Notch Oncoproteins Depend on γ-Secretase/Presenilin Activity for Processing and Function

Abstract

During normal development Notch receptor signaling is important in regulating numerous cell fate decisions.Mutations that truncate the extracellular domain of Notch receptors can cause aberrant signaling and promote unregulated cell growth. We have examined two types of truncated Notch oncoproteins that arise from proviral insertion into the Notch4 gene (Notch4/int-3) or a chromosomal translocation involving the Notch1 gene (TAN-1). Both Notch4/int-3 and TAN-1 oncoproteins lack most or all of their ectodomain. Normal Notch signaling requires ␥-secretase/presenilin-mediated proteolytic processing, but whether Notch oncoproteins are also dependent on ␥-secretase/presenilin activity is not known. We demonstrate that Notch4/int-3-induced activation of the downstream transcription factor, CSL, is abrogated in cells deficient in presenilins or treated with a pharmacological inhibitor of ␥-secretase/presenilins. Furthermore, we find that both Notch4/int-3 and TAN-1 accumulate at the cell surface, where presenilindependent cleavage occurs, when ␥-secretase/presenilin activity is inhibited. ␥-Secretase/presenilin inhibition effectively blocks cellular responses to Notch4/int-3, but not TAN-1, apparently because some TAN-1 polypeptides lack transmembrane domains and do not require ␥-secretase/presenilin activity for nuclear access. These studies highlight potential uses and limitations of ␥-secretase/presenilin inhibitors in targeted therapy of Notch-related neoplasms.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2

“…Since PI might also affect the g-secretase-mediated cleavage of membrane bound wT-Notch2 receptors, 37 B-CLL were treated with the potent g-secretase inhibitor compound E as a control. 38 As shown in Figure 3c, inhibition of g-secretase had no influence on the amount of DNA-bound Notch2IC complexes.…”

“…23 Another possible explanation is based on the observation that the PI also inhibit g-secretase activity, thereby preventing the generation of the Notch2IC fragment. 37 This possibility was ruled out by a series of experiments which show that blocking g-secretase activity with specific inhibitors 38 had no influence on the presence and the amount of the Notch2IC complex ( Figure 3c, and Hubmann et al, manuscript in preparation). The functional inactivation of Notch2IC as a transcriptional activator could be demonstrated by the downregulation of CD23a mRNA levels.…”

Hepatitis B virus X protein activates Notch signaling by its effects on Notch1 and Notch4 in human hepatocellular carcinoma

“…Notch2 and Notch3 were equally expressed between the two cell lines. Notch4 showed two bands, one of which with a higher molecular mass was its NTM form, and the lower one was its intracellular region of Notch (ICN) form (24). Although the NTM form of Notch4 was equally expressed between HepG2X and HepG2-pc cells, the ICN form appeared only in HepG2X (Fig.…”

Sonic Hedgehog regulates Hes1 through a novel mechanism that is independent of canonical Notch pathway signalling

“…Notch pathway activity was monitored using a reporter construct with 12 RBP-Jk binding motifs driving luciferase expression (pGA981-6; Minoguchi et al, 1997;Das et al, 2004).…”