It has been shown that a physiologically optimal level of NS is essential for normal cell homeostasis. Both aberrant overexpression and depletion of NS inhibit cell proliferation (46
). In this study, we have further illustrated the mechanisms underlying the role of NS in cell cycle regulation. We show that both aberrantly high and low levels of NS induce G1
arrest and reduce cell proliferation rates through the activation of p53 as a result of the inhibition of MDM2. Upon ectopic overexpression, excess NS molecules bind directly to MDM2 and inhibit MDM2-mediated p53 ubiquitylation and degradation. When the level of NS is significantly reduced by siRNA, a stress occurs, leading to the association of ribosomal proteins L5 and L11 with MDM2 and subsequently the inhibition of MDM2 function. As a result, p53 is activated in both cases (Fig. ). Therefore, p53 acts as a key cell cycle checkpoint that senses the aberrant cellular levels of NS via distinct mechanisms that funnel into the inhibition of MDM2.
Schematic model illustrating potential mechanisms underlying p53 activation by aberrantly high levels of NS upon overexpression or by siRNA-caused low levels of NS (see text for further discussion).
Our domain mapping studies show that NS binds to the central acidic region of MDM2. This region has been shown to be critical for mediating p53 degradation (19
). Indeed, many MDM2 regulatory proteins, such as L5, L23, and ARF, target this region and regulate MDM2-mediated p53 turnover (10
). Similar to the effect of these proteins on MDM2 function, the binding of NS to MDM2 also leads to the inhibition of E3 ligase activity of MDM2 toward p53, and thus, consequent p53 activation. Also, overexpression of NS markedly inhibits MDM2 degradation. Hence, these effects are reminiscent of the role of ARF in regulating the p53-MDM2 pathway.
Since MDM2 locates mostly in the nucleoplasm (36
), while NS resides predominantly in the nucleolus (45
), how do these proteins interact in cells? It is possible that under normal growth conditions, the steady-state nucleoplasmic NS is extremely low and would not significantly interact with MDM2. That is why we can detect only a weak binding for the endogenous proteins in normally cultured cells. However, when NS is overexpressed, a significant portion of the protein is accumulated in the nucleoplasm and thus is able to interact with MDM2. Consistent with this, the GTP-binding-defective mutant GFP-NSG1dm
, which was expressed mostly in nucleoplasm while retaining partial nucleolus localization (data not shown), interacted with MDM2 somewhat more efficiently than did wt NS (Fig. ). The dynamic nature of both proteins in nuclear shuttling would allow their interaction to respond to cell growth and stress signals. It is conceivable that in response to certain cellular stresses, NS may be redistributed from the nucleolus to the nucleoplasm, where it can target MDM2.
It has been shown that p53 can bind to NS at the N-terminal basic domain (46
), whereas here we show that MDM2 interacts with the coiled-coil domain of NS. Together, these studies suggest that NS could bind to p53 and MDM2 simultaneously. Indeed, we detected a ternary complex that contains all three proteins (data not shown). Thus, MDM2 does not compete with p53 for NS binding. This result is similar to the regulation of the MDM2-p53 pathway by other nucleolar proteins, such as ARF and ribosomal proteins, which do not disrupt the p53-MDM2 binding. Likewise, they associate with both p53 and MDM2, and inhibit MDM2-mediated p53 ubiquitylation and degradation (8
Since the role of NS in regulating the p53-MDM2 circuit is similar to that of ARF, one question would be whether NS possesses intrinsic tumor suppressor activity. NS is highly expressed in proliferative cells, and it is conceivably responsive to high proliferative signals. Our data suggest that abnormally high levels of NS are detrimental to cells due to p53 activation by NS via inhibition of MDM2. Thus, it is possible that NS may provide cells with a surveillance mechanism to check uncontrolled cell growth and tumorigenesis. However, to our knowledge, no genetic alterations such as mutations or deletions in the NS
gene have been documented so far. This might be partially because that loss of NS function also activates p53, and mice deleted of NS are embryonic lethal (see discussion below). Chromosome translocation, mutation, and deletion in the B23
locus have been reported in leukemias and lymphomas (14
). Thus, it would be interesting and worthwhile to screen genetic alterations of the NS
gene in primary cancers in the future.
Similar to the overexpression of NS, depletion of NS by siRNA also induces p53. While the manuscript for this report was in preparation, Ma and Pederson also showed that knocking down NS by siRNA induced p53-dependent G1
cell cycle arrest (24
). Our further analysis reveals that ribosomal proteins L5 and L11 are required for the NS depletion-induced p53 levels, activation, and cell cycle arrest, suggesting that depletion of NS triggers a stress that activates p53. Unlike other nucleolar stresses such as those induced by treatment of 5-FU, actinomycin D, or the genetic knockdown of TIA-IA (23
), but like the case of S6
), the knockdown of NS by siRNA did not apparently disrupt the nucleolus, which is consistent with the observation that NS deficiency does not cause nucleolar disruption in embryonic blastocysts (3
). This raises a question whether the nucleolar disruption is absolutely required for a nucleolar stress to occur. Because NS is localized at the distinct region of the nucleolus that is not actively involved in the ribosomal biogenesis (34
), how NS depletion causes nucleolar stress remains to be determined. Alternatively, it is possible that the reduction of NS by siRNA may cause a nucleolus-independent stress that also activates the L5/L11-MDM2-p53 pathway, as our results clearly show that the ribosomal proteins L5 and L11 are required for NS depletion-induced p53 activation. In addition, NS depletion by siRNA enhances the interaction of MDM2 with L5 and L11. Another speculation would be that NS may regulate the nucleolar import of ribosomal proteins for ribosome assembly and that the depletion of NS would lead to the accumulation of free L5 and L11 in the nucleoplasm, where they find MDM2 to interact with. These speculations remain to be investigated in the future.
Intriguingly, heterozygous deletion of NS in mice did not trigger significant p53 activation (54
). This result suggests that the loss of one copy of the NS
gene might not reduce NS to a level that would sufficiently trigger a stress. Also, a p53 knockout did not rescue the lethality by the NS knockout in mice (3
), suggesting that NS may have p53-independent function essential for early embryogenesis. Consistently, the knockdown of L5 and L11 only partially rescued the NS knockdown-induced cell cycle arrest and inhibition of cell proliferation (Fig. ), although the induction of p53 by NS siRNA treatment was completely abolished by knockdown of L5 or L11 (Fig. ). Uncovering this p53-independent function would provide a complete picture for better understanding the role of NS in cell proliferation.