated into pCMV-TAG 4, and their correct orientation was verified by enzymatic digestion.
Cell Culture and Differentiation Induction-Mouse C2C12 myoblasts were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (growth medium) (Invitrogen) at 37°C and 5% CO 2 . Cells at 80% confluence were cultured for the time indicated in serum-free medium supplemented with 100 nM insulin (differentiation medium).
Fractionation of Nuclei and Nucleoli-Nuclei were purified as described previously (7). Briefly, 5 ϫ 10 6 cells were suspended in 500 l of nuclear isolation buffer (10 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 10 mM ␤-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 15 g of calpain inhibitors I and II/ml, 5 mM NaF, protease and phosphatase inhibitor mixtures (Roche Applied Science)) for 8 min on ice. Milli-Q water (500 l) was then added to swell the cells for 3 min. The cells were sheared by eight passages through a 23-gauge hypodermic needle. Nuclei were recovered by centrifugation at 400 ϫ g at 4°C for 6 min and washed once in 500 l of washing buffer (10 mM Tris-HCl, pH 7.4, 2 mM MgCl 2 , protease inhibitors as described above). The purity of the isolated nuclei was assessed by detection of ␤-tubulin. Nucleoli were purified using a variation on a method described by Scherl et al. (17). Cells were lysed in 5 ml of Buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 15 g of calpain inhibitors I and II/ml, and protease and phosphatase inhibitors mixtures (Roche Applied Science)) and incubated on ice for 5 min. The cells were sheared by three passages through a 23-gauge hypodermic needle. Nuclei were recovered by centrifugation at 218 ϫ g and 4°C for 5 min. The pellet contained enriched, but not highly pure, nuclei. Nuclei were resuspended with 3 ml of S1 solution (0.25 M sucrose, 10 mM MgCl 2 ) and then left to layer over 3 ml of S2 solution (0.35 M sucrose, 0.5 mM MgCl 2 ). Nuclei were cleaned by centrifugation at 1430 ϫ g for 5 min at 4°C. Purified nuclei were resuspended with 3 ml of S2 solution and sonicated for 6 ϫ 10 s. The nucleolar suspension was layered over 3 ml of S3 solution (0.88 M sucrose, 0.5 mM MgCl 2 ) and centrifuged at 3000 ϫ g at 4°C for 10 min. To obtain purified nucleoli, the pellet was washed with 0.5 ml of S2 solution and then centrifuged at 1430 ϫ g at 4°C for 5 min.
Immunoprecipitation, Pulldown of GST Fusion Protein, and Western Blotting-Nuclear lysates in radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 140 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 50 mM NaF, protease and phosphatase inhibitor mixtures) were precleared for 30 min at 4°C with 20 l of protein A/G-agarose (Santa Cruz Biotechnology) and centrifuged, and the supernatants were incubated at 4°C overnight with primary antibody followed by 1-h incubation with 30 l of protein A/G-agarose at 4°C with agitation. For GST pulldown, nuclear lysates obtained as described above were incubated for 2 h at 4°C with 40 l of GST resin. Pellets were washed three times with radioimmune precipitation assay buffer, washed once with Tris-HCl, pH 7.5, and boiled in Laemmli sample buffer. Immunoprecipitates and proteins from nucleus or nucleolus purifications were separated by 12% SDS-PAGE. Proteins were transferred to nitrocellulose membrane, blotted with the specific antibody, and detected using a chemiluminescence eEF1A Is a Novel Substrate for Protein Kinase C ␤I method (Pierce). Blots were visualized in an Eastman Kodak Co. Digital Image Station 2000R.
In Vitro Kinase Assay-PKC␤I and eEF1A were immunoprecipitated as described above and then phosphorylated in vitro in kinase reaction mixture (25 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 5 mM ␤-glycerophosphate, 100 M Na 3 VO 4 , 100 mM ATP, 5 Ci/assay [␥-32 P]ATP (3000 Ci/mmol) (PerkinElmer Life Sciences)) for 20 min at 30°C. In the case of assays using only PKC␤I, the immunoprecipitate was incubated in a kinase reaction mixture containing 1 g of myelin basic protein as substrate. In a second experiment, FLAG-tagged eEF1A wild type (WT) or FLAG-eEF1A S53A mutant was expressed in C2C12 cells, immunoprecipitated, and washed twice with PBS plus 1% Nonidet P-40 and twice in TNE (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA). Pellets were subjected to an in vitro kinase assay as stated above with 2 g of recombinant PKC␤I (Cell Signaling Technology), boiled in Laemmli sample buffer, and centrifuged. Supernatants were separated by 12% SDS-PAGE, blotted on nitrocellulose membranes, and Western blotted with the indicated antibodies. Radioactive bands were localized by autoradiography.
LC-MS/MS Analysis-Bands of interest were excised; washed in 100 mM ammonium bicarbonate, pH 8, 50% acetonitrile (ACN) until complete destaining; and finally digested with sequencing grade trypsin (Promega, Madison, WI) at 37°C overnight. Peptides were extracted sequentially three times with 50% ACN, 0.1% trifluoroacetic acid (TFA) in water. Each extraction involved 5 min of stirring followed by centrifugation and removal of the supernatant. The original supernatant and those obtained from sequential extractions were combined and dried down. Peptides were resuspended in 20 l of 2% ACN, 0.1% formic acid, and 1 l was injected into an LC system (LC Packings) coupled on line with a 4000Q-TRAP instrument (Applied Biosystem, Framingham, MA). Samples were loaded onto a 15-cm ϫ 75-m-inner diameter PepMap C 18 3-m column using a standard LC Packings UltiMate pump and FAMOS autosampler. Samples were desalted on line prior to separation using a microprecolumn (5-mm ϫ 300-m-inner diameter) cartridge. The washing solvent was 0.1% formic acid delivered at a flow rate of 30 l/min for 3 min. Peptides were separated using a linear gradient from 95% buffer A (0.1% formic acid in 2% ACN) to 90% buffer B (0.1% formic acid in 80% ACN) in 50 min. The mass spectrometer was operated in data-dependent MS/MS mode where an MS scan was taken, and then the two highest ions were selected for fragmentation followed by dynamic exclusion for 1 min (m/z full-scan acquisition range from 400 to 1700 Da/e and m/z tandem mass spectrum acquisition range from 70 to 1700 Da/e). For peptide sequence searching, the mass spectra were processed and analyzed using the ProteinPilot v.2 (Applied Biosystems, Framingham, MA) against the murine International Protein Index database version 3.13 (50,489 entries), allowing for cysteine modification with methyl methanethiosulfate and oxidation of methionines with mass tolerances of 0.15 Da for MS and 0.1 Da for MS/MS, allowing for one missed (trypsin) cleavage, and including peptides with a minimum confidence score of 95%. The unused parameter calculated by the software is a confidence percentage that reflects the summed score of all the peptides that are unique to that protein and are not found in other proteins. Each individual peptide score is log 10 (Confidence), so a peptide with 99% confidence contributes 2 to the unused score.
Immunofluorescence Analysis on in Situ Enriched Nuclei-Nuclei were extracted, adapting a protocol for nuclear matrix extraction. Briefly, C2C12 cells growing on glass coverslips were permeabilized using a cytoskeletal buffer (10 mM Pipes, pH 6.8, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl 2 , 1 mM EGTA, 0.5% Triton X-100, 2 mM vanadyl ribonucleoside complex, 1.0 mM PMSF, 10 g/ml protease inhibitor mixture (Roche Applied Science) supplemented with 2 mM Na 3 VO 4 ) on ice for 5 min. Cytoskeletal debris were removed by washing with 0.1% Tween 20 in PBS. In situ enriched nuclei were fixed for 30 min at room temperature with 4% freshly prepared paraformaldehyde in PBS. The coverslips were blocked using PBS containing 3% BSA for 1 h at room temperature. Primary antibodies were incubated for 3 h at 4°C in PBS with 3% BSA. Samples were washed with PBS and incubated with the fluorescent secondary antibodies (1:200 for FITC-conjugated and TRITC-conjugated IgG) for 1 h at room temperature in PBS containing 3% BSA. Slides were mounted with an antifade reagent in glycerol and observed with a Nikon E 600 fluorescence microscope equipped with a digital camera. Images were acquired and analyzed with NIS-Elements BR 2.20 software.
Real Time PCR-Total cellular RNA was extracted using the RNeasy minikit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. After the measurement of RNA concentration, cDNA was synthesized starting from 2 g of total RNA using 200 units of Moloney murine leukemia virus retrotranscriptase (Promega), 0.5 g of oligo(dT) primers, 25 units of ribonuclease inhibitor, and a 10 mM concentration of each dNTP. The reactions were incubated for 1 h at 42°C. The expression of eEF1A1 and eEF1A2 genes was determined by using a TaqMan-based real time PCR method. Their expression levels were analyzed and quantified by means of TaqMan-specific probes (assay numbers Mm01966122_u1 and Mm00514649_m1, respectively, Applied Biosystems, Foster City, CA). GAPDH was used as the reference housekeeping gene (assay number Mm99999915_g1, Applied Biosystems, Foster City, CA). All real time PCRs were performed in a MicroAmp Optical 96-well reaction plate (Applied Biosystems, Foster City, CA) in a total reaction volume of 20 l using 12.5 l of TaqMan PCR Universal Master Mix (Applied Biosystems, Foster City, CA), 1.25 l of the desired gene expression assay containing the primers and probes, and 1 l of cDNA. Each reaction was repeated in triplicate. Reactions were carried out using the ABI PRISM 7300 sequence detection system (Applied Biosystems, Foster City, CA) with the following thermal conditions: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The ⌬⌬Ct method was used to quantitate amounts of each gene relative to the GAPDH amount in each reaction according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). The results of different sets of experiments were statistically analyzed by GraphPad Prism 3.02 software.

Identification of Novel Putative cPKC Phosphosubstrates by
Mass Spectrometry Analysis-To study the early myogenically mediated signaling events related to nuclear cPKC activation, the mouse C2C12 skeletal cell line was induced to differentiate with insulin. Immunoprecipitation experiments, from nuclei of control and insulin-stimulated C2C12 cells, were performed with the phospho(Ser)-PKC substrate antibody or nonspecific IgG. The immunoprecipitated samples were electrophoretically resolved by SDS-PAGE and Coomassie-stained. Bands that appeared to be visibly present in the control and insulintreated lanes were excised from the gel, digested with trypsin, and subjected to mass spectrometry analysis (LC-MS/MS) (Fig. 1). ProteinPilot software led to the identification of 69 proteins with an unused score Ͼ2, although only 16 contained the specific epitope recognized by the antibody (Table I). Of particular interest because of its presence in the nucleus was a 50-kDa protein identified as eEF1A, which carries the aminoacyl-tRNA onto the ribosome during translation and is also eEF1A Is a Novel Substrate for Protein Kinase C ␤I involved in several cellular processes, including oncogenic transformation and cell proliferation. The analysis of the amino acid sequence of eEF1A revealed the presence of one potential cPKC phosphorylation site within the identified consensus cPKC phosphorylation motif of (K/R)XS*H(K/R) (where S* is phosphoserine) recognized by the phospho-cPKC substrate antibody. The exact sequence recognized is shown in Table I. The potential phosphorylated serine that was identified by the cPKC corresponded to Ser 53 of the eEF1A protein.
eEF1A Is a Nuclear Target for Conventional PKCs-To validate the results obtained by MS/MS spectrometry analysis that identified eEF1A as a phosphosubstrate of cPKC, lysates from nuclei of control and insulin-stimulated cells were immunoprecipitated with anti-cPKC phosphosubstrate antibody. Immunoprecipitation was clearly evident and was increased after 1 h of insulin stimulation, suggesting that insulin treatment resulted in a significant increase in expression and phosphorylation of eEF1A (as shown by densitometric analysis) that was potentially mediated by cPKC ( Fig. 2A). Therefore, we investigated the effect of insulin stimulation on the subcellular localization and expression of endogenous eEF1A using immunofluorescence microscopy (Fig. 2B). We observed an increase of eEF1A localization after insulin treatment in several distinct foci in the nucleus. These foci were identified as nucleolus because they were co-localized with nucleolin, a marker protein of the nucleolus (Fig. 2B). To further verify the subcellular localization of eEF1A and to determine whether its expression increased after insulin administration, we analyzed different subcellular fractions of C2C12 cells by Western blot. Nucleoli and the nucleoplasmic fraction from total nuclei of C2C12 cells were purified by subcellular fractionation. As shown in Fig. 2C, eEF1A was detected both in the purified nucleoplasmic fraction and in the nucleolar preparation. Analysis of insulin-stimulated C2C12 subcellular fractions showed an increase of eEF1A in the nucleus.
PKC␤I Is Present in Nucleus of Myoblasts and Associates with eEF1A-Because eEF1A localized within the nucleolus, we determined which cPKC isozymes were present in this cellular compartment in control and insulin-stimulated cells. Western blotting revealed that the nuclear fraction of C2C12 myoblasts possessed cPKC␣ and -␤I (Fig. 3A) but not cPKC␤II and -␥ (data not shown). Based on these findings, we examined whether PKC␣ and/or -␤I and eEF1A could interact in vivo. Nuclear lysates from C2C12 cells, stimulated with 100 nM insulin for 1 h, were immunoprecipitated with anti-eEF1A antibody, and the pellets were probed with anti-PKC␤I or with anti-PKC␣ (Fig. 3B). PKC␣ did not retain the ability to form a complex with eEF1A, whereas the presence of endogenous PKC␤I in the IP suggested that the two proteins interact in vivo. Likewise, immunoprecipitation of nuclear lysate from insulin-stimulated C2C12 cells with anti-PKC␤I co-immunoprecipitated endogenous eEF1A (Fig. 3B).
eEF1A2 Transcription Is Activated by Insulin-In mammalian cells, two tissue-and development-specific isoforms, eEF1A1 and eEF1A2, are present. Interestingly, eEF1A2 is present normally only in muscle and neurons. This prompted us to evaluate the expression of both isoforms after insulin administration by real time PCR (Fig. 4). Mouse C2C12 were first cultured to confluence and then induced to differentiate into multinucleated myotubes by changing the culture medium from a high serum to a serum-free medium or to a serum-free medium with 100 nM insulin. Real time PCR analysis of total RNA extracted from unstimulated C2C12 cells did not demonstrate a significant change in eEF1A1 mRNA levels when compared with the expression of the insulin-stimulated sample. The expression level of eEF1A2 remained low during growth in high serum medium but was enhanced 1.5-fold in response to insulin, similar to the increase of eEF1A protein observed in the nucleus ( Fig. 2A). Taken together, these data indicate that the expression of eEF1A2 protein is up-regulated upon insulin administration, whereas eEF1A1 remains stable.
PKC␤I Phosphorylates eEF1A2 on Ser 53 -Because the transcription of eEF1A2 is responsive to insulin, we chose to determine whether eEF1A2 was a substrate for PKC␤I. Nuclei of C2C12 myoblasts were immunoprecipitated with anti-eEF1A; contemporarily, an aliquot of nuclear lysate from C2C12 myoblasts treated or untreated with insulin was immunoprecipitated with anti-PKC␤I antibody (Fig. 5A). The immunocomplexes were mixed together, and eEF1A phosphorylation was evaluated by an in vitro kinase assay. In the FIG. 1. C2C12 isolated nuclei immunoprecipitated with antiphospho-PKC substrate antibody. Nuclei purified from C2C12 growth medium and differentiating medium cells (100 nM insulin (ins) for 1 h) were immunoprecipitated with an anti-phospho(Ser)-PKC (pPKC) substrate antibody (lanes 3 and 4, respectively). 50 g of growth medium and differentiating medium nuclear lysates (NL) were loaded as control (lanes 1 and 2, respectively). Samples were separated by 12% SDS-PAGE and Coomassie-stained overnight. Visible bands were excised from the gel, digested with trypsin, and analyzed by LC-MS/MS.

eEF1A Is a Novel Substrate for Protein Kinase C ␤I
presence of [␥-32 P]ATP, insulin-activated PKC␤I was able to phosphorylated eEF1A. To examine whether Ser 53 was a specific site for PKC␤I, we mutated Ser 53 to alanine and examined whether PKC␤I was able to phosphorylate this mutant (Fig. 5B). C2C12 cells were transfected with FLAG-eEF1A2 or FLAG-eEF1A2 S53A, and the anti-FLAG immunoprecipitates from insulin-stimulated C2C12 cells were combined with recombinant, active PKC␤I in the presence of [␥-32 P]ATP. Although wild type eEF1A2 could be phosphorylated by PKC␤I in vitro, PKC␤I failed to phosphorylate the Ser 53 mutant. A weak phosphorylation of eEF1A2 was detected in the control sample, most probably due to endogenous PKC␤I, which, as shown above in Fig. 3B, co-immunoprecipitates with eEF1A2 (Fig. 5B). As a control, the filter was probed with polyclonal antibodies against eEF1A2, PKC␤1, and phospho(Ser)-PKC substrate. Thus, these data demonstrate that PKC␤I phosphorylates eEF1A2 at Ser 53 in vitro and that Ser 53 was the site recognized by the anti-phospho-cPKC substrate antibody.
To assess how Ser 53 phosphorylation affected eEF1A2 localization, C2C12 cells were transiently transfected with FLAG-eEF1A2 WT or FLAG-eEF1A2 S53A, and the localization of eEF1A2 was determined by immunofluorescence using anti-FLAG antibody. The FLAG-tagged proteins were observed to localize in a manner similar to endogenous eEF1A ( Fig. 2C and data not shown).
Interaction of eEF1A2 with PKC␤I Is Dependent on Phosphorylation Status of eEF1A2-To test whether the interaction of eEF1A2 with PKC␤I was dependent on the phosphorylation status of eEF1A2, we performed GST pulldown experiments (Fig. 6). A recombinant eEF1A2 and eEF1A2 S53A expressed as GST fusion proteins were purified from bacteria and immobilized on glutathione-Sepharose. We incubated nuclear lysates from untreated and insulin-stimulated C2C12 cells with GST, GST-eEF1A2, or GST-eEF1A2 S53A after which PKC␤I binding was assessed by Western blotting with a polyclonal antibody against PKC␤I and with the cPKC phosphosubstrate-specific antibody. We found that endogenous PKC␤I associated specifically with GST-eEF1A2 WT, whereas it did not associate with GST-eEF1A2 S53A. As a control, the blot was stripped and reprobed with a polyclonal antibody against eEF1A2, which reveals the presence of each GST fusion protein. Moreover, the blot was stripped and reprobed with a monoclonal antibody against GST, demonstrating that equal amounts of GST protein were used in each case. Thus, these results confirmed the interaction between eEF1A2 and PKC␤I. In addition, the binding of PKC␤I to eEF1A2 was dependent on the phosphorylation status at Ser 53 .

DISCUSSION
In contrast to general proteomics, functional proteomics does not aim to identify or characterize every protein in the cell but rather to provide information about a small number of proteins that are directly relevant to the biological question being studied. To achieve this goal, functional proteomics often is combined with complementary techniques, such as protein biochemistry, molecular biology, and cell physiology. In our laboratory, we use functional proteomics to answer questions about signal transduction in a variety of systems (19 -22). Kinase substrate antibodies have been useful in identifying cellular phosphorylation states associated with specific signaling pathways and can be used together with bioinformatics analysis to predict and identify new phosphoproteins and new sites of protein phosphorylation. In this work, we used a proteomics-based approach to explore new nuclear substrates of cPKC.

eEF1A Is a Novel Substrate for Protein Kinase C ␤I
inositide-PLC␤1 appears to be one of the main players of this signaling system not only in normal but also in pathological conditions (23,24). We have previously (7,8) shown that nuclear PLC␤1 is required for C2C12 differentiation and that it might play a crucial role in the initiation of the genetic program responsible for muscle differentiation. Because PLC activation results in the production of DAG, it is plausible that DAG-sensitive members, such as conventional isoforms ␣, ␤⌱, ␤⌱⌱, and ␥ of the PKC family, may be involved in insulinstimulated nuclear signaling pathways.
Using a co-immunoprecipitation-and mass spectrometrybased methodology, we identified a new protein, i.e. eEF1A, interacting with and phosphorylated by a cPKC. We present evidence that PKC␤I can directly phosphorylate eEF1A on Ser 53 .
Among the 69 proteins identified by spectrometry analysis, only 16 contained the specific epitope recognized by the antibody (Table I). Most of these proteins are localized at the nuclear level, or they can be present in different compartments either in the subnuclear or in the cytoplasmic fraction (Sorbs3 and interferon-activable protein 204). Except for the myosin heavy chain IX (25), none of the proteins included in the table were previously known to be a substrate of conventional PKCs. For instance, the DNA topoisomerase 1 (26,27) is phosphorylated by PKC, but the type of PKC and the amino acid residue involved has not been identified yet; in other cases, even if the phosphorylation occurs on the serine included in the antibody epitope, such as for the interferonactivable protein 204 (28) and the nuclear receptor coactivator 5 (29), there is still no evidence that relates the phosphorylative event to a conventional PKC isoform.
Among the proteins identified, we chose to further investigate the eEF1A. Elongation factors play an important role in the translation elongation mechanism (30); eEF1A1 and eEF1A2 are variants of the protein elongation factor eEF1A with eEF1A1 being expressed ubiquitously, whereas eEF1A2 is normally expressed only in heart, muscle, and brain. The canonical role for these proteins involves regulation of ribosomal polypeptide elongation by binding of amino-acylated tRNAs for transport to the ribosomes. eEF1A2 has also been found to have a number of non-canonical functions, including phosphatidylinositol signaling, apoptosis, cytoskeletal modifications, targeting proteins for degradation, and participation in the heat shock response. It has also been shown that eEF1A2 can transform cells and give rise to tumors in nude mice. Notably, eEF1A2 has antiapoptotic functions in certain systems, whereas eEF1A1 is a proapoptotic protein (31,32). Moreover, recent findings have suggested a link between eEF1A and lipid signaling (18).
As eEF1A2 is present normally only in muscles and neurons, we hypothesized that eEF1A2 could be involved in the phosphospecific signaling process in our cellular model. Thus, the elucidation of the presence of cPKC phosphorylated form of eEF1A in the nuclear fraction could yield new insights in nuclear PLC␤1 signaling, nuclear function, gene expression, and myogenesis. We demonstrated that eEF1A is phosphorylated at a putative cPKC motif and that eEF1A is clearly detectable in nuclear lysates. Both expression and phosphorylation markedly increased in the presence of insulin treatment. Similarly, it was recently demonstrated that eEF1A is involved in cultured myogenic differentiation where the expression of eEF1A2 protein is activated upon myogenic differentiation (33). We observed a marked presence of eEF1A expression after insulin treatment in several distinct foci in the nucleus. These foci of eEF1A were identified as nucleolus because they were co-localized with nucleolin, a marker protein of the nucleolus (Fig. 3C). eEF1A is present mainly in the cytoplasm of cells (34), but a small population of eEF1A has been identified previously in the nucleus (35)(36)(37)(38). These two populations of eEF1A may play different physiological roles. A very interesting study demonstrated that nuclear eEF1A includes complexes of eEF1A with ZPR1 in nucleoli of proliferating cells (39). Previous studies of eEF1A indicate that it can bind RNA (40) and that it interacts with RNA polymerase (41). One possible role of the nucleolar complex of eEF1A with ZPR1 is to functionally interact, directly or indirectly, with RNA (39). The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication, and tumor suppression. Knowledge of the human nucleolar proteome has been expanded dramatically by the two recent mass spectrometry studies on isolated nucleoli from HeLa cells (17,42). Nearly 400 proteins were identified within the nucleolar proteome so far in humans, among them eEF1A (43). Moreover, the nucleolar presence of eEF1A was identified in a proteomics analysis of the nucleolus of adenovirus-infected cells (44). It is important to bear in mind that the protein profile of nucleoli is not static because it is modulated by changes in cell physiology. Thus, the composition of nucleoli varies during cell cycle progression and can be altered by stress, tumor development, signaling events, and viral infections. Interaction with other proteins can change the cellular localization of a particular protein, especially when this interaction is critical for cellular function. It was our objective to search for cPKC substrates that might be phosphorylated by one of the cPKC isozymes. By co-immunoprecipitation and in vitro kinase assay, we demonstrated that endogenous eEF1A and PKC␤I proteins interact in vivo and that eEF1A is phosphorylated by PKC␤I in vitro. Moreover, by phosphoamino acid analysis and mutagenesis, we further demonstrated that PKC␤I phosphorylates eEF1A on Ser 53 in the KGSFK PKC␤I motif. Large scale proteomics studies revealed that conserved tyrosine residues (Tyr 29 , Tyr 85 , Tyr 86 , Tyr 141 , Tyr 162 , and Tyr 254 ) in both human eEF1A variants (45)(46)(47)(48) are phosphorylated. Lamberti et al. (49) confirmed serine and threonine phosphorylation of eEF1A and suggested other likely serine and threonine phosphorylation sites based on a bioinformatics study of eEF1A1. In their investigation on the phosphorylation status of non-conserved Thr and Ser residues of eEF1A1 and eEF1A2, Soares et al. (50) adopted a structural approach (in conjunction with sequence-based phosphorylation predictors) and examined whether the residues in question were exposed and accessible to kinases or buried and inaccessible.
Our data indicated that the expression of eEF1A2 protein is activated upon insulin administration, whereas eEF1A1 remains stable at a low level. In recent years, two lines of evidence have implicated eEF1A2 in disease, suggesting that there may indeed be subtle differences in the functions of the two forms of eEF1A. First, EEF1A2 has been shown to be a potential oncogene; it is overexpressed in a proportion of ovarian tumors but is not expressed in normal ovary (51), and it is similarly overexpressed in two-thirds of breast tumors but not normal breast tissue (52,53). Phosphorylation has been determined to modify some of the activities of mammalian eEF1A, typically resulting in a stimulatory effect (32,54). We observed that among the two isoforms only the mRNA expression of eEF1A2 is slightly up-regulated upon insulin administration, whereas eEF1A1 remained stable. Recently, a physical and functional relationship between eEF1A2 and PI4KIII␤ has been identified. In fact, human eEF1A2 can directly bind and activate PI4KIII␤ (18). The involvement of eEF1A in nuclear processes largely remains to be established. An LC-MS/MS analysis showed that eEF1A interacts with the transcriptional repressor FBI-1 (also called Pokemon or ZBTB7A) in the nuclei of HEK293T cells. Translation initiation factors are a significant component to oncogenic cell transformation and malignancy. The protein interaction between eEF1A and the proto-oncogene FBI-1 might initiate a cellular regulatory mechanism that couples translation and cell proliferation (55).
To conclude, our proteomics exploration of cPKC signaling in the nuclei of C2C12 cells demonstrated that the up-regulation of eEF1A intranuclear content, evoked by insulin, is associated with an increase in the phosphorylation of the Ser 53 residue of the protein. Our findings indicate that the phosphorylation is elicited by PKC␤I and that the binding of PKC␤I to eEF1A2 is dependent on its interaction with its specific eEF1A2 phosphorylation site. eEF1A Is a Novel Substrate for Protein Kinase C ␤I