NIRF appears to represent an intermodular hub protein that interacts with the core components of the cell cycle machinery and exhibits a unique centrality in the hierarchical modular architecture of the cell cycle network. NIRF ubiquitinates the two different classes of G1 cyclins (D1 and E1), and induces G1 arrest. DNA copy number loss in various malignancies was found for the NIRF gene, which was the target of a recurrent microdeletion in non-small cell lung carcinoma. In addition, the NIRF gene was closely linked with a susceptibility locus of colorectal carcinoma. These observations suggest that NIRF is a novel component of the core cell cycle machinery and is also a novel tumor suppressor.
Hub proteins hold the whole network together and are therefore important in both the topology maintenance and functioning of the network.2,4
The analysis of the NIRF PPI network indicated its scale-free property, and demonstrated the presence of a few highly connected hubs in the cell cycle network. Applying the top 20% as a general threshold,23
the degree and centrality indices for NIRF were sufficiently high that NIRF can be judged to be a hub protein with high betweenness. While the degree and centrality indices of NIRF were not among the top-ranked ones, it is often mentioned that more extensively studied proteins are prone to investigative bias to have more interactions than less intensely studied proteins.72–74
In addition, the centrality indices of a node tend to be correlated with its degree,44,75
indicating that there are also biases in the centrality indices of the well-known proteins in the network. Accordingly, it is feasible that the centrality indices as well as the degree determined for NIRF in this study are all underestimated. More interacting partners will probably be found for NIRF in the near future, which will raise its centrality indices even higher.
The cell cycle network directs not only cell cycle progression, but also diverse cellular processes,1
in which NIRF behaves as an intermodular hub protein.5,7
NIRF is a hub protein that characteristically interacts with the principal components of the cell cycle network, i.e., cyclins and their CDKs, pRB and p53. Each of these molecules is also a hub protein, with profound influences on different aspects of the cell cycle. These hub proteins individually interact with many other proteins composing the respective functional modules, which as a whole constitute a huge PPI network.6
Eventually, the cell cycle network organized in this way not only regulates the cell cycle but also orchestrates versatile cellular functions such as cell growth, death, differentiation and senescence.1
In this sense, the cell cycle network itself can be viewed as occupying a pivotal position in the cellular interactome network comprising a multitude of signaling pathways. On the other hand, NIRF is an intermodular hub protein with high betweenness, and is thereby supposed to occupy a unique critical position not only in the cell cycle network but also in the whole information network of the cell. As an intermodular hub protein connecting network modules, NIRF may be involved in the coordination of diverse information flows related to cell cycle control. Importantly, mutations of intermodular hubs are frequently associated with carcinogenesis.7,76
This is because such aberrations in intermodular hubs result in abnormalities in both network organization and information flow, culminating in global dysfunction of the network.7,8
Accordingly, it is possible that abnormalities in the NIRF gene may abrogate the integrity of the cell cycle network, which might ultimately bring about tumorigenesis.
NIRF is capable of ubiquitinating both cyclins D1 and E1 that cooperate with each other to control the G1
This is consistent with the idea that NIRF is a cell cycle protein involved in G1
The ubiquitination of these G1
cyclins has been assigned to the SCF-type ubiquitin ligases.12,17
Our findings demonstrate that NIRF is another ubiquitin ligase for cyclins D1 and E1. Intriguingly, there are some differences in the ubiquitinating properties between NIRF and the SCF ubiquitin ligases. First, the SCF ubiquitin ligases are only able to target either cyclin D1 or E, but not both,18
thereby allowing individual control for each cyclin. In contrast, NIRF ubiquitinates both G1
cyclins, which could provide coordinated regulation at the G1
/S boundary. Second, NIRF seems to directly bind to the G1
cyclins independently of phosphorylation, as shown by our in vitro assay results that NIRF binds to cyclin D1 and E1 and directly promotes their ubiquitination. In contrast, the SCF ligases recognize the cyclins only when they are phosphorylated.13–16
It is noteworthy that, in addition to the phosphorylation-dependent ubiquitination, phosphorylation-independent ubiquitination pathways have been proposed for both cyclins D113
To the best of our knowledge, NIRF is the first example of a single-subunit ubiquitin ligase grouped into the latter category. These differences between NIRF and the SCF ligases suggest a unique mechanism underlying the substrate recognition by NIRF. In addition, our results highlight a possible characteristic property of NIRF as a hub-type ubiquitin ligase targeting multiple substrates for ubiquitination. Thus, in cases with abnormalities of NIRF, the control for both cyclins D1 and E might become coincidentally abrogated, which could readily result in impaired control at the G1
/S boundary. This is one possible scenario in which defects in an intermodular hub with ubiquitin ligase activity disrupt the integrity of the cell cycle control, ultimately leading to tumor formation. A similar related example has been proposed for BRCA1.7
From the above considerations, we hypothesized that NIRF may be involved in the genesis of malignancies as a novel tumor suppressor. In support of this, the NIRF gene is localized in 9p24, which is deleted in numerous types of tumors at the highest frequency46
and where many publications have proposed the presence of various tumor-associated genes.60–66
Indeed, our database analyses provided findings compatible with our hypothesis. A significant loss of the DNA copy number of the NIRF gene was shown in several types of tumors. In particular, a recurrent microdeletion targeting NIRF was found in non-small cell lung carcinoma. Consistently, the NIRF mRNA was reduced in lung adenocarcinoma and squamous cell lung carcinoma, both of which are subtypes of non-small cell lung carcinoma. These analytic findings suggest that loss of the NIRF gene is associated with tumor formation, supporting the concept that NIRF is a tumor suppressor. This is in good agreement with the observation that NIRF induces G1
arrest, since many tumor suppressors function at the G/S boundary to inhibit inappropriate cell cycle progression.1,37
To our surprise, the SNP rs719725, a reported susceptibility locus for colorectal carcinoma,25
is present in the immediate vicinity of the NIRF gene, further strengthening the possible relationship of NIRF with carcinogenesis. Of note, this SNP is localized just within the commonly deleted region observed in the non-small cell lung carcinoma data set. This observation indicates that this SNP locus is really a target of chromosomal aberration in a particular type of cancer. It also suggests that a single deletion event can simultaneously affect both the NIRF gene and this SNP locus. Although the SNP rs719725 has not been known to be linked with the risk of non-small cell lung carcinoma, it is not surprising that a single locus is associated with predispositions to multiple types of tumors. Therefore, the above observations meet our expectation that a chromosomal deletion of the NIRF gene might be involved in the formation of tumors, again raising the probability that NIRF functions in tumor suppression.
To characterize NIRF as a unique hub protein in the cell cycle network, several more points should be taken into account. First, NIRF interacts with the two major tumor suppressors (pRB and p53) as well as the two different G1
cyclins (cyclins D1 and E1), all of which are the critical regulators of the G1
checkpoint system. The G1
checkpoint is the most critical barrier that hinders cells from undergoing malignant transformation.78
Given that NIRF exhibits several properties compatible with a tumor suppressor, these interactions suggest an intense functional interplay between NIRF and all of the above cell cycle proteins to ensure the precise functioning of the G1
checkpoint control. NIRF might cooperate with pRB and p53 to effectively induce G1
arrest in response to various insults that trigger DNA damage. Second, NIRF also interacts with the mitotic cyclins (cyclins A2 and B1), suggesting other roles for NIRF in the intra-S and G2
/M controls. These are novel findings that demonstrate a distinguishing characteristic of NIRF, since no other ubiquitin ligases interact with all the members of the cyclin family. These observations also suggest the possibility that a common mechanism is responsible for the binding between NIRF and the different classes of cyclins (manuscript in preparation). Lastly, NIRF has a paralogous ubiquitin ligase, Np95/ICBP90/UHRF1, with opposing effects on cell cycle progression.31,32
This paralog has been reported to be involved in the epigenetic inheritance of DNA methylation through an association of its SRA domain with DNMT1.79
While our study characterized the relationships of NIRF with the PPI network, ubiquitination and genetic aberration, corresponding studies regarding Np95/ICBP90/UHRF1 have not yet been reported. A comparative study will be informative to better define the role for this family in cell cycle control, in light of their associations with cancer. In addition, there is a high degree of sequence conservation between the SRA domains of NIRF and Np95/ICBP90/UHRF1,21,39
suggesting that NIRF also functions in epigenetic regulation. In this regard, a further study should be performed to examine the possible biological couplings among the cell cycle, ubiquitination and epigenetic regulations by NIRF and Np95/ICBP90/UHRF1.
In summary, based on the characteristic centrality within the hierarchical modular architecture of the cell cycle network, NIRF is supposed to influence multiple signaling modules related to cell cycle control. It is also suggested that NIRF induces G1 arrest by ubiquitinating both cyclins D1 and E1 and thereby acts as a tumor suppressor. We propose that NIRF occupies a pivotal position within the global information network of the cells, thus defining a critical nodal point connecting multiple signaling modules whose defect is associated with tumorigenesis.