Can you imagine racing vehicles where the cars occasionally stop and get serviced by a pit crew? In the photo, a pit crew is quickly refueling and adjusting a race car. A pit crew member may also change a tire or make other modifications in order to improve the performance of the vehicle. Since the pit crew typically has only 10 seconds, they must coordinate with each other, work quickly and accurately so that the car can get back in the race. Everything about this pit stop is well orchestrated. The stop itself is strategically timed, accounting for rate of fuel consumption, weight of fuel and even the weather and lighting conditions! In the photo, you can see that the pit crew members each have separate jobs. What would happen if the pit crew is not well coordinated? If each of the crew member fought over a particular site for modification? What if the pit stop was not planned?
Diseases first begin with small changes at the molecular level. Just like a pit crew, we have proteins (enzymes) that modify other proteins. These modifications must be well executed for proper cellular function. In a healthy person, the modification serve to signal proper function. However, sometimes, a mistake in these modfications will lead to greater errors. When this happens, proteins fail to function properly and in turn unleash a cascade of effects that ultimately lead to diseases. Is it possible to detect diseases as soon as changes occur at the molecular level? Before painful symptoms arise? Before the disease becomes widespread? To do this, we must first understand the early molecular changes that occur inside proteins. We must first understand these initial modifications and their meaning.
Our research focuses on two post-translational modifications (PTMs): arginine methylation and serine phosphorylation.
Posttranslational modifications (PTMs) describe a change that occurs after protein synthesis. While there are many types of PTMs, including phosphorylation, acetylation, ubiquitination, sumoylation and neddylation, we are particularly interested in understanding the role of arginine methylation, the addition of a methyl group onto the arginine residues in proteins.
The enzymatic addition of a methyl group to arginine residues in proteins is essential to our overall knowledge of cellular biochemistry and physiological function. The enzymes responsible for arginine methylation, protein arginine methyltransferases (PRMTs), have been found in nearly all eukaryotic organisms including protozoa, fungi, flies, plants and animals. To date, there are nine confirmed human methyltransferases, PRMT1-9.
Protein arginine methylation was initially observed in 1967. Since then, many arginine-methylated proteins or substrates have been discovered. These include estrogen receptor alpha (ER-α), the transcription factor and tumor suppressor protein p53, spliceosomal proteins (i.e. SMN, snRNPs) and the DNA repair protein MRE11. PRMTs also methylate histone proteins. Thus, like phosphorylation, methylation affects regulatory mechanisms in the cell and is now implicated in a variety of diseases including cancer, lupus and diabetes.
Does arginine methylation block neighboring phosphorylation?
In cells, information is transmitted via signal transduction by phosphorylation cascades that are misregulated in many diseases. To ensure efficient and accurate cell signaling, kinases, the enzymes responsible for phosphorylation, must first recognize a specific signal sequence or motif on a protein. One such motif is known as the Akt motif. Studies have shown that protein arginine methyltransferases methylate arginine and that this modification blocks serine phosphorylation (Figure 2). It is our hypothesis that there are many motifs like the Akt motif, whose phosphorylation can be modulated by methylation. Testing the limits of these phosphorylation motifs as they respond to arginine methylation will provide a complete catalogue of validated methylation/phosphorylation crosstalk substrates, thereby aiding in deciphering the molecular codes by which proper cell signaling occurs.
Our laboratory is currently investigating crosstalk in two proteins: histone H3, a protein that is implicated in cancer and PGC-1alpha, a protein implicated in metabolism and obesity. We propose that arginine methylation can inhibit phosphorylation of neighboring serine residues in these proteins regardless of the kinase phosphorylating the substrate. This work is fundamental to understanding the effects of crosstalk between posttranslational modifications (PTMs) and protein regulation. In addition, abnormal phosphorylation is a cause of many diseases. This work will aid in providing a novel approach to regulating phosphorylation and can potentially serve as a novel therapeutic target.
Bedford, M.T., and S.G. Clarke. 2009. Protein arginine methylation in mammals: who, what, and why. Molecular cell. 33:1-13.
Buhr, N., C. Carapito, C. Schaeffer, E. Kieffer, A. Van Dorsselaer, and S. Viville. 2008. Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells. Electrophoresis. 29:2381-2390.
Bungard, D., B.J. Fuerth, P.Y. Zeng, B. Faubert, N.L. Maas, B. Viollet, D. Carling, C.B. Thompson, R.G. Jones, and S.L. Berger. 2010. Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science. 329:1201-1205.
Cheung, P., K.G. Tanner, W.L. Cheung, P. Sassone-Corsi, J.M. Denu, and C.D. Allis. 2000. Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Molecular cell. 5:905-915.
Choi, S., C.R. Jung, J.Y. Kim, and D.S. Im. 2008. PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex. Biochimica et biophysica acta. 1780:1062-1069.
Cote, J., and S. Richard. 2005. Tudor domains bind symmetrical dimethylated arginines. The Journal of biological chemistry. 280:28476-28483.
Dhar, S., V. Vemulapalli, A.N. Patananan, G.L. Huang, A. Di Lorenzo, S. Richard, M.J. Comb, A. Guo, S.G. Clarke, and M.T. Bedford. 2013. Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Scientific reports. 3:1311.
Dhar, S.S., S.H. Lee, P.Y. Kan, P. Voigt, L. Ma, X. Shi, D. Reinberg, and M.G. Lee. 2012. Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4. Genes & development. 26:2749-2762.
Feng, Y., J. Wang, S. Asher, L. Hoang, C. Guardiani, I. Ivanov, and Y.G. Zheng. 2011. Histone H4 acetylation differentially modulates arginine methylation by an in Cis mechanism. The Journal of biological chemistry. 286:20323-20334.
Gonsalvez, G.B., L. Tian, J.K. Ospina, F.M. Boisvert, A.I. Lamond, and A.G. Matera. 2007. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins. The Journal of cell biology. 178:733-740.
Guccione, E., C. Bassi, F. Casadio, F. Martinato, M. Cesaroni, H. Schuchlautz, B. Luscher, and B. Amati. 2007. Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature. 449:933-937.
Guo, Z., L. Zheng, H. Xu, H. Dai, M. Zhou, M.R. Pascua, Q.M. Chen, and B. Shen. 2010. Methylation of FEN1 suppresses nearby phosphorylation and facilitates PCNA binding. Nature chemical biology. 6:766-773.
Gwinn, D.M., D.B. Shackelford, D.F. Egan, M.M. Mihaylova, A. Mery, D.S. Vasquez, B.E. Turk, and R.J. Shaw. 2008. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Molecular cell. 30:214-226.
Hsu, J.M., C.T. Chen, C.K. Chou, H.P. Kuo, L.Y. Li, C.Y. Lin, H.J. Lee, Y.N. Wang, M. Liu, H.W. Liao, B. Shi, C.C. Lai, M.T. Bedford, C.H. Tsai, and M.C. Hung. 2011. Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation. Nature cell biology. 13:174-181.
Hyllus, D., C. Stein, K. Schnabel, E. Schiltz, A. Imhof, Y. Dou, J. Hsieh, and U.M. Bauer. 2007. PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation. Genes & development. 21:3369-3380.
Iberg, A.N., A. Espejo, D. Cheng, D. Kim, J. Michaud-Levesque, S. Richard, and M.T. Bedford. 2008. Arginine methylation of the histone H3 tail impedes effector binding. The Journal of biological chemistry. 283:3006-3010.
Kirmizis, A., H. Santos-Rosa, C.J. Penkett, M.A. Singer, M. Vermeulen, M. Mann, J. Bahler, R.D. Green, and T. Kouzarides. 2007. Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation. Nature. 449:928-932.
Kowenz-Leutz, E., O. Pless, G. Dittmar, M. Knoblich, and A. Leutz. 2010. Crosstalk between C/EBPbeta phosphorylation, arginine methylation, and SWI/SNF/Mediator implies an indexing transcription factor code. The EMBO journal. 29:1105-1115.
Lau, A.T., S.Y. Lee, Y.M. Xu, D. Zheng, Y.Y. Cho, F. Zhu, H.G. Kim, S.Q. Li, Z. Zhang, A.M. Bode, and Z. Dong. 2011. Phosphorylation of histone H2B serine 32 is linked to cell transformation. The Journal of biological chemistry. 286:26628-26637.
Lee, Y.H., M.T. Bedford, and M.R. Stallcup. 2011. Regulated recruitment of tumor suppressor BRCA1 to the p21 gene by coactivator methylation. Genes & development. 25:176-188.
Lee, Y.H., and M.R. Stallcup. 2011. Roles of protein arginine methylation in DNA damage signaling pathways is CARM1 a life-or-death decision point? Cell cycle. 10:1343-1344.
Li, X., X. Hu, B. Patel, Z. Zhou, S. Liang, R. Ybarra, Y. Qiu, G. Felsenfeld, J. Bungert, and S. Huang. 2010. H4R3 methylation facilitates beta-globin transcription by regulating histone acetyltransferase binding and H3 acetylation. Blood. 115:2028-2037.
Lu, H., J.Y. Cui, S. Gunewardena, B. Yoo, X.B. Zhong, and C.D. Klaassen. 2012. Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing. Epigenetics : official journal of the DNA Methylation Society. 7:914-929.
Mahajan, K., B. Fang, J.M. Koomen, and N.P. Mahajan. 2012. H2B Tyr37 phosphorylation suppresses expression of replication-dependent core histone genes. Nature structural & molecular biology. 19:930-937.
Miranda, T.B., J. Sayegh, A. Frankel, J.E. Katz, M. Miranda, and S. Clarke. 2006. Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A. The Biochemical journal. 395:563-570.
Paik, W.K., and S. Kim. 1980. Studies of the identity of the unknown in protein hydrolysates. Biochemical and biophysical research communications. 97:8-16.
Pal, S., S.N. Vishwanath, H. Erdjument-Bromage, P. Tempst, and S. Sif. 2004. Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes. Molecular and cellular biology. 24:9630-9645.
Sakamaki, J., H. Daitoku, K. Ueno, A. Hagiwara, K. Yamagata, and A. Fukamizu. 2011. Arginine methylation of BCL-2 antagonist of cell death (BAD) counteracts its phosphorylation and inactivation by Akt. Proceedings of the National Academy of Sciences of the United States of America. 108:6085-6090.
Wang, H., Z.Q. Huang, L. Xia, Q. Feng, H. Erdjument-Bromage, B.D. Strahl, S.D. Briggs, C.D. Allis, J. Wong, P. Tempst, and Y. Zhang. 2001. Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science. 293:853-857.
Weekes, J., K.L. Ball, F.B. Caudwell, and D.G. Hardie. 1993. Specificity determinants for the AMP-activated protein kinase and its plant homologue analysed using synthetic peptides. FEBS letters. 334:335-339.
Wuchty, S., D. Arjona, A. Li, Y. Kotliarov, J. Walling, S. Ahn, A. Zhang, D. Maric, R. Anolik, J.C. Zenklusen, and H.A. Fine. 2011. Prediction of Associations between microRNAs and Gene Expression in Glioma Biology. PloS one. 6:e14681.
Yamagata, K., H. Daitoku, Y. Takahashi, K. Namiki, K. Hisatake, K. Kako, H. Mukai, Y. Kasuya, and A. Fukamizu. 2008. Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Molecular cell. 32:221-231.
Yang, Y., Y. Lu, A. Espejo, J. Wu, W. Xu, S. Liang, and M.T. Bedford. 2010. TDRD3 is an effector molecule for arginine-methylated histone marks. Molecular cell. 40:1016-1023.
Zurita-Lopez, C.I., Sandberg, T., Kelly, R., and Clarke, S.G. Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues. J. Biol. Chem. 2012, 287(11), 7859-7870.