Abstract

Notch signaling controls diverse eukaryotic differentiation processes in multiple cell types, thus demanding versatile tools with which Notch triggers downstream events. Ubiquitin-mediated proteolysis has previously been shown to be one such tool with which Notch regulates the turnover of the basic helix-loophelix transcription factor, E47. Here, we show that Notch signaling also accelerated the degradation of Tal1/SCL (T cell acute leukemia 1/stem cell leukemia) protein, a basic helix-loop-helix protein involved in the development of hematopoietic, vascular, and neuronal tissues. Notch-induced Tal1/SCL degradation was mediated by ubiquitination and proteasomes. The sequence responsible for Tal1 degradation was localized to a region in the C terminus of Tal1, which is evolutionarily conserved, thus suggesting a functional significance. Analogous to the situation for E47, Notch-induced Tal1/SCL degradation not only required Skp2, a substrate-binding subunit of SCF ubiquitin ligase complexes, but also relied on CHIP, a chaperone-binding protein with a ubiquitin ligase activity. In contrast to the fact that the N-terminal tetratricopeptide region (TPR) domain of CHIP is necessary and sufficient for E47 ubiquitination and degradation, CHIP promoted Tal1 degradation with both chaperone binding and ubiquitin ligase activities, which are mediated by its TPR domain and U box, respectively. Although the TPR domain was not involved in Tal1/SCL binding, it was required for enhancing its degradation. Likewise, the ubiquitin ligase activity of CHIP was dispensable for Tal1/SCL binding but essential for degradation. These findings provide both novel mechanistic insights into the operation of cullin-based ubiquitin ligase complexes and potential means by which Notch and Tal1/SCL regulate eukaryotic development.

Highlights

  • Cellular domain and its translocation into the nucleus

  • Expression of shRNA against RBP-J␬ restored the level of Tal1 in cells cotransfected with Tal1 and N1-IC to that in cells expressing Tal1 alone (Fig. 1C)

  • We evaluated the role of CHIP in Tal1 ubiquitination

Read more

Summary

Introduction

Cellular domain and its translocation into the nucleus. The intracellular domain of Notch receptors acts as a transcription coactivator by associating with the DNA-binding subunit, RBP-J␬ ( referred to as CSL), and stimulates transcription of genes, many of which are still unknown (4 –7). Notch Signaling Induces Ubiquitin-mediated and Proteasome-dependent Degradation of Tal1—To examine the effect of activated Notch1 receptor on Tal1 degradation, we cotransfected into NIH3T3 cells a construct expressing human Tal1 cDNA with increasing amounts of plasmids producing the N1-IC. This reduction was reversed by addition of the MG-132 proteasome inhibitor to transfected cells, suggesting that Tal1 proteins were degraded by the proteasome in the presence of Notch signaling (Fig. 1B).

Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call