Characterization of a high-molecular-weight Notch complex in the nucleus of Notchic-transformed RKE cells and in a human T-cell leukemia cell line

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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 (Nic) 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). Nic 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, Nic exists exclusively in the largest Nic-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 Nic. Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notchic.

Original languageEnglish (US)
Pages (from-to)3927-3941
Number of pages15
JournalMolecular and cellular biology
Volume22
Issue number11
DOIs
StatePublished - 2002

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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