This work is the first demonstration that two posttranslational modifications can affect the same sites in a protein, and the first comprehensive analysis of arginylation in the nucleus that reveals the identity of a significant number of previously unknown protein arginylation targets and outlines a likely function for protein arginylation in chromatin structure and gene expression. Our result that ATE1 is less abundant and more active in the nucleus suggest that this enzyme undergoes an additional level of regulation that could increase or silence its activity dependent on its intracellular localization. Such regulation could be achieved by modulating the interaction of ATE1 with different binding partners in the cytoplasm and the nucleus that may form either activatory or inhibitory complexes with this enzyme and/or regulate its activity by changing its conformation or posttranslationally modified state. Different chemical environments in these two intracellular compartments may also play a role, by creating favorable or unfavorable ionic conditions for arginylation.
It has been previously shown that some ATE1 isoforms showed preferential nuclear localization under certain conditions, suggesting that this localization depends on the cell’s physiological state (Kwon et al., 1999
; Rai and Kashina, 2005
; Wang et al., 2011
). It is possible that higher arginylation activity in nucleus could be necessary because of the existence of higher number and/or amount of natural substrates of ATE1 in nucleus. It was previously found that partially purified ATE1 preparation can arginylate nuclear proteins in vitro (Kaji, 1976
). Our data demonstrates for the first time that ATE1 preferentially arginylates nuclear proteins in vivo and that the activity of this enzyme can be differentially regulated in different intracellular compartments.
Many of the proteins identified in the cytosolic and chromatin fractions have a demonstrated role in the corresponding compartments, such as HSP90 in the cytosol and DNA- and RNA-binding proteins involved in chromatin structure, gene expression, and RNA processing in vivo. For such proteins, regulation by arginylation with or without subsequent methylation can conceivably facilitate their structural interactions and affect their in vivo functions. For nucleic acid-binding proteins, the modifications likely affect their ability to interact with the nucleic acids (which should be facilitated by the addition of the positively charged Arg), or other proteins, which could be repelled or attracted by the chemical groups on the matching surfaces of the interaction partners. A number of proteins found in the nucleosolic and chromatin fractions, however, have been previously characterized as cytoplasmic proteins. For some of those, including actin, GAPDH, filamin, and spectrin, recent data demonstrate their involvement in gene expression and transcriptional regulation (Grummt, 2006
; Loy et al., 2003
; Sawa et al., 1997
; Tang et al., 2003
). It is likely that arginylation and arginylation/methylation of these proteins in the nucleus can facilitate this specific function. For others, such as collagen, no such function has been previously demonstrated. It is possible that their interaction with the chromatin is a non-specific effect of the purification, resulting from the presence of the surface Arg residues that may induce their interaction with DNA after the cell and nuclear lysis. It is also possible, however, that their presence in the nuclear fractions found in this study reflects their previously unknown function in the chromatin and/or transcriptional control.
Structural and sequence analysis shows that arginylated sites for most of the identified proteins are located in the middle of their polypeptide chains, in conserved domains predicted to affect key molecular interactions (, S5
). It has been previously found that for many arginylated proteins sites for the posttranslational addition of Arg are located in the middle of the polypeptides, far away from the N-terminus, suggesting a novel regulatory mechanism that couples arginylation with partial regulatory proteolysis without disrupting the protein’s quarternary structure (Wong et al., 2007
). In agreement with this, all the available structural data presented in our current study (, Fig. S4
) show that arginylated sites are exposed within the structurally important conserved domains on the protein surface, suggesting that arginylation on these sites affects fully synthesized and folded proteins and plays an important regulatory role. The measured distances of the added Arg or methyl-Arg residues in histones to the phosphate groups in the DNA backbone are particularly encouraging, as these distances with the added Arg are short enough to significantly affect the interaction of histones with DNA, and thus can affect DNA packaging and/or transcriptional regulation. Conceivably, addition of methyl groups onto Arg in these positions should stabilize the arginylation sites and ensure their longer-term action in the nucleosome. Further studies of the individual arginylation sites identified in this study will reveal the functional significance of their regulation by arginylation and advance the knowledge about this poorly understood posttranslational modification to the next level of understanding.
Our finding that posttranslationally added Arg can be methylated constitutes a proof of principle and the first demonstration that posttranslational modifications can double on the same sites within the proteins. It has been previously shown that a negative posttranslational regulation of methylation by phosphorylation can occur by modifying the neighboring residues to change the charge and conformation of the methylation sites (Bedford and Richard, 2005
). We show for the first time a positive regulation by a posttranslational modification that can facilitate methylation by creating an acceptor site for methyl groups. While many of the functions of such double modifications remain to be determined, our study shows a clear correlation between arginylation/methylation and the nuclear size. As a smaller nucleus implies a higher compaction, which can affect nuclear functions significantly, it is reasonable to suggest that arginylation/methylation will have a significant effect on nuclear function. It also appears likely that arginylation with subsequent methylation of the added Arg residues can serve as a second-order regulatory mechanism that could affect those major metabolic processes in which arginylation and methylation have been implicated. Methylation may also act to protect posttranslationally added Arg from chemical modifications in vivo that could destroy or inactivate them (for example, by methylglyoxal – a cytotoxic agent linked to disease), in line with a recently proposed model that methylation acts as a ‘guardian’ of Arg (Fackelmayer, 2005
). Finally, methylation may be necessary to protect proteins against degradation by the N-end rule pathway, shown to affect some proteins with Arg in the N-terminal position (Varshavsky, 1997
). It appears likely that the posttranslationally added Arg residues within a protein that further serve as targets of methylation should be especially important and/or long-lived to allow their further modification by Arg methyltransferases, an additional level of regulation or protection of these proteins and their important functional sites in vivo.