Notch Regulation by the Fbw7/hcdc4/Sel-10 Ubiquitin Ligase.

T-cell neoplasms frequently sustain mutations in the Notch1 gene, leading to the expression of constitutively active Notch proteins. Such mutations often target the C-terminal PEST domain, which is known to be involved in protein stability. The ubiquitin ligase Fbw7/hdcd4/Sel-10 is a tumor suppressor that negatively regulates Notch function by targeting the Notch protein for ubiquitination and proteasomal degradation. Although the PEST domain is known to be important for Fbw7/Notch interactions, the specific residues that regulate binding of Notch to Fbw7 have not been defined. Based on the structural motifs (known as phosphodegrons) common to known substrates of Fbw7, we have identified two candidate peptide sequences within the Notch protein and have generated a series of mutants in these regions. Using co-immunoprecipitation assays, we show that one potential phosphodegron that is outside of the PEST domain does not appear to influence Notch binding to Fbw7. However, a second potential phosphodegron is present within the PEST domain and contains a conserved threonine residue (T2512) which is central to binding of Fbw7 to Notch. A mutant in which this residue is replaced by alanine (T2512A) shows a prolonged half life when compared to wild type Notch ICD, supporting its role in Notch stability. To evaluate the role of Fbw7 mediated Notch degradation in vitro and in vivo, we used lentiviral vectors to transfect hematopoietic cells with shRNA targeting Fbw7. These studies demonstrate that Fbw7 knockdown leads to phenotypes consistent with increased Notch activity. Because Notch is commonly mutated in human leukemias, we hypothesized that Fbw7 may also sustain mutations that lead to loss of Notch regulation. We evaluated primary human T cell leukemias for mutations in Fbw7 and found that 1 of 23 samples contains a heterozygous mutation in the Fbw7 common region (R505C). We show that this mutant is deficient in binding to Notch, suggesting that Fbw7 mutation may contribute to the deregulation of Notch that is commonly seen in T-cell neoplasms. Together, this work shows that Fbw7 is an important regulator of Notch function whose mutation may be an important step in leukemogenesis.