Although the mechanisms of Notch/LIN-12 signal transmission are being defined through genetic and biochemical analyses, little is known about the mechanisms involved in down-regulating the Notch signal. Notch signaling mediates numerous key developmental decisions in both vertebrates and invertebrates. As such, mechanisms to down-regulate Notch signaling are likely critical to maintain proper developmental programs or to prevent oncogenic functions of Notch proteins. sel-10
was originally identified genetically in C. elegans
as a negative regulator of lin-12
). The fact that the SEL-10 protein is related to the F-box/WD40 repeat family of proteins suggested that SEL-10 down-regulates Notch/LIN-12 signaling by targeting these proteins for ubiquitin-mediated protein turnover (10
). This proposed function of SEL-10 would represent a key mechanism by which Notch signaling is reduced in physiological settings. Based on the paradigm established by analysis of budding yeast Cdc4, F-box/WD40 proteins are predicted to bind their target proteins in a phosphorylation-dependent fashion.
Here, we demonstrate that Notch1 signaling is negatively regulated by SEL-10. Interference with SEL-10 function by expression of the WD repeat region enhances steady-state levels of Notch1 proteins by reducing the rate of turnover, thus increasing Notch1-mediated signaling, indicating that SEL-10 is directly involved in mediating Notch ubiquitination and degradation. We also found the Notch4 proteins interact efficiently with SEL-10 but that the levels and activity of the intracellular domain of Notch4, Notch4(int-3), are relatively resilient to interference with SEL-10 function. Thus, these two Notch proteins behave differently in response to blocking of SEL-10 function.
The ease with which we could detect Notch4(int-3)–SEL-10 protein complexes prompted us to choose Notch4(int-3) proteins as a focus for detailed binding studies. This proved an effective way of dissecting the biochemical interactions in greater detail than could be achieved with substrates that would be tremendously labile when complexed to SEL-10. We demonstrated that the WD40 repeats of SEL-10 bind to the C-terminal domain of Notch4, a domain important for Notch4 phosphorylation. Moreover, SEL-10 binds preferentially to phosphorylated forms of Notch4 and shields the phosphate groups from nonspecific dephosphorylation by CIP, suggesting that the interaction is directly mediated by phosphorylated amino acids within the C-terminal domain of Notch4. We also found that several forms of Notch proteins, Notch1ICD and Notch4(int-3)C, are very unstable as a result of rapid turnover via the proteasome pathway. However, full-length SEL-10 expression did not have an appreciable effect on Notch protein levels or activities (data not shown). This result may indicate that sufficient SEL-10 activity exists in these cells to mediate Notch protein turnover.
Finally, recombinant SEL-10 assembles into SCF ubiquitin ligase complexes in insect cells. These complexes bind coexpressed Notch and mediate highly processive (and efficient) ubiquitination of bound Notch proteins. Given that other SCF complexes (including SCFCdc4, SCFGrr1, SCFSkp2, and SCFβ-TRCP) have been shown to be extremely selective for phosphorylated substrates, we presume that recombinant Notch proteins are targeted to SCFSEL-10 by an endogenous protein kinase in insect cells. Although the exact mechanism by which Notch proteins are targeted for ubiquitination remains unclear, it is evident from the in vitro experiments that Notch proteins can serve as excellent substrates for SCFSEL-10. Given the extraordinary substrate specificity that is evinced by all other SCF ubiquitin ligases that have been evaluated to date, the efficient and highly processive in vitro ubiquitination that we observed (Fig. C) indicates that Notch is a physiological substrate for SCFSEL-10. Based on all of the data, the most parsimonious hypothesis is that the phosphorylation of Notch by an unidentified protein kinase targets it to SCFSEL-10, which in turn extensively ubiquitinates Notch as a prelude to its degradation. Conclusive proof of this hypothesis in vivo will ultimately require the mapping of phosphorylation sites and the construction of nonphosphorylatable point mutant versions of Notch.
We conclude that the C-terminal domain of Notch4 distal to the CDC10/ankyrin repeats is a negative regulatory domain because it is responsible for interactions with SEL-10. This notion is consistent with the fact that the C-terminal domain contains a PEST sequence, which is characteristic of many short-lived proteins and which is thought to be a target for phosphorylation and ubiquitination (28
). It has also been observed that a C-terminal deletion can activate GLP-1, a C. elegans
Notch protein (22
). The C-terminal domain of Notch proteins is also where some other regulatory proteins bind. For example, Drosophila
protein Dishevelled has been reported to bind to this region and may thus mediate the interaction between the Wingless and Notch signaling pathways (2
). Our results predict that Notch levels and activity may be controlled by a kinase(s) that phosphorylates the C terminus of Notch proteins. This phosphorylation would mediate SEL-10 binding and thus ubiquitination and degradation by the 26S proteasome. Little is known about kinases that phosphorylate and regulate Notch, but one would predict that the kinase that phosphorylates the C terminus has a negative regulatory function in Notch signaling.
An interesting observation is that Notch4(int-3) proteins show strong and specific interactions with SEL-10 but do not seem to be readily degraded by the proteasome pathway, in contrast to Notch1IC. Overexpression of the WD40 repeat region or treatment by lactacystin failed to increase the steady-state levels of Notch4(int-3) (data not shown). Pulse-chase analysis indicated that Notch4(int-3) has a much longer half-life in cells than does Notch4(int-3)C, the C-terminal domain of Notch4(int-3) (data not shown). However, Notch4(int-3) still seems to be ubiquitinated in cells because a Western blot of Notch4(int-3) often displays a very high-molecular-weight smear in addition to the main Notch4(int-3) signal at the predicted molecular weight (unpublished observations). This smear can be seen even without proteasome inhibitor treatment and is very typical of proteins that are ubiquitinated. Notch4(int-3) also serves as a substrate for SEL-10-dependent in vitro ubiquitination (Fig. A). The fact that Notch4(int-3) protein levels are relatively unaffected by interference with SEL-10 activity is consistent with the fact that in signaling assays, Notch4(int-3) activity was not elevated by coexpression with a dominant-negative form of SEL-10 (data not shown). One possible explanation for these observations is that the extracellular sequence, the transmembrane domain, or the ankyrin repeats in Notch4(int-3) can function to prevent the protein from being degraded by the proteasome even after ubiquitination. The resultant increased stability of Notch4(int-3) may also contribute to the potent oncogenic activity of this variant of Notch4, whose gene was originally defined as a mammary oncogene (12
). In contrast, our studies show that Notch4(int-3)C and Notch1IC, both lacking a transmembrane domain and extracellular sequence, can be readily stabilized by proteasome inhibitors or overexpression of the WD40 repeat region of SEL-10. This issue can be further addressed by biochemical studies using a Notch4(int-3) fragment containing only the intracellular domain or possibly chimeric proteins of Notch4(int-3) and Notch1IC.
Other reports also suggest that Notch proteins are likely turned over by ubiquitination. For example, it has been reported that the steady-state level of the Notch1 intracellular domain can be elevated by lactacystin, a proteasome inhibitor (32
). In addition, Notchless
, a novel Drosophila
gene identified as a modulator of Notch
activity, encodes a WD40 repeat-containing protein that binds to the intracellular domain of Notch (31
). However, the function of Notchless
is not clear because both loss-of-function mutations and overexpression of the gene lead to increased Notch activity. These results, once again, suggest that regulation of the Notch pathway is very complex. A recent report suggests that Notch proteins are targets for ubiquitination and provides biochemical evidence that the Itch protein may participate in mediating Notch ubiquitination (27
). However, this study did not establish that Itch is responsible for or participates in the ubiquitination of Notch in vivo or that Notch ubiquitination, stability, or activity is altered in mice with the Itch
Ubiquitin-mediated protein degradation is a highly regulated and selective process used to down-regulate several signaling pathways (1
). F-box/WD40 family proteins can bind to multiple target proteins (23
). We report that Notch signaling also utilizes ubiquitin-mediated protein turnover to down-regulate the Notch/LIN-12 signal. This is evident both in C. elegans
) and in Notch signaling in mammalian cells (Fig. C and D). Thus, clear evidence from sequence homology, functional studies, and binding studies points to the high level of conserved function of SEL-10 as a negative regulator of Notch signaling from worms to humans. SEL-10 also interacts genetically and physically with the C. elegans
presenilin, SEL-12 (45
), and may target both Notch and presenilin for degradation. The data presented here suggest that hSEL-10 may serve a similar role in the down-regulation of presenilin function as in the down-regulation of Notch. Presenilin is required for the activation of Notch, probably by mediating the proteolytic cleavage of the transmembrane domain of Notch, enabling the nuclear access of the Notch intracellular domain (5
). It is not clear whether SEL-10 has effects on the presenilin-Notch interaction or whether it targets each protein separately. It will be interesting to define how SEL-10 is directed to distinct targets, such as Notch and presenilins. Understanding how Notch proteins are regulated by SEL-10 may also provide new approaches to controlling Notch activity. For example, as constitutive activation of Notch can lead to tumorigenesis, SEL-10 activity could be used to reverse Notch activity in these circumstances.