Characterization of a High-Molecular-Weight Notch Complex in the Nucleus of Notch^ic-Transformed RKE Cells and in a Human T-Cell Leukemia Cell Line

Abstract

Notch genes encode a family of transmembrane proteins that are involved in many cellular processes, such as differentiation, proliferation, and apoptosis. It is well established that all four Notch genes can act as oncogenes; however, the mechanism by which Notch proteins transform cells remains unknown. Previously, we reported that both nuclear localization and transcriptional activation are required for neoplastic transformation of RKE cells. Furthermore, we identified cyclin D1 as a direct transcriptional target of constitutively active Notch molecules. In an effort to understand the mechanism by which Notch functions in the nucleus, we sought to determine if Notch formed stable complexes using size exclusion chromatography. Herein, we report that the Notch intracellular domain (N^ic) forms distinct high-molecular-weight complexes in the nuclei of transformed RKE cells. The largest complex is approximately 1.5 MDa and contains both endogenous CSL (for CBF1, Suppressor of Hairless, and Lag-1) and Mastermind-Like-1 (Maml). N^ic molecules that do not have the high-affinity binding site for CSL (RAM) retain the ability to associate with CSL in a stable complex through interactions involving Maml. However, Maml does not directly bind to CSL. Furthermore, Maml can rescue ΔRAM transcriptional activity on a CSL-dependent promoter. These results indicate that deletion of the RAM domain does not equate to CSL-independent signaling. Moreover, in SUP-T1 cells, N^ic exists exclusively in the largest N^ic-containing complex. SUP-T1 cells are derived from a T-cell leukemia that harbors the t(7;9)(q34;q34.3) translocation and constitutively express N^ic. Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notch^ic.

We thank members of the Capobianco lab for intellectual support and technical assistance during this work. We also thank Melanie Stegman and other members of the Robbins lab for assistance with the fast protein liquid chromatography. We are grateful to Emery Bresnick (University of Wisconsin—Madison) for providing anti-CSL antibody and to James D. Griffin (Dana Farber Cancer Institute, Harvard Medical School) for supplying anti-Maml antibody and the cDNA for human Maml. We also thank Spryos Artavanis-Tsakonas (Harvard University) for providing the bTAN15A hybridoma.

This work was supported by NIH grant RO1CA83736-02 (to A.J.C.). A.J.C. is a scholar of the Leukemia and Lymphoma Society (award 1298-02). This work was also supported in part by predoctoral awards to S.J. from the DOD Breast Cancer Research Program, DAMD17-01-1-0202, and the Albert J. Ryan Foundation.