The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors on the basis of reports describing their down-regulation and mislocalization in a variety of cancer types. In addition to their regulation by transcriptional mechanisms, understanding the range of PTMs (post-translational modifications) of INGs is important in understanding how ING functions are fine-tuned in the physiological setting and how they add to the repertoire of activities affected by the INGs. In the present paper we review the different PTMs that have been reported to occur on INGs. We discuss the PTMs that modulate ING function under normal conditions and in response to a variety of stresses. We also describe the ING PTMs that have been identified by several unbiased MS-based PTM enrichment techniques and subsequent proteomic analysis. Among the ING PTMs identified to date, a subset has been characterized for their biological significance and have been shown to affect processes including subcellular localization, interaction with enzymatic complexes and ING protein half-life. The present review aims to highlight the emerging role of PTMs in regulating ING function and to suggest additional pathways and functions where PTMs may effect ING function.

DISCOVERY OF THE ING GENE FAMILY

The ING (INhibitor of Growth) family of proteins was discovered in 1996, using a subtractive hybridization-based tumour suppressor isolation strategy involving normal and cancer epithelial cells followed by an in vivo functional screen [1]. Overexpression of the first member of the ING family, ING1, was shown to effect cell-cycle arrest, primarily in the G0/G1-phase of the cell cycle [1]. The ING1 gene is located at the chromosomal location 13q34 and encodes four potential alternatively spliced isoforms, all containing a highly conserved PHD (plant homeodomain), commonly found in chromatin regulatory proteins [2,3]. ING1a and ING1b are expressed in almost all cell types, including primary fibroblasts and epithelial cells [4,5] and differ only in their N-termini (Figure 1). Several reports have suggested that ING1b can efficiently induce apoptosis when overexpressed [68]. In contrast, ING1a overexpression induces cellular senescence in primary fibroblasts [9]. A second member of the ING family, p33ING2 (ING2) was identified on the basis of sequence homology with ING1 and was found to also regulate cell growth [10]. The three remaining members of the ING family, p47ING3, p28ING4 and p28ING5, were identified through bioinformatic analyses of the human genome [4]. ING3 was later reported to modulate cell growth and p53-mediated transcription [11], whereas ING4 and ING5 were shown to associate with HAT (histone acetyltransferase) complexes and, like ING1 and ING2, regulate the acetylation status of p53 [12,13]. Phylogenetic analyses across taxonomically diverse organisms have shown that the ING family is evolutionarily well conserved from unicellular yeast to humans [14]. Bioinformatic analysis has revealed the existence of three ING genes in fungi, nematodes and Drosophila, whereas mammalian genomes encode five ING genes. ING1 and ING2 bear close resemblance structurally and functionally, as do ING4 and ING5 [14,15]. Although the biochemical function of ING proteins is still being actively investigated, ING proteins have been implicated in diverse cellular processes. These include apoptosis [68], the DNA damage response [16,17], chromatin remodelling [1820], hormonal responses [21,22], DNA repair and nucleotide excision repair [2325], senescence [9,13,26,27], cell-cycle regulation [28,29], TGFβ (transforming growth factor β) signalling [30], hypoxia-inducible pathways [3133], angiogenesis and cell migration [3436] and NF-κB (nuclear factor κB) signalling [37,38]. Roles for ING2 and ING5 have also been demonstrated in muscle differentiation [39] and stem cell maintenance [40] respectively.

Domains of the ING family of proteins

Figure 1
Domains of the ING family of proteins

The ING family is encoded by five genes (ING1ING5) and each of these generates several isoforms. ING1 and ING2 are closely related phylogenetically as are ING4 and ING5, whereas ING3 is comparatively distinct. UR represents unique regions of the ING proteins. Domains of the INGs include the PBD whose function is not well understood but it may contribute to the ability of ING1 to interact with the Sin3a HDAC complex. The LID is exclusive to the ING family of proteins in the human proteome. Lamin A/C interacts with the LID of ING1 and tethers it to the nucleus. The NLS is essential for translocation of INGs to the nucleus by binding to karyopherins. The PHD form of zinc finger is the most conserved domain of the ING family of proteins and binds to trimethylated Lys4 on histone 3 (the H3K4Me3 mark) with high affinity. The PBR binds bioactive phosphoinositides to activate ING1 and ING2 in response to stress and it overlaps with a UIM. Ubiquitin, therefore, competes with phosphoinositides at the PBR/UIM, linking stress-inducible phosphoinositide signalling with ubiquitin metabolism. NTS, nucleolar translocation sequence; PIP, PCNA-interacting protein. Arrows indicate major isoforms.

Figure 1
Domains of the ING family of proteins

The ING family is encoded by five genes (ING1ING5) and each of these generates several isoforms. ING1 and ING2 are closely related phylogenetically as are ING4 and ING5, whereas ING3 is comparatively distinct. UR represents unique regions of the ING proteins. Domains of the INGs include the PBD whose function is not well understood but it may contribute to the ability of ING1 to interact with the Sin3a HDAC complex. The LID is exclusive to the ING family of proteins in the human proteome. Lamin A/C interacts with the LID of ING1 and tethers it to the nucleus. The NLS is essential for translocation of INGs to the nucleus by binding to karyopherins. The PHD form of zinc finger is the most conserved domain of the ING family of proteins and binds to trimethylated Lys4 on histone 3 (the H3K4Me3 mark) with high affinity. The PBR binds bioactive phosphoinositides to activate ING1 and ING2 in response to stress and it overlaps with a UIM. Ubiquitin, therefore, competes with phosphoinositides at the PBR/UIM, linking stress-inducible phosphoinositide signalling with ubiquitin metabolism. NTS, nucleolar translocation sequence; PIP, PCNA-interacting protein. Arrows indicate major isoforms.

ING PROTEINS SERVE AS TARGETING MODULES FOR HAT AND HDAC (HISTONE DEACETYLASE) COMPLEXES

Like ING1, most of the other ING genes encode multiple splicing isoforms (Figure 1). The sequence homology displayed across ING family members [14] is also conserved at the functional level with ING1 and ING2 being stoichiometric members of Sin3a HDAC complexes (HDAC1 and 2). ING3 is a stoichiometric member of the NuA4/Tip60 [Tat (transactivator of transcription)-interactive protein 60 kDa] HAT complex, whereas ING4 and ING5 can be found in the HBO1 HAT complex. ING5, which plays a role in stem cell differentiation [40] can also be found in the MOZ/MORF HAT complex [15]. All of the ING proteins are believed to act as the targeting module of these HAT and HDAC complexes, by virtue of specifically recognizing the histone ‘mark’ H3K4Me3 (histone H3 trimethylated on Lys4) [4143].

KO (KNOCKOUT) MODELS OF THE INGs

ING1- and ING2-KO models have corroborated the role of ING proteins as bona fide tumour suppressors. ING1-KO mice develop B-cell lymphomas and are more sensitive to ionizing radiation [44,45]. Although ING1-KO mice are smaller in size compared with their wild-type counterparts, no significant differences in general morphology or physiology were noted. Unlike ING1-KO mice, male ING2-KO mice are defective in spermatogenesis, have degenerated seminiferous tubules and are infertile, whereas females appear relatively unaffected [46]. This is consistent with a role for ING2 in regulating chromatin structure as a member of HDAC complexes that serve to compact chromatin in developing sperm cells. An increase in the incidence of soft tissue sarcomas was also noted in ING2-deficient mice, further supporting their classification as tumour suppressors. KO of ING4 has indicated that it plays a role in the innate immune response [47], whereas KO of ING3 in Caenorhabditis elegans has identified a role for ING proteins in DNA damage-induced germ-line apoptosis [48].

THE ING TUMOUR SUPPRESSORS IN CANCER

Since the discovery of ING1 as a tumour suppressor [49] that is actively targeted to the nucleus by karyopherins [50], the mutational and expression status of the INGs has been studied in a wide variety of tissues. Each of the ING family members has been found to be altered in sequence, localization or amount in multiple cancer types. The first study of ING proteins in clinical cancer samples reported a marked reduction of ING1 mRNA expression in 44% of primary breast cancers and in all ten breast cancer cell lines examined. However, one germ-line missense and three silent mutations in the sequence were seen [51]. Reduced ING1 levels also correlated with an increased propensity for nodal metastasis. Decreased ING1 expression has been reported in lymphoid malignancies, human gastric cancer, non-small-cell lung carcinoma, astrocytoma, neuroblastoma and sporadic colorectal cancer [5257]. In contrast, increased expression of the ING1 isoform ING1b has been reported in melanoma cell lines, along with silent and possible missense mutations of the ING1 gene [58].

ING2 has also been reported to be down-regulated in a variety of lung cancer cell lines, hepatocellular carcinoma and non-small-cell lung carcinoma [5961]. Mislocalization of ING2 is observed in cutaneous melanomas [62] and ING2 deletions have been reported in a subset of advanced HNSCC (head and neck squamous cell carcinoma) [63]. In contrast with the majority of studies reporting inactivation of ING2 by various mechanisms, up-regulation of ING2 was seen in colon cancer where it regulates MMP (matrix metalloproteinase) 13, promoting cancer metastasis and invasion [64].

ING3 levels have been reported to have some utility in cancer prognosis. Significant loss of ING3 expression was seen in HNSCC and was later correlated with significantly reduced overall survival within a follow-up period of 5 years [65,66]. Similarly, ING3 was mislocalized in malignant melanoma where decreased nuclear protein levels combined with increased cytoplasmic ING3 was noted. Nuclear ING3 levels correlated with a decreased propensity of melanomas to progress and with improved 5-year disease-specific survival [67].

The role of ING4 in tumour progression was first reported in brain tumours [38]. ING4 expression was highly down-regulated in gliomas. Gliomas expressing the least ING4 were more aggressive and scored as higher-grade tumours. ING4 down-regulation resulted in higher expression of NF-κB-responsive genes that promote angiogenesis and tumorigenesis. ING4 deletion and down-regulation of ING4 were subsequently reported in HNSCC, hepatocellular carcinoma, gastric adenocarcinoma, human astrocytomas, lung cancer and HER2 (human epidermal growth factor receptor 2)-positive breast cancer [6871].

Cytoplasmic mislocalization of ING5 has been reported in HNSCC [72]. Deletion of the ING5 locus was first reported in OSCC (oral squamous cell carcinoma), one of the most common forms of HNSCC [73]. Also, significant reductions in ING5 mRNA levels and missense mutations in the LZL (leucine zipper-like region), NCR (novel conserved region) or LID (lamin-interacting domain) domains of ING5 have all been reported in OSCC [74].

Owing to their classification as tumour suppressors and their effect on cell growth and apoptosis, ING proteins have been examined for their efficacy in gene therapy or as agents in combinatorial cancer therapy [75]. The first studies of this kind showed that adenoviral ING1b in combination with p53 was extremely potent in killing glioma cells and human oesophageal carcinoma cells [8,76]. Adenovirus-mediated ING4 expression was also effective against human non-small-cell lung carcinoma, hepatocarcinoma, breast carcinoma and pancreatic carcinoma [7781].

In general ING proteins appear to be down-regulated and/or mislocalized in a broad range of cancer types. However, in some instances, overexpression also correlates with carcinogenesis and tumour aggressiveness. Since all ING proteins serve as stoichiometric members of HAT and HDAC complexes, both increased and reduced levels would be expected to alter the epigenome of cancer cells, particularly since some INGs share occupancy in the same chromatin remodelling complex.

DOMAINS OF THE ING PROTEINS

ING1–ING5 are multidomain proteins (Figure 1), with the PHD being the most highly conserved domain [2,14]. ING PHDs specifically recognize the N-terminus of histone H3 at the H3K4 residue in a manner sensitive to the degree of methylation of Lys4. This observation has led to the designation of INGs as ‘readers’ of the histone epigenetic code [41,42]. Since ING proteins are also stoichiometric members of HAT and HDAC complexes [15], their binding to methylated H3K4 residues targets chromatin remodelling activity to DNA. Therefore ING proteins may be considered important targeting components of histone code ‘writers’ as well.

All human ING proteins contain an NLS (nuclear localization signal) that targets them to the nucleus by binding to karyopherins [50]. Several of the ING proteins also contain NTS (nucleolar translocation sequence) motifs within the NLS that target ING1, at least, to the nucleoli under conditions of stress [82].

ING proteins also contain a sequence that is unique in the entire human proteome. This sequence was named the LID since it interacted most avidly with lamin A [83]. When mutated, lamin A is responsible for the devastating HGPS (Hutchinson–Gilford progeria syndrome), a form of premature aging in which children die of old age at ~13 years [84]. The interaction between the LID and lamin A has been hypothesized to help tether ING1 in the nucleus and regulate its functions as an epigenetic modifier. Since HGPS cells show marked alterations in their chromatin structure and ING1 does not interact with the mutant form of lamin called progerin, it has been proposed that ING proteins may play a role in the transduction of the HGPS phenotype, through altering the epigenetic status of lamin A mutant cells [83].

The PIP [PCNA (proliferating-cell nuclear antigen)-interacting protein] motif is unique to the ING1b isoform. ING1b interacts with PCNA in a UV-dependent manner to initiate a DNA repair response and/or induce apoptosis [16]. ING1b may also be important for E3 ubiquitin ligase Rad18-mediated PCNA mono-ubiquitination [25]. Mono-ubiquitinated PCNA associates with Polη (DNA polymerase η) to ensure proper lesion bypass and error-free DNA replication [25].

ING proteins also contain a PBD (partial bromodomain) that was identified by bioinformatic analysis [14]. Although a clear function of this domain has not been identified, this region interacts with SAP30 of the Sin3-HDAC1 and HDAC2 complexes [20] and thereby might recruit HAT or HDAC components for chromatin remodelling [15].

A PBR (polybasic region), closely juxtaposed to the PHD, is conserved between ING1 and ING2, but is absent in the other ING proteins (Figure 1). ING2 was shown to be the protein in the human proteome that was best able to bind to bioactive phosphoinositides such as phosphatidylinositol 5-phosphate in the nucleus. This interaction regulated the growth inhibitory effects of ING2 in a p53-sensitive manner [85,86]. The biological significance of this interaction is reflected by the fact that the rare phospholipids that bind specifically to ING1 and ING2 are stress inducible and highly bioactive, suggesting that the INGs that target HDAC complexes to chromatin are strongly induced in response to stress. Recently, an additional novel function for the PBR was identified. Analysis showed that the PBR overlaps with a UIM (ubiquitin-interaction motif) in the C-terminal region of ING1b [87]. Ubiquitin and phosphorylated lipid species competed for binding to this site, establishing a link between bioactive lipid signalling and ubiquitin-mediated proteosomal degradation [87]. The PBR/UIM of ING1b interacts with mono-ubiquitinated p53 and stabilizes it, most likely by blocking polyubiquitination through targeting deubiquitination.

An LZL is found in ING2, ING3, ING4 and ING5, but is absent from ING1. This was first identified by bioinformatic analysis of INGs from various species [14]. The LZL appears to be important for nucleotide-excision-repair-associated functions of ING2 and in UV-mediated apoptosis. On the basis of the spacing of leucine residues at ~7 amino acid intervals along the N-terminus of ING3 and ING4, it was proposed that these residues might promote homo- or hetero-dimer formation, or might confer upon ING proteins an ability to interact with a variety of other leucine-zipper-containing proteins [14]. Consistent with this prediction, X-ray crystallographic studies have reported the dimerization of ING4 via its N-terminal region that contains the LZL region [88]. The LZL of ING2 also interacts with components of HDAC1 complexes and was a critical domain in influencing muscle differentiation by ING2 [39].

The complete NMR or X-ray crystallographic structure for any of the ING proteins is yet to be solved, but atomic resolution crystal structures of ING-PHD fingers in complex with H3K4me3 have been determined using X-ray crystallography (reviewed in [89]). Structures of the soluble ligand-free states of the PHD fingers have also been obtained by NMR spectroscopy [89a]. The PHD is composed of three loops with two zinc-binding sites, four β-sheets and two α-helices, which recognize H3K4 in a methylation-sensitive manner. This aids in chromatin remodelling, allowing the ING proteins to function as histone code ‘readers’ as noted previously, binding with increasing avidity as the methylation state of H3K4 increases. ING family members can, thus, regulate chromatin by selective recruitment of different HAT and HDAC complexes, therefore targeting histone code ‘writers’ to the chromatin. Such chromatin alterations probably play a role in the mechanism by which ING family proteins induce apoptosis and act as tumour suppressors, but the details of these mechanisms are yet to be defined.

RAPID ALTERATION OF ING FUNCTIONS BY PTM (POST-TRANSLATIONAL MODIFICATION)

PTMs are a group of very dynamic processes effected by thousands of proteins whose functions are evolutionarily well conserved, helping to ensure proper PTM dynamics and fidelity. One protein can be conferred multiple functions through its ability to be post-translationally modified in various ways and many PTMs may act as ‘rheostats’ such as the progressive methylation of histone H3K4 promoting the increasing binding of ING PHD fingers [19]. A very large and fast growing literature is developing to describe the myriad of PTMs that contribute to regulating protein function in various ways. A list of PTMs whose functions or potential functions have been previously described as targeting members of the ING family are presented in Table 1. With the increased use of high-throughput analytical techniques such as MS, global proteome analysis has become a reality. This has added copious amounts of data to many fields of biological inquiry. These techniques have been useful in detecting very labile PTMs which otherwise are very challenging to identify using conventional biochemical procedures. The description of global phosphorylation during mitosis [90], SUMOylation in response to heat shock [91], ubiquitination [92], acetylation [93], O-linked glycosylation during cytokinesis [94], N-linked glycosylation [95] and several other PTMs under varied cellular conditions, have significantly added to our understanding of PTMs in different biological contexts (reviewed in [96]). In the present review, we identify all of the PTMs that have been reported for ING1–ING5 proteins to date. As described below, some of these PTMs have been well studied, whereas others that have been identified by high-throughput proteomic strategies are not yet well characterized.

Table 1
PTMs that modify members of the ING family and also modify other functionally related proteins

Non-ING proteins that are known to physically or functionally interact with the INGs and are modified by the indicated PTM are listed. Rb, retinoblastoma.

PTM Target substrate(s) Proposed function 
Acetylation ING2, ING3, ING4, ING5, p53, histones, ATM, Rb Stability, PTM cross-talk, subcellular localization, activity, protein–protein/DNA interaction 
Citrullination ING4 Protein stability, activity 
Methylation ING3, ING5, histones, Rb Gene activation or repression, chromatin remodelling, mRNA splicing, protein–DNA/RNA interaction, activity 
Phosphorylation ING1, RTKs, ATM, ATR, Chk1, Chk2 Protein–protein interaction and signal transduction, activity, localization, PTM cross-talk 
SUMOylation ING2, p53, RNF168 Stability, PTM cross-talk, subcellular localization, activity, protein–protein/DNA interaction, binding to SIMs 
Ubiquitination ING1, ING2, ING3, PCNA, histones Degradation, PTM cross-talk, binding to UIMs, signal transduction, activity 
PTM Target substrate(s) Proposed function 
Acetylation ING2, ING3, ING4, ING5, p53, histones, ATM, Rb Stability, PTM cross-talk, subcellular localization, activity, protein–protein/DNA interaction 
Citrullination ING4 Protein stability, activity 
Methylation ING3, ING5, histones, Rb Gene activation or repression, chromatin remodelling, mRNA splicing, protein–DNA/RNA interaction, activity 
Phosphorylation ING1, RTKs, ATM, ATR, Chk1, Chk2 Protein–protein interaction and signal transduction, activity, localization, PTM cross-talk 
SUMOylation ING2, p53, RNF168 Stability, PTM cross-talk, subcellular localization, activity, protein–protein/DNA interaction, binding to SIMs 
Ubiquitination ING1, ING2, ING3, PCNA, histones Degradation, PTM cross-talk, binding to UIMs, signal transduction, activity 

PHOSPHORYLATION OF THE INGs

Phosphorylation is arguably the best-described of all PTMs and is known to contribute to regulating the majority of cellular processes and have an impact on virtually all fundamental processes [97]. Phosphorylation involves the addition of phosphate groups from high-energy donors such as ATP (Figure 2). Phosphoproteomic enrichment coupled with high-resolution MS analysis suggests that more than 70% of the proteome is phosphorylated to varying degrees [90]. The ING1 protein has also been shown to be phosphorylated at several residues. The first phosphorylation target residue identified for any ING protein was Ser199 of ING1b. Once phosphorylated, Ser199 and adjacent amino acid residues serve as a docking site for 14-3-3 proteins [98]. Upon binding, 14-3-3 regulates both the subcellular localization of ING1 and its activity. For example phospho-serine-mediated 14-3-3 binding and nuclear export regulate the ability of ING1b to transcriptionally regulate the p21 cyclin-dependent kinase inhibitor and subsequent induction of apoptosis in response to DNA damage [98]. The evolutionarily conserved counterpart of Ser199 on ING1a is Ser342. This residue is also phosphorylated [90], suggesting a conserved role for this modification in both ING1 isoforms. Another residue on ING1b, Ser126, is phosphorylated in response to genotoxic stress [99]. Chk1 (checkpoint kinase 1) may be the predominant kinase that phosphorylates ING1b in response to stress at this site. Chk1 is a downstream effector in the ATM (ataxia telangiectasia mutated)/ATR (ATM- and Rad3-related) phosphorylation cascade. Ser126 phosphorylation alters the half-life (t1/2) of ING1b. Wild-type ING1b has a t1/2 of ~17 h in the human melanoma cell line MMRU. In contrast, the S126A mutant which cannot be phosphorylated at this residue shows a very short t1/2 of ~6 h. It was further reported that in the absence of stress, Ser126 phosphorylation is cell-cycle-dependent and is maximum during the G2/M-phase of the cell cycle. In contrast, in UV-treated cells, phosphorylated Ser126 peaks during late S-phase followed by a gradual decrease. Phosphorylated Ser126-ING1b has the ability to down-regulate cyclin B1 expression either directly or as a consequence of cell-cycle arrest and thereby might contribute to regulating cell cycle and proliferation by this target gene [99]. ING1b is degraded in the 20S proteasome. In response to stress, phosphorylated Ser126-ING1b binds to NAD(P)H quinone oxidoreductase 1, which inhibits the degradation of phosphorylated ING1b [100].

Major PTMs in eukaryotes that target the ING proteins

Figure 2
Major PTMs in eukaryotes that target the ING proteins

(i) Serine, tyrosine or threonine residues on ING proteins serve as phosphate group acceptors. The CDK1 (cyclin-dependent kinase 1) and Chk1 (checkpoint kinase 1) serine/threonine kinases and Src tyrosine kinase have been implicated in phosphorylating the INGs, but the phosphatases that reverse these phosphorylations are currently unknown. (ii) Acetylation usually occurs on internal lysine residues; however, N-terminal acetylation also occurs as a co-translational modification and is rarely a PTM. Acetylation is mediated by several HATs that are more broadly referred to as KATs. This is also a reversible PTM by HDACs, also referred to as KDACs. In humans, mice and other eukaryotes KDACs are classified on the basis of sequence identity and the need for the cofactor NAD+ for catalysing removal of acetyl groups. Some evidence suggests that INGs may be modified by members of the KAT and KDAC complexes that they participate in as stoichometeric members. (iii) Citrullination is the conversion of arginine or trimethylated arginine residues into citrulline by calcium-dependent PADs, releasing amine or methylamine respectively. ING4 has been reported to be citrullinated. (iv) Ubiquitination is addition of ubiquitin (Ub) protein moieties on lysine residue(s) of a protein and is mediated by a three-stage enzymatic cascade consisting of ubiquitin E1 activation enzyme, ubiquitin E2 conjugation enzyme and E3 ubiquitin ligase. ING1–ING5 have been reported to be ubiquitinated and ING1 interacts with the de-ubiquitination (DUB) enzyme HAUSP, to regulate p53 turnover. (v) SUMOylation is similar to ubiquitination and is catalysed in a similar three-step manner. However, unlike ubiquitin, three different SUMO proteins can be added to proteins and SUMOylation has been reported to affect the binding affinity of ING2 to the Sin3a–HDAC complex. (vi) Protein methylation of lysine and arginine residues is catalysed by the transfer of methyl groups from S-adenosylmethionine (AdoMet) by methyl transferases. AdoHcy, S-adenosylhomocysteine. ING3 has been reported to be dimethylated on Arg19, but the functional consequences of this modification are unknown. HMT, histone methyltransferase; PRMT, protein arginine N-methyltransferase.

Figure 2
Major PTMs in eukaryotes that target the ING proteins

(i) Serine, tyrosine or threonine residues on ING proteins serve as phosphate group acceptors. The CDK1 (cyclin-dependent kinase 1) and Chk1 (checkpoint kinase 1) serine/threonine kinases and Src tyrosine kinase have been implicated in phosphorylating the INGs, but the phosphatases that reverse these phosphorylations are currently unknown. (ii) Acetylation usually occurs on internal lysine residues; however, N-terminal acetylation also occurs as a co-translational modification and is rarely a PTM. Acetylation is mediated by several HATs that are more broadly referred to as KATs. This is also a reversible PTM by HDACs, also referred to as KDACs. In humans, mice and other eukaryotes KDACs are classified on the basis of sequence identity and the need for the cofactor NAD+ for catalysing removal of acetyl groups. Some evidence suggests that INGs may be modified by members of the KAT and KDAC complexes that they participate in as stoichometeric members. (iii) Citrullination is the conversion of arginine or trimethylated arginine residues into citrulline by calcium-dependent PADs, releasing amine or methylamine respectively. ING4 has been reported to be citrullinated. (iv) Ubiquitination is addition of ubiquitin (Ub) protein moieties on lysine residue(s) of a protein and is mediated by a three-stage enzymatic cascade consisting of ubiquitin E1 activation enzyme, ubiquitin E2 conjugation enzyme and E3 ubiquitin ligase. ING1–ING5 have been reported to be ubiquitinated and ING1 interacts with the de-ubiquitination (DUB) enzyme HAUSP, to regulate p53 turnover. (v) SUMOylation is similar to ubiquitination and is catalysed in a similar three-step manner. However, unlike ubiquitin, three different SUMO proteins can be added to proteins and SUMOylation has been reported to affect the binding affinity of ING2 to the Sin3a–HDAC complex. (vi) Protein methylation of lysine and arginine residues is catalysed by the transfer of methyl groups from S-adenosylmethionine (AdoMet) by methyl transferases. AdoHcy, S-adenosylhomocysteine. ING3 has been reported to be dimethylated on Arg19, but the functional consequences of this modification are unknown. HMT, histone methyltransferase; PRMT, protein arginine N-methyltransferase.

Recent findings also suggest that the Src kinase phosphorylates ING1b and relocalizes it to the cytoplasm. Src kinase also significantly alters the t1/2 of ING1b. In the presence of active Src the t1/2 of ING1b is 8 h which is significantly less than the wild-type ING1b t1/2 of 17–18 h. It was further noted that in the presence of Src, ING1b has an attenuated ability to induce ING1-mediated apoptosis (L. Yu, S. Thakur, R.Y.Y. Leong-Quong, K. Suzuki, A. Pang, J.D. Bjorge, K. Riabowol and D.J. Fujita, unpublished work). Thus interaction with Src and subsequent phosphorylation might regulate ING1b function by relocating it to the cytoplasm and eventually targeting it for degradation. This is in contrast with the Ser126 phosphorylation that increases the t1/2 of ING1b.

High-resolution MS-based phosphoproteome analysis of the human cell cycle has also identified several sites in all five ING proteins that can be phosphorylated in a cell-cycle-dependent or -independent manner [90]. Although the relevant kinases that target these residues can be predicted by sequence analysis, most have not been confirmed by independent biochemical analyses. Despite this, some of these residues are likely to be significant targets. For example, ING1 is phosphorylated on Tyr44 [90] and this residue is specific to the ING1b isoform of ING1. However, this tyrosine residue is also conserved in ING2 and the corresponding amino acid has been reported to also be phosphorylated (Tyr56) as suggested by MS data [90]. The identified residue and peptide is listed in PHOSIDA [101,102]. This is of special interest because both ING1b and ING2 interact with the Sin3a–HDAC1 complex through their N-termini [28,39,103]. Therefore this modification might influence ING1 and ING2 residency in the Sin3a–HDAC complex. Another interesting modification on ING1a, the longest isoform of ING1, is the phosphorylation of Ser293, corresponding to Ser150 in ING1b. Phosphorylated Ser293-ING1a was identified in a screen for phospho-PKC (protein kinase C) substrates as listed in the PhosphoSitePlus® database (http://www.phosphosite.org) [104], suggesting that PKC might also regulate ING1 function. Figure 3 shows the phosphorylation sites that have been identified on ING proteins, and Figure 4 lists the various kinases INGs interact with. As noted above, in most cases the physiological roles and biological significance of the phosphorylated forms of ING proteins are yet to be determined.

PTMs of ING1–ING5

Figure 3
PTMs of ING1–ING5

Different PTMs that have been reported to occur on the five members of ING family. Target residues have been biochemically validated or identified in unbiased proteomic analyses. AcK, acetylated lysine; meR, methylated arginine; PolyUbK, polyubiquitinated lysine; pS, phosphorylated serine; pY, phosphorylated tyrosine; pT, phosphorylated threonine; RCitrullin, arginine into citrullin conversion; SUMO1K, SUMOylated lysine; UbK, ubiquitinated lysine. Residues in red are biochemically validated PTMs.

Figure 3
PTMs of ING1–ING5

Different PTMs that have been reported to occur on the five members of ING family. Target residues have been biochemically validated or identified in unbiased proteomic analyses. AcK, acetylated lysine; meR, methylated arginine; PolyUbK, polyubiquitinated lysine; pS, phosphorylated serine; pY, phosphorylated tyrosine; pT, phosphorylated threonine; RCitrullin, arginine into citrullin conversion; SUMO1K, SUMOylated lysine; UbK, ubiquitinated lysine. Residues in red are biochemically validated PTMs.

ING-interacting proteins that mediate ING modification

Figure 4
ING-interacting proteins that mediate ING modification

A global protein interactome analysis of yeast [124] revealed that proteins found in several PTM pathways interacted with the three yeast ING proteins, Yng1, Yng2 and Pho23. (i) Given that INGs are part of HAT and HDAC complexes, they interact with many proteins that acetylate and de-acetylate histones and non-histone substrates. Thus it is tempting to speculate that any of these acetylation or deacetylation complexes could also target INGs. CBP, CREB (cAMP-response-element-binding protein)-binding protein; SIRT1, sirtuin1. (ii) ING1 interacts with several phosphorylation pathway proteins [124]; however, few kinases have been biochemically validated to interact with or phosphorylate INGs. p38MAPK (mitogen-activated protein kinase) interacts with ING1 in a UV-dependent manner, but the function of this interaction is unknown. Chk1 and CDK1 regulate ING1 phosphorylation on Ser126 and could be regulated in a DNA-damage-responsive manner. Src also phosphorylates ING1 and translocates it to the cytoplasm. (iii) Only ING2 has been reported to be SUMOylated on its NLS and this regulates its interaction with the Sin3a–HDAC complex. (iv) PAD4 interacts with ING4 and citrullinates the NLS domain on ING4 and inhibits its ability to interact with, and acetylate p53. Citrullination of other family members has not been reported. (v) ING1 and ING2 proteins harbour a UIM motif that binds ubiquitin and stabilized mono-ubiquitinated forms of p53; however, ING proteins also interact with several E3 ubiquitin ligases that mediate ubiquitination and degradation in a proteasome-dependent manner. Yeast interactome analysis identified several other E1, E2 and E3 ubiquitin pathway proteins that interact with yeast INGs; however, the functional consequences of these interactions remain to be clarified.

Figure 4
ING-interacting proteins that mediate ING modification

A global protein interactome analysis of yeast [124] revealed that proteins found in several PTM pathways interacted with the three yeast ING proteins, Yng1, Yng2 and Pho23. (i) Given that INGs are part of HAT and HDAC complexes, they interact with many proteins that acetylate and de-acetylate histones and non-histone substrates. Thus it is tempting to speculate that any of these acetylation or deacetylation complexes could also target INGs. CBP, CREB (cAMP-response-element-binding protein)-binding protein; SIRT1, sirtuin1. (ii) ING1 interacts with several phosphorylation pathway proteins [124]; however, few kinases have been biochemically validated to interact with or phosphorylate INGs. p38MAPK (mitogen-activated protein kinase) interacts with ING1 in a UV-dependent manner, but the function of this interaction is unknown. Chk1 and CDK1 regulate ING1 phosphorylation on Ser126 and could be regulated in a DNA-damage-responsive manner. Src also phosphorylates ING1 and translocates it to the cytoplasm. (iii) Only ING2 has been reported to be SUMOylated on its NLS and this regulates its interaction with the Sin3a–HDAC complex. (iv) PAD4 interacts with ING4 and citrullinates the NLS domain on ING4 and inhibits its ability to interact with, and acetylate p53. Citrullination of other family members has not been reported. (v) ING1 and ING2 proteins harbour a UIM motif that binds ubiquitin and stabilized mono-ubiquitinated forms of p53; however, ING proteins also interact with several E3 ubiquitin ligases that mediate ubiquitination and degradation in a proteasome-dependent manner. Yeast interactome analysis identified several other E1, E2 and E3 ubiquitin pathway proteins that interact with yeast INGs; however, the functional consequences of these interactions remain to be clarified.

METHYLATION OF ING3 AND ING5

The only reports of ING protein methylation to date are for ING3 and ING5. A high-throughput screen identified Arg19 of ING3 as a dimethylated residue and Arg126 of ING5 as a monomethylated residue [104], but the functional consequences of these modifications are unknown.

ACETYLATION OF ING PROTEINS

The addition of acetyl groups, primarily on lysine residues located on the N-termini of proteins or on internal amino acids, alters their charge significantly. N-terminal acetylation usually occurs during translation, and rarely after translation. The N-termini of newly synthesized proteins is modified by NATs (N-acetyl transferases) during translation and this occurs after the initiator methionine residue is cleaved from the protein (reviewed in [105]). Serine, threonine, glycine, alanine, valine, aspartic acid and glutamic acid are often modified by NATs [105]. However, internal acetylation happens primarily on lysine residues and is facilitated by several HATs or HDACs also called KATs (lysine acetyltransferases) or KDACs (lysine deacetylases). Acetylation is considered to be one of the most important modifications regulating chromatin-based gene transcription (reviewed in [106]). It is well established that INGs are members of HAT and HDAC complexes (Figure 4), so it is tempting to speculate that they may also be substrates for acetylation. Previously, a human acetylome project identified ING3, ING4 and ING5 as novel substrates for acetylation on more than one lysine residue [93]. PhosphoSitePlus® also lists ING1 and ING2 as proteins likely to be acetylated. Although more than 20 acetylation sites have been identified (Figure 3), consequences of ING protein acetylation have not been determined for any site. This is particularly interesting given the fact that the INGs reside in complexes that function to increase (HAT) or decrease (HDAC) acetylation levels [2,15]. ING3, the third member of the family, is acetylated on several lysine residues [93], all belonging to the unique region of the protein. These observations suggest that acetylation might be a mechanism by which this protein is distinctively regulated in the cell.

UBIQUITINATION OF ING PROTEINS

The conjugation of ubiquitin to the lysine residues of proteins involves several proteins, including E1, E2 and E3 ligases. The ubiquitin protein itself consists of 76 amino acids (~8.5 kDa). Proteins can be mono-ubiquitinated (addition of a single ubiquitin moiety on one lysine residue), multi-monoubiquitinated (addition of single ubiquitin moieties on multiple lysine residues) or polyubiquitinated (multiple ubiquitin moieties tandemly repeated on a single lysine residue). The first identified function of ubiquitination was to target proteins for proteasome-dependent degradation. Recent work has highlighted the role of ubiquitination and the ubiquitin-binding motif in cell signalling, DNA damage repair, autophagy and other cellular processes [107]. It is estimated that at least tetra-ubiquitination of a protein is required for its efficient proteasome-dependent degradation [108]. Interestingly, ING1b has been reported to be degraded in the 20S proteasome, but the specific number of ubiquitin residues added to ING1 was unclear since multi-ubiquitinated forms of ING1 were not observed under the experimental conditions used [100]. ING2 and ING3 were also reported to be degraded through a ubiquitin-dependent proteosomal system. The PHD of ING2 was crucial for its proteosome-mediated degradation [109], whereas Lys96 was the major ubiquitin acceptor site identified in ING3 [110]. It is worth noting that mono-ubiquitination of ING1b might have a role distinct from serving as a tag for degradation since ING1b is involved in the DNA damage response and inducibly binds PCNA with high avidity [16]. Despite ING1–ING3 being reported to be ubiquitinated, the target residues of these proteins are unknown. Although ubiquitination of ING4 and ING5 has not been well characterized to date, two unbiased proteomics strategies identified Lys66 of ING4 and Lys67 of ING5 as ubiquitin acceptors, as shown in Figure 3 [111,112].

CITRULLINATION OF ING PROTEINS

The PTM of citrullination occurs when arginine is converted into citrulline by PAD (peptidylarginine deaminase) as shown in Figure 2. A high-density protein array identified ING4 as a non-histone substrate of PAD4, a major histone PAD [113]. The NLS of ING4 was citrullinated on more than one residue with preferred sites being Arg133 and Arg166 and this was reported to regulate the ING4–p53 interaction and the ability of p53 to induce p21 expression. Citrullination of ING4 also decreased its ability to acetylate p53. Citrullination was also reported to alter the t1/2 of ING4 and promote its degradation [113]. Therefore it will be interesting to see whether other members of the ING family can serve as substrates for citrullination.

SUMOYLATION OF ING PROTEINS

SUMOylation is another PTM in which SUMO (small ubiquitin-related modifier) polypeptides of ~100 amino acids and 12 kDa (this may vary among species) are attached to the lysine residues of proteins. The general mechanism of SUMOylation is similar to that of ubiquitination (Figure 2). SUMO derives its name from ubiquitin with which it shares structural similarities and also the presence of a ubiquitin fold; although there is only approximately 18% sequence identity between ubiquitin and SUMO. Three different SUMO protein isoforms (SUMO 1–3) have been identified in vertebrates, including humans [114]. RanGTPase (RanGap1) was the first protein that was shown to be SUMOylated [115]. Over the last 20 years, more than 1000 protein substrates have been shown to be the target of SUMO 1–3 isoforms. Proteome-wide analyses by several groups [91,116,118–120] have implicated SUMOylated protein function in processes such as DNA repair, nucleosome remodelling, RNA binding, transcription, tumour suppression and the heat-shock response, among others.

The second member of the ING family, ING2, was shown to be SUMOylated in vitro and in vivo on Lys195 [121]. The SUMOylation of ING2 did not play a role in ING2-mediated cell-cycle regulation [28]; however, SUMOylation of ING2 enhanced its association with the Sin3a–HDAC complex, thereby modulating HDAC-mediated alterations in gene expression. SUMOylation of ING2 also regulates its binding to the promoter of the TMEM71 gene which encodes transmembrane protein 71. SUMOylation-deficient mutants of ING2 are not able to significantly up-regulate TMEM71 and several other genes owing to their lower affinity for the Sin3a–HDAC complex [121]. Consistent with this study in human cells, an MS-based global analysis of SUMO targets in Saccharomyces cerevisiae revealed that pho23, one of the yeast ING counterparts, was a SUMOylated protein [122]. Several SUMOylation prediction software packages also predict the ability of ING1 and ING3 to be SUMOylated [123]; however, it would be interesting to test the biological relevance and consequences of SUMOylation of these and other ING family members.

CONCLUDING REMARKS

Since the discovery of the first ING protein, several groups have independently identified diverse biological roles that ING proteins fulfil. PTM of these proteins has only recently begun to be studied. The modifications of the INGs that have been identified appear to alter localization, activate them for entering multiprotein complexes of various types and alter their half-lives. Since the INGs appear to play central roles as stoichiometric members of chromatin-modifying complexes and as tumour suppressors, altering their levels within cells is likely to have significant biological repercussions. Understanding the fine tuning of these proteins through various PTMs should help us to determine how these epigenetic regulators function as tumour suppressors, and how they may be exploited as tools or as future therapeutic targets.

Abbreviations

     
  • ATM

    ataxia telangiectasia mutated

  •  
  • ATR

    ATM- and Rad3-related

  •  
  • Chk1

    checkpoint kinase 1

  •  
  • HAT

    histone acetyltransferase

  •  
  • HDAC

    histone deacetylase

  •  
  • HGPS

    Hutchinson–Gilford progeria syndrome

  •  
  • HNSCC

    head and neck squamous cell carcinoma

  •  
  • ING

    inhibitor of growth

  •  
  • KAT

    lysine acetyltransferase

  •  
  • KDAC

    lysine deacetylase

  •  
  • KO

    knockout

  •  
  • LID

    lamin-interacting domain

  •  
  • LZL

    leucine zipper-like region

  •  
  • NAT

    N-acetyl transferase

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NLS

    nuclear localization signal

  •  
  • OSCC

    oral squamous cell carcinoma

  •  
  • PAD

    peptidylarginine deaminase

  •  
  • PBD

    partial bromodomain

  •  
  • PBR

    polybasic region

  •  
  • PCNA

    proliferating-cell nuclear antigen

  •  
  • PHD

    plant homeodomain

  •  
  • PKC

    protein kinase C

  •  
  • PTM

    post-translational modification

  •  
  • SUMO

    small ubiquitin-related modifier

  •  
  • UIM

    ubiquitin-interaction motif

FUNDING

K.R. is a Scientist of the Alberta Heritage Foundation for Medical Research. This work was supported by the Canadian Institutes of Health Research and the Canadian Breast Cancer Foundation (to K.R.).

We thank Dr Pinaki Bose for his insightful comments and for reviewing the paper before submission.

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