Abstract
High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i. e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64∼Lys-70 > Lys-66 > Lys-14∼Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70∼Lys-73 > Lys-64∼Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bustin, M.; Reeves, R. High-mobility-group chromosomal proteins: Architectural components that facilitate chromatin function. Prog. Nucleic Acid Res. Mol. Biol. 1996, 54, 35–100.
Reeves, R.; Nissen, M. S. The AT-DNA-binding domain of mammalian high mobility group I chromosomal proteins: A novel peptide motif for recognizing DNA structure. J. Biol. Chem. 1990, 265, 8573–8582.
Elton, T. S.; Nissen, M. S.; Reeves, R. Specific AT-DNA sequence binding of RP-HPLC purified HMG-I. Biochem. Biophys. Res. Commun. 1987, 143, 260–265.
Johnson, K. R.; Lehn, D. A.; Reeves, R. Alternative processing of mRNAs encoding mammalian chromosomal high-mobility-group proteins HMG-I and HMG-Y. Mol. Cell. Biol. 1989, 9, 2114–2123.
Johnson, K. R.; Lehn, D. A.; Elton, T. S.; Barr, P. J.; Reeves, R. Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y). J. Biol. Chem. 1988, 263, 18338–18342.
Reeves, R.; Beckerbauer, L. HMGI/Y proteins: Flexible regulators of transcription and chromatin structure. Biochim. Biophys. Acta. 2001, 1519, 13–29.
Reeves, R. Molecular biology of HMGA proteins: Hubs of nuclear function. Gene. 2001, 277, 63–81.
Banks, G. C.; Li, Y.; Reeves, R. Differential in vivo modifications of the HMGI(Y) nonhistone chromatin proteins modulate nucleosome and DNA interactions. Biochemistry. 2000, 39, 8333–8346.
Edberg, D. D.; Adkins, J. N.; Springer, D. L.; Reeves, R. Dynamic and differential in vivo modifications of the isoform HMGA1a and HMGA1b chromatin proteins. J. Biol. Chem. 2005, 280, 8961–8973.
Reeves, R.; Edberg, D. D.; Li, Y. Architectural transcription factor HMGI(Y) promotes tumor progression and mesenchymal transition of human epithelial cells. Mol. Cell. Biol. 2001, 21, 575–594.
Lund, T.; Holtlund, J.; Laland, S. G. On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells. FEBS Lett. 1985, 180, 275–279.
Reeves, R.; Langan, T. A.; Nissen, M. S. Phosphorylation of the DNA-binding domain of nonhistone high-mobility group I protein by cdc2 kinase: Reduction of binding affinity. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1671–1675.
Lund, T.; Laland, S. G. The metaphase specific phosphorylation of HMG I. Biochem. Biophys. Res. Commun. 1990, 171, 342–347.
Xiao, D. M.; Pak, J. H.; Wang, X.; Sato, T.; Huang, F. L.; Chen, H. C.; Huang, K. P. Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C γ and neurogranin/RC3 genes. J. Neurochem. 2000, 74, 392–399.
Palvimo, J.; Linnala-Kankkunen, A. Identification of sites on chromosomal protein HMG-I phosphorylated by casein kinase II. FEBS Lett. 1989, 257, 101–104.
Wang, D. Z.; Ray, P.; Boothby, M. Interleukin 4-inducible phosphorylation of HMG-I(Y) is inhibited by rapamycin. J. Biol. Chem. 1995, 270, 22924–22932.
Schwanbeck, R.; Gymnopoulos, M.; Petry, I.; Piekielko, A.; Szewczuk, Z.; Heyduk, T.; Zechel, K.; Wisniewski, J. R. Consecutive steps of phosphorylation affect conformation and DNA binding of the chironomus high mobility group A protein. J. Biol. Chem. 2001, 276, 26012–26021.
Piekielko, A.; Drung, A.; Rogalla, P.; Schwanbeck, R.; Heyduk, T.; Gerharz, M.; Bullerdiek, J.; Wisniewski, J. R. Distinct organization of DNA complexes of various HMGI/Y family proteins and their modulation upon mitotic phosphorylation. J. Biol. Chem. 2001, 276, 1984–1992.
Edberg, D. D.; Bruce, J. E.; Siems, W. F.; Reeves, R. In vivo posttranslational modifications of the high mobility group A1a proteins in breast cancer cells of differing metastatic potential. Biochemistry. 2004, 43, 11500–11515.
Sgarra, R.; Diana, F.; Bellarosa, C.; Dekleva, V.; Rustighi, A.; Toller, M.; Manfioletti, G.; Giancotti, V. During apoptosis of tumor cells HMGA1a protein undergoes methylation: Identification of the modification site by mass spectrometry. Biochemistry. 2003, 42, 3575–3585.
Sgarra, R.; Diana, F.; Rustighi, A.; Manfioletti, G.; Giancotti, V. Increase of HMGA1a protein methylation is a distinctive characteristic of leukaemic cells induced to undergo apoptosis. Cell Death Differ. 2003, 10, 386–389.
Miranda, T. B.; Webb, K. J.; Edberg, D. D.; Reeves, R.; Clarke, S. Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a. Biochem. Biophys. Res. Commun. 2005, 336, 831–835.
Sgarra, R.; Lee, J.; Tessari, M. A.; Altamura, S.; Spolaore, B.; Giancotti, V.; Bedford, M. T.; Manfioletti, G. The AT-hook of the chromatin architectural transcription factor high mobility group A1a is arginine-methylated by protein arginine methyltransferase 6. J. Biol. Chem. 2006, 281, 3764–3772.
Zou, Y.; Wang, Y. Tandem mass spectrometry for the examination of the posttranslational modifications of high-mobility group A1 proteins: Symmetric and asymmetric dimethylation of Arg25 in HMGA1a protein. Biochemistry. 2005, 44, 6293–6301.
Zou, Y.; Wang, Y. Mass spectrometric analysis of high-mobility group proteins and their post-translational modifications in normal and cancerous human breast tissues. J. Proteome Res. 2007, 6, 2304–2314.
Munshi, N.; Agalioti, T.; Lomvardas, S.; Merika, M.; Chen, G.; Thanos, D. Coordination of a transcriptional switch by HMGI(Y) acetylation. Science. 2001, 293, 1133–1136.
Munshi, N.; Merika, M.; Yie, J.; Senger, K.; Chen, G.; Thanos, D. Acetylation of HMG I(Y) by CBP turns off IFN β expression by disrupting the enhanceosome. Mol. Cell. 1998, 2, 457–467.
Jiang, X.; Wang, Y. Acetylation and phosphorylation of high-mobility group A1 proteins in PC-3 human tumor cells. Biochemistry 2006, 45, 7194–7201.
Meng, F.; Forbes, A. J.; Miller, L. M.; Kelleher, N. L. Detection and localization of protein modifications by high resolution tandem mass spectrometry. Mass Spectrom. Rev. 2005, 24, 126–134.
Larsen, M. R.; Trelle, M. B.; Thingholm, T. E.; Jensen, O. N. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. Biotechniques 2006, 40, 790–798.
Goshe, M. B.; Smith, R. D. Stable isotope-coded proteomic mass spectrometry. Curr. Opin. Biotechnol. 2003, 14, 101–109.
Julka, S.; Regnier, F. Quantification in proteomics through stable isotope coding: A review. J. Proteome Res. 2004, 3, 350–363.
Tao, W. A.; Aebersold, R. Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. Curr. Opin. Biotechnol. 2003, 14, 110–118.
Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591–6596.
Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteom. 2002, 1, 376–386.
Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 1999, 17, 994–999.
Lemmel, C.; Weik, S.; Eberle, U.; Dengjel, J.; Kratt, T.; Becker, H. D.; Rammensee, H. G.; Stevanovic, S. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling. Nat. Biotechnol. 2004, 22, 450–454.
Goshe, M. B.; Veenstra, T. D.; Panisko, E. A.; Conrads, T. P.; Angell, N. H.; Smith, R. D. Phosphoprotein isotope-coded affinity tags: Application to the enrichment and identification of low-abundance phosphoproteins. Anal. Chem. 2002, 74, 607–616.
Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal. Chem. 2001, 73, 2578–2586.
Xiong, L.; Andrews, D.; Regnier, F. Comparative proteomics of glycoproteins based on lectin selection and isotope coding. J. Proteome Res. 2003, 2, 618–625.
Zhang, H.; Li, X. J.; Martin, D. B.; Aebersold, R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 2003, 21, 660–666.
Smith, C. M.; Gafken, P. R.; Zhang, Z.; Gottschling, D. E.; Smith, J. B.; Smith, D. L. Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4. Anal. Biochem. 2003, 316, 23–33.
Elton, T. S.; Reeves, R. Purification and postsynthetic modifications of Friend erythroleukemic cell high mobility group protein HMG-I. Anal. Biochem. 1986, 157, 53–62.
Reeves, R. HMGA proteins: Isolation, biochemical modifications, and nucleosome interactions. Methods Enzymol. 2004, 375, 297–322.
Zou, Y.; Webb, K.; Perna, A. D.; Zhang, Q.; Clarke, S.; Wang, Y. A mass spectrometric study on the in vitro methylation of HMGA1a and HMGA1b proteins by PRMTs: Methylation specificity, the effect of binding to AT-rich duplex DNA and the effect of C-terminal phosphorylation. Biochemistry 2007, 46, 7896–7906.
Gu, W.; Roeder, R. G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997, 90, 595–606.
Brownell, J. E.; Allis, C. D. An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6364–6368.
Goodman, R. H.; Smolik, S. CBP/p300 in cell growth, transformation, and development. Genes Dev. 2000, 14, 1553–1577.
Bannister, A. J.; Kouzarides, T. The CBP co-activator is a histone acetyltransferase. Nature 1996, 384, 641–643.
Ogryzko, V. V.; Schiltz, R. L.; Russanova, V.; Howard, B. H.; Nakatani, Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 1996, 87, 953–959.
Sterner, D. E.; Berger, S. L. Acetylation of histones and transcription-related factors. Microbiol. Mol. Biol. Rev. 2000, 64, 435–459.
Bhakat, K. K.; Hazra, T. K.; Mitra, S. Acetylation of the human DNA glycosylase NEIL2 and inhibition of its activity. Nucleic Acids Res. 2004, 32, 3033–3039.
Bhakat, K. K.; Izumi, T.; Yang, S. H.; Hazra, T. K.; Mitra, S. Role of acetylated human AP-endonuclease (APE1/Ref-1) in regulation of the parathyroid hormone gene. EMBO J. 2003, 22, 6299–6309.
Hasan, S.; Stucki, M.; Hassa, P. O.; Imhof, R.; Gehrig, P.; Hunziker, P.; Hubscher, U.; Hottiger, M. O. Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. Mol. Cell. 2001, 7, 1221–1231.
Bhakat, K. K.; Mokkapati, S. K.; Boldogh, I.; Hazra, T. K.; Mitra, S. Acetylation of human 8-oxoguanine-DNA glycosylase by p300 and its role in 8-oxoguanine repair in vivo. Mol. Cell. Biol. 2006, 26, 1654–1665.
Herrera, J. E.; Sakaguchi, K.; Bergel, M.; Trieschmann, L.; Nakatani, Y.; Bustin, M. Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes. Mol. Cell. Biol. 1999, 19, 3466–3473.
Bergel, M.; Herrera, J. E.; Thatcher, B. J.; Prymakowska-Bosak, M.; Vassilev, A.; Nakatani, Y.; Martin, B.; Bustin, M. Acetylation of novel sites in the nucleosomal binding domain of chromosomal protein HMG-14 by p300 alters its interaction with nucleosomes. J. Biol. Chem. 2000, 275, 11514–11520.
Ferranti, P.; Malorni, A.; Marino, G.; Pucci, P.; Goodwin, G. H.; Manfioletti, G.; Giancotti, V. Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma: Identification of phosphorylation sites. J. Biol. Chem. 1992, 267, 22486–22489.
Grunstein, M. Histone acetylation in chromatin structure and transcription. Nature. 1997, 389, 349–352.
Struhl, K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 1998, 12, 599–606.
Bannister, A. J.; Miska, E. A.; Gorlich, D.; Kouzarides, T. Acetylation of importin-α nuclear import factors by CBP/p300. Curr. Biol. 2000, 10, 467–470.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online June 13, 2007
Rights and permissions
About this article
Cite this article
Zhang, Q., Zhang, K., Zou, Y. et al. A quantitative study on the in vitro and in vivo acetylation of high mobility group A1 proteins. J Am Soc Mass Spectrom 18, 1569–1578 (2007). https://doi.org/10.1016/j.jasms.2007.05.020
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2007.05.020