The protein Nα-terminal acetyltransferase hNaa10p (hArd1) is phosphorylated in HEK293 cells
© Arnesen et al; licensee BioMed Central Ltd. 2008
Received: 07 January 2009
Accepted: 02 March 2009
Published: 02 March 2009
The hNaa10p (hArd1) protein is the catalytic subunit of the human NatA Nα-terminal acetyltransferase complex. The NatA complex is associated with ribosomes and cotranslationally acetylates human proteins with Ser-, Ala-, Thr-, Val-, and Gly- N-termini after the initial Met- has been removed. In the flexible C-terminal tail of hNaa10p, there are several potential phosphorylation sites that might serve as points of regulation.
Using 2D-gel electrophoresis and hNaa10p specific antibodies, we have investigated whether hNaa10p is phosphorylated in HEK293 cells. Several differently charged forms of hNaa10p are present in HEK293 cells and treatment with Calf Intestine Alkaline Phophatase (CIAP) strongly suggests that hNaa10p is phosphorylated at multiple sites under various cell culture conditions. A direct or indirect role of GSK-3 kinase in regulating hNaa10p phosphorylation is supported by the observed effects of Wortmannin and LiCl, a GSK-3 activator and inhibitor, respectively.
We demonstrate that hNaa10p protein is phosphorylated in cell culture potentially pointing at phosphorylation as a means of regulating the function of one of the major Nα-terminal acetyltransferases in human cells.
Protein Nα-terminal acetylation is a very common protein modifications in eukaryotic cells, and approximately 80% of soluble human proteins is estimated to carry this modification. In humans, the hNatA complex is the major Nα-terminal acetyltransferase, acetylating Ser-, Thr-, Val-, Ala-, Gly- N-termini after the initial Met- has been removed by Methionine aminopeptidases [, Arnesen T and Van Damme P et al., submitted]. The recently described human NatB and NatC complexes are composed of distinct subunits and acetylate different subsets of Met-termini [[3, 3], Starheim et al., submitted]. The hNatA complex composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1) [4, 5] is functionally conserved from yeast displaying almost identical substrate specificity (Arnesen T and Van Damme P et al., submitted). Naa10p, Naa11p, Naa15p and Naa16p represent the novel names of Ard1, Ard2, Nat1 and Nat2, respectively, and will be officially presented later this year when the nomenclature of this enzyme class is revised (Polevoda B, Arnesen T and Sherman F, unpublished). hNaa10p and/or hNaa15p has been demonstrated to be important for cell survival [6–8] suggesting important functions for hNaa10p or hNatA mediated acetylation in human cells. In humans, there are two paralogues for each NatA subunit, hNaa10p and hNaa11p , and hNaa15p and hNaa16p (Arnesen et al., submitted), potentially creating a more flexible and complex system for NatA mediated N-terminal acetylation as compared to lower eukaryotes.
The C-terminus of hNaa10p is flexible  and contains many Ser- and Thr- amino acid residues that potentially might serve as a phosphorylation sites that depending on the phosphorylation state may modify the function of the protein. To investigate the possible phosphorylation of hNaa10p and the kinase involved, we analyzed hNaa10p by 2D-gel electrophoresis and Western blotting using anti-hNaa10p. Indeed, the 2-D electrophoresis pattern of hNaa10p indicated multiple isoforms with different charges which is consistent with the hNaa10p protein being phosphorylated at multiple residues. Furthermore, GSK-3 kinase, a highly conserved regulatory serine/threonine protein kinase, is involved in these phosphorylation events.
Unless otherwise stated, HEK293 Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 2% L-Glutamine in 60 mm dishes (Nunc) at 37°C and 5% CO2.
After cell harvesting, the proteins were precipitated by addition of 1 ml cold (4°C) 7% TCA, centrifuged at 13000 g for 5 minutes, and the supernatant was poured off. The proteins were resuspended and rinsed in 1 ml cold (4°C) 5% TCA, centrifuged at 13000 rpm for 5 minutes, and the supernatant was poured off again. The protein pellet was rinsed three times in H2O-saturated ether (to remove TCA) and centrifuged at 13000 rpm for 5 minutes after each washing step. The pellets were dried at room temperature until they detached from the tube walls and were free of ether, for approximately one hour. Each pellet was dissolved in 100 μl rehydration buffer (6 M urea, 2 M thiourea, 4% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) Triton X-100, trace amounts of bromophenol blue) and sonicated for 5 × 10 seconds to improve solubilisation. The protein samples were stored at -80°C until analysis by 2D-PAGE. Typically 50 μl of protein sample (from approximately 5 × 106 cells) was mixed with 300 μl and 200 μl rehydration buffer for 18 and 13 cm IPG strips respectively, and then 1.75 μl ampholytes (0.5%) were added. pH range of the ampholytes varied according to the IPG strips pH-range. The solution was centrifuged at 13000 rpm (Heraeus Biofuge 13), for 10 minutes to separate and remove any insoluble aggregates. The whole volume of the rehydration solution was applied on an Immobiline DryStrip Reswelling Tray (Pharmacia Biotech) with an Immobilized pH Gradient gel DryStrip (IPG strip) (Pharmacia). After rehydration at room temperature for 12–15 hours, isoelectric focusing (IEF) was performed with either Multiphor II™ Flat Bed Electrophoresis Unit (Pharmacia), connected to an EPS 3501 XL Power Supply (Pharmacia), or the Ettan IPGphor™ Isoelectric Focusing Unit (Amersham Biosciences). After isoelectric focusing the IPG strips were put in a Petri dish for direct equilibration by SDS equilibration buffer with 100 mM DTT and thereafter SDS equilibration buffer containing 55 mM iodoacetamide, before analysis by second dimension SDS-PAGE (12.5% polyacrylamide) using the PROTESN II xi 2-D system (20 cm) from Bio-Rad.
SYPRO Ruby protein gel stain (BioRad) was used (according to instruction manual) to visualize proteins in general. To detect the hNaa10p protein, we performed Western blotting of 2D-PAGE gels utilizing the Multiphore II NovaBlot Electrophoretic Transfer Unit. After protein blotting, the nitrocellulose membrane was incubated in 5% dry milk diluted in PBS-Tween overnight at 4°C. The membrane was incubated with anti-hNaa10p antibody , diluted (1:500) in 1% dry milk, for 1 hour in room temperature. After washing, 3 × 10 minutes with PBS-Tween, the membrane was incubated for 1 hour with anti-rabbit conjugated to HRP (Amersham Biotech) diluted 1:2000 in PBS-Tween. After washing 3 × 10 minutes with PBS-Tween and once with PBS, ECL technology was used to develop the protein blot (Amersham Pharmacia).
Results and discussion
Potential hNaa10p phosphorylation sites predicted by ELM.
Amino acid position #
(P-site in bold)
(P-site in bold)
Proline-Directed kinase (e.g. MAPK)
The presence of endogenous hNaa10p protein has been demonstrated in several human cell lines, using a specific antibody . 1D-SDS-PAGE and immunoblotting with specific antibody demonstrated the endogenous hNaa10p protein to be approximately 30 kDa as expected from the predicted open reading frame resulting in a protein of 235 amino acid residues.
Due to the ability of 2D-PAGE to separate and visualize a very large number of proteins in complex mixtures of polypeptides according to their pI value and molecular weight, it is suitable for the investigation of posttranslational modifications (PTMs) which result in changes in molecular mass and pI, such as phosphorylation.
Then, cells were stimulated in vivo with arsenic oxide (As2O3), which acts on cells through a variety of mechanisms, influencing numerous signal transduction pathways and resulting in a vast range of cellular effects that include apoptosis induction, growth inhibition, promotion or inhibition of differentiation, and angiogenesis inhibition . Interestingly, cell stimulation with 0.3 mM arsenic induced an acidic shift of the hNaa10p protein (Figure 4D) while 0.1 mM arsenic did not have any significant effect on hNaa10p (Figure 4C). It should be noted that all arsenic treated cells in these experiments did not show any signs of apoptosis.
In conclusion, our results suggest that hNaa10p is phosphorylated in vivo and that GSK-3 kinase is phosphorylating and possibly regulating the hNaa10p protein. Since GSK-3 may phosphorylate Ser/Thr-residues if the n+4 position is primed/pre-phosphorylated (Table 1) , there are most likely one or more additional kinases responsible for hNaa10p phosphorylation. Further studies are needed to determine whether and which additional kinases are involved in hNaa10p phosphorylation, to reveal the modification sites within hNaa10p and the functional consequences of such phosphorylations.
These studies were supported by Norwegian Cancer Society and Norwegian Health Region West.
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