Citrullination of Inhibitor of Growth 4 (ING4) by Peptidylarginine Deminase 4 (PAD4) Disrupts the Interaction between ING4 and p53

Abstract

Gene expression is regulated by a number of interrelated posttranslational modifications of histones, including citrullination. For example, peptidylarginine deminase 4 (PAD4) converts peptidyl arginine to citrulline in histone H3 and can repress gene expression. However, regulation of gene expression through citrullination of non-histone proteins is less well defined. Herein, we identify a tumor suppressor protein, inhibitor of growth 4 (ING4), as a novel non-histone substrate of PAD4. ING4 is known to bind p53 via its nuclear localization signal (NLS) region and to enhance transcriptional activity of p53. We show that PAD4 preferentially citrullinates ING4 in the same NLS region and thereby disrupts the interaction between ING4 and p53. A citrulline-mimicking Arg-NLS-Gln ING4 mutant, which has all Arg residues in the NLS mutated to Gln, loses its affinity for p53, can no longer promote p53 acetylation, and results in repression of downstream p21 expression. In addition, we found that citrullination leads to increased susceptibility of ING4 to degradation, likely impacting p53-independent pathways as well. These findings elucidate an interaction between posttranslational citrullination, acetylation, and methylation and highlight an unusual mechanism whereby citrullination of a non-histone protein impacts gene regulation.Protein arginine deiminases (PADs 2 or PADIs) catalyze the posttranslational modification of peptidyl arginine and, more slowly, peptidyl monomethylarginine to form peptidyl citrulline. This citrullination reaction is a hydrolytic deimination that eliminates the positive charge of the arginine side chain, can lead to changes in inter-and intraprotein interactions, and can even cause localized protein unfolding (1, 2). The human PAD4 isoform is calcium-regulated, nuclear-targeted, and is of particular interest because of its role in gene regulation as a catalyst of histone modification and chromatin remodeling (3). PAD4 expression is up-regulated in a variety of human malignant cancers, and PAD4-specific inhibitors can kill a selection of cancerous cells lines including HL-60, MCF7, and HT-29 (4). Accordingly, PAD4 has been suggested as a possible target for anticancer therapeutics (5).Some PAD4-interacting non-histone proteins have also been identified, including the tumor suppressor p53 (6). PAD4 binds p53 and inhibits p53-regulated cell cycle arrest and apoptosis. Through this interaction, PAD4 gets recruited to gene promoters that are targeted by p53 and, in the case of p21, can downregulate expression. Reciprocally, p53 can regulate the expression of PAD4 through a p53-response element located in a PAD4 gene intron (7). These findings suggest one mechanism whereby PAD4 can promote cancerous growth through its interactions with p53, although other unidentified pathways may also contribute.PAD4 also acts through citrullination of non-histone proteins, such as p300 (8). The p300 protein, histone methyltransferase coactivator-associated arginine methyltransferase-1 (CARM1) and glucocorticoid rec...

Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells

“…PAD4 was recently also shown to citrullinate the nonhistone proteins p300, nucleophosmin (NPM1), ING4, Elk-1 and RPS2, thereby partially influencing the activity or localization of these proteins (16)(17)(18)(19). Despite this progress, in-depth studies linking experiments that examine cell morphology, mouse xenograft, and human tissue array analyses to elucidate the role of PAD4 in breast cancer progression have largely been lacking.…”

“…Citrullination has been linked to either transcriptional repression or activation, depending on the context (12)(13)(14)(15)(16). Recently PAD4 was also shown to citrullinate nonhistone proteins, raising important issues as to what other substrates may be targets of citrullination in different biological contexts (16)(17)(18)(19). Because PAD4 is expressed in breast cancer cells, we aimed to investigate whether PAD4 activity might play a role in breast cancer initiation or progression.…”

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

“…PAD4 is the only PAD family member containing a nuclear localization signal and citrullinates many substrates including histones (e.g. H3, H2A, and H4), p300/CREBbinding protein (CBP), nucleophosmin, ING4, and nuclear lamin C to exert various functions (15)(16)(17)(18)(19). Genome-wide association and pathology studies have implicated PAD4 in the etiology of rheumatoid arthritis and cancers in human patients (20 -23).…”

ING4 inhibits the translation of proto‐oncogene MYC by interacting with AUF1

“…The PHD finger serves as an important effector domain for its binding to the H3K4me3 and HAT complex [13]. ING4 associates with p53 via its NLS region, leading to enhanced acetylation of p53 on Lys-382 and transcriptional activity of p53 [24,27]. ING4 dimerization through its N-terminal region is essential for apoptosis and growth inhibition [28].…”