On the relationship status for Arf and NPM1–it’s complicated

Abstract

Arf levels are tightly regulated in cells and correlate with the level of ribosome biogenesis and proliferative status of cells. Through multivalent interactions with NPM1 – a regulator of ribosome biogenesis, and Mdm2 – a regulator of cellular fate, Arf integrates within the nucleolar matrix, altering its structure, dynamics and function and therefore modulates the cell cycle.

Graphical abstract

Arf levels are tightly regulated in cells and correlate with the level of ribosome biogenesis and proliferative status of cells. Through multivalent interactions with NPM1 – a regulator of ribosome biogenesis, and Mdm2 – a regulator of cellular fate, Arf integrates within the nucleolar matrix, altering its structure, dynamics and function and therefore modulates the cell cycle.

The nucleolar protein nucleophosmin (NPM1) and tumor suppressor protein p14Arf interact and function together in tumor suppression and cellular stress sensing pathways. Although known for more than a decade, characterization of the structural mechanism of this interaction has been challenging. In this issue of The FEBS Journal, Luchinat, et al. [1], provide new insights into this elusive interaction.

Human p14Arf protein (p19Arf in mice) is intrinsically disordered [1, 2], the translational product of an alternative reading frame within the Ink4a locus on chromosome 9p21, which also encodes the cyclin-dependent kinase inhibitor, p16^Ink4A [3]. These two structurally and functionally unrelated proteins are the product of alternative first exons and share second and third exons (Fig. 1A), and they mediate tumor suppression through independent mechanisms [4].

Figure 1. An alternative reading frame within the Ink4a locus encodes a disordered, polycationic protein.

(A) Ink4a locus encodes for two unrelated tumor suppressor proteins, p14Arf and p16^Ink4A; exons encoding for Arf and p16^Ink4A are color-coded blue and purple, respectively. Dual colored exons represent overlap coding regions. (B) Net charge per residue analysis (prepared using CIDER, http://pappulab.wustl.edu/CIDER/) of the human (p14Arf) and mouse (p19Arf) protein sequences. Charge distribution in the C-terminal region of p19Arf is more uniform compared to p14Arf, and thus the location of a possible R2 motif is unclear.

The cellular levels of p14Arf and p19Arf (referred to generally as ”Arf”) are tightly regulated during the cell life cycle through transcriptional, translational and degradative mechanisms. Arf expression is suppressed in normal, dividing cells through the transcription repressor activity of p53 [3] and is induced in response to hyper-proliferative signals resulting from abnormal expression of oncogenes, such as Ras, c-Myc, v-Abl, E1A and E2F1 [5]. Upon expression, Arf functions as a bona fide tumor suppressor [3]; Arf^−/− mice develop sarcomas and other tumors due to failure of p53-dependent checkpoints that control cell cycle arrest and apoptosis [4, 6]. Arf activates p53 by directly binding and inhibiting Mdm2 (also known as Hdm2), the E3 ubiquitin ligase that mediates p53 proteasomal degradation; interestingly, this mechanism is associated with co-localization of Arf and Mdm2 in the nucleolus [5]. Furthermore, a study in mice showed that triple knockout animals, lacking the entire Arf-p53-Mdm2 axis, developed more tumors than p53^−/−/Mdm2^−/− and p53^−/− animals [7], revealing additional, p53-independent tumor suppressor roles for Arf [4]. One such p53-independent, anti-proliferative function of Arf is inhibition of ribosome biogenesis through interactions, in the nucleolus, with NPM1 [8].

Maintenance of basal Arf expression is required for nucleolar homeostasis; for example, deletion of p19Arf resulted in NPM1-dependent hyperactivation of ribosome biogenesis and protein synthesis, and increased nucleolar size [9]. Interestingly, enlarged nucleoli and a consequential increase in rates of ribosome biogenesis and protein synthesis are a phenotypic hallmark of cancer cells [10]. Furthermore, a common feature of the p53-dependent and -independent mechanisms of Arf tumor suppressor function is the involvement of the nucleolus.

The nucleolus is a membrane-less organelle formed via phase separation of ribosomal RNA with ribosomal and non-ribosomal proteins [11], creating a liquid-like milieu for ribosome biogenesis and stress sensing and signaling. The hallmarks of proteins that undergo phase separation include structural disorder, multivalency of binding motifs, and in particular for the nucleolus, multivalency of arginine-rich short linear motif (R-motifs) [12].

The intrinsically disordered Arf protein exhibits a highly basic character (pI ~ 12; Fig. 1B), due to enrichment in arginine residues (25/132 amino acids, 18.9% for p14Arf and 37/169, 21.9% for p19Arf), which are clustered within several multivalent arginine-rich short linear motifs (R-motifs; Fig. 1B) [12]. Consequently, electrostatic forces influence interactions between Arf and its partners [2, 13–15]. When expressed above basal levels in response to mitogenic or other stress stimuli, Arf interacts with the disordered acidic region of Mdm2 via its N-terminal segment, encompassing the first R-motif (termed R1, Fig. 1B), to form high order, fibrillar complexes in vitro and in cultured cells [2] and targets Mdm2 to the nucleolus [16]. Notably, the N-terminal 20 residues from human p14Arf [17, 18] and 37 residues from the mouse homologue, p19Arf [8], are sufficient to recapitulate the p53-dependent tumor suppressor function of Arf, including Mdm2 binding, p53 activation and nucleolar localization. The same region of Arf encompassing the R1 motif also interacts with NPM1, leading to co-localization of the two proteins within the nucleolus and formation of supramolecular complexes [8, 15]. In fact, Arf co-precipitates with myriad partners within MDa-sized complexes when isolated from cultured fibroblast cells [19, 20]. To date, 163 interactors of p14Arf have been curated in the Biogrid repository (https://thebiogrid.org); 39/163 of these are associated with a “nucleolus” gene ontology term, including the non-ribosomal, highly abundant nucleolar proteins NPM1 and Nucleolin. Collectively, these data suggest that the multifarious tumor suppressor functions of Arf are mediated through integration within the liquid-like nucleolar matrix.

In this issue of The FEBS Journal, Luchinat, et al. [1], report a new interaction site for NPM1 in the less highly conserved C-terminal region of p14Arf. The authors showed using NMR spectroscopy that this site, which we term the R2-motif (residues 84-103, Fig. 1B), interacts with the folded N-terminal oligomerization domain of NPM1 by engaging the same acidic binding grooves shown previously to bind a short peptide derived from Arf’s R1 motif [1, 15]. Interestingly, this newly described interaction site is also engaged in interactions with Mdm2 [17], as discussed above for the N-terminal R1-motif of Arf. In the current study, the region containing R1 within full-length ¹⁵N-labeled p14Arf was undetectable by NMR due to resonance broadening even in the presence of a moderate concentration of a chaotropic agent (4 M urea), and, therefore, was hypothesized to cause Arf self-association and poor solubility [1]. Interestingly, addition of excess NPM1 prevented p14Arf aggregation in in vitro turbidity assays, possibly by inhibiting p14Arf-p14Arf self-association. Together, these observations introduce additional complexities regarding Arf’s interactions within the nucleolus and their roles in p53-dependent and –independent tumor suppression. Our understanding of nucleolar structure and function was advanced recently through studies demonstrating the roles of the proteins Fibrillarin and NPM1 in organization of the dense fibrillar component and granular component regions, respectively, through phase separation with ribosomal components [12, 21]. Based upon these insights, we envision several scenarios for Arf-mediated interactions within the nucleolus: (1) R1 of Arf promiscuously interacts with itself (Arf-Arf self-interaction), NPM1 and Mdm2 (Fig. 2A), (2) R2 of Arf similarly interacts with Mdm2 and NPM1 (Fig. 2A), and (3) R1 and R2 of Arf, through their multivalency, nucleate expanded networks via the previous interactions, forming supramolecular complexes (Fig. 2B). These promiscuous, weak and transient interactions could underlie formation of heterogeneous, percolated networks, causing retention of p14Arf, NPM1 and Mdm2 through phase separation within the liquid-like environment of the nucleolus [1, 15] (Fig. 2C). Additional interactions involving the polyampholytic intrinsically disordered region of NPM1, which contains 2 acidic tracts [22], and the two R-motifs of p14Arf likely promote formation of the intermolecular network and phase separation. While speculative, these scenarios provide mechanistic hypotheses describing the interplay between the Arf tumor suppressor and two of its regulatory targets for testing in the future.

Figure 2. Arf: a signaling hub that engages in multivalent interactions with NPM1 and Mdm2, and modulates nucleolar function.

(A) p14Arf interacts with NPM1, itself, and Mdm2 via the R1-motif, and with NPM1 and Mdm2 via the R2- motif; (B) The interactions depicted in (A) can, through multivalency, form heterogeneous interaction networks and mediate integration of Arf within nucleoli (C), altering their structure, dynamics and function (i.e., the processes of ribosome biogenesis and stress signaling), depending on the expression levels of the Arf protein. Complete suppression of Arf expression leads to uncontrolled ribosome biogenesis and protein synthesis, providing cells a proliferative advantage, while its overexpression supresses ribosome biogenesis and triggers p53-dependent cell cycle arrest or apoptosis.

Accumulation of Arf within the nucleolus integrates upstream stress signals with downstream effector pathways (cell cycle arrest or apoptosis through Mdm2 inhibition and p53 activation, and inhibition of ribosome biogenesis through interactions with NPM1). We propose that integration of Arf within the nucleolar matrix modulates the structure, dynamics and function of this liquid-like microenvironment. This hypothesis is based on the known role of NPM1 in organizing the outer region of the nucleolus through phase separation with ribosomal components, and the ability of Arf to bind to the same regions of NPM1 involved in phase separation with ribosomal and non-ribosomal proteins [12, 22]. The observations that Arf and Mdm2 co-localize within the nucleolus and that these proteins form supramolecular structures in vitro provide further support. Loss or silencing of Arf is observed in many human cancers [4]; in cultured cells, the same Arf loss led to enlarged nucleoli, enhanced ribosome biogenesis and elevated protein synthesis, all of which are dependent upon NPM1 [9]. Thus, the cancer phenotypes may arise, at least in part, due to the effect of Arf loss on nucleolar homeostasis. Conversely, oncogenic signaling-dependent activation of Arf expression inhibits ribosomal RNA processing in a manner dependent on NPM1’s ability to integrate within nucleoli [8, 12]. Together, these data support a mechanism wherein Arf incorporation into the nucleolar matrix disrupts the compositional balance of the nucleolar matrix through competitive interactions with NPM1, thereby altering ribosome biogenesis.

The work of Luchinat et al., provides new insights into the interactions of Arf with itself and its cellular partners. When placed in the context of phase separation, these insights inspire hypotheses regarding how Arf mediates the nucleolar localization and inhibition of Mdm2, and inhibition of ribosome biogenesis through interactions with NPM1. We propose a new role of Arf in regulating the dynamic and compositional properties of the nucleolus by altering nucleolar homeostasis, via integration within the nucleolar matrix.