PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

Sebastian K.J. Landor, Niina M. Santio, William B. Eccleshall, Valeriy M. Paramonov, Ellen K. Gagliani, Daniel Hall, Shao Bo Jin, Käthe M. Dahlström, Tiina A. Salminen, Adolfo Rivero-Müller, Urban Lendahl, Rhett A. Kovall, Päivi J. Koskinen (Corresponding author), Cecilia Sahlgren (Corresponding author)

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

Original languageEnglish
Article number100593
Number of pages15
JournalJournal of Biological Chemistry
Volume296
DOIs
Publication statusPublished - 30 Mar 2021

Bibliographical note

Funding Information:
Funding and additional information—This study was financially supported by Academy of Finland (grants 309373 to C. S. and 287040 to P. J. K.), Jane and Aatos Erkko Foundation (to C. S.), Drug Research Doctoral Programme (to N. M. S. and W. B. E.), Turku Doctoral Programme of Molecular Medicine (to V. M. P.), Swedish Cultural Foundation of Finland and Finnish-Norwegian Medical Foundation (to S. K. J. L.), Emil Aaltonen Foundation (to N. M. S.), Maud Kuistila Memorial Foundation (W. B. E.), K. Albin Johansson Foundation (to S. K. J. L. and V. M. P.), European Community Mobility Programme EMA2, Ida Montin Foundation, Turku University Foundation and Instrumentarium Science Foundation (to V. M. P.), Sigrid Juselius Foundation (K. M. D. and T. A. S.) and Tor, Joe, and Pentti Borg’s Foundation (T. A. S.), the Swedish Cancer Society (to U. L.), and the National Institutes of Health (CA178974 to R. K.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

Funding Information:
This study was financially supported by Academy of Finland (grants 309373 to C. S. and 287040 to P. J. K.), Jane and Aatos Erkko Foundation (to C. S.), Drug Research Doctoral Programme (to N. M. S. and W. B. E.), Turku Doctoral Programme of Molecular Medicine (to V. M. P.), Swedish Cultural Foundation of Finland and Finnish-Norwegian Medical Foundation (to S. K. J. L.), Emil Aaltonen Foundation (to N. M. S.), Maud Kuistila Memorial Foundation (W. B. E.), K. Albin Johansson Foundation (to S. K. J. L. and V. M. P.), European Community Mobility Programme EMA2, Ida Montin Foundation, Turku University Foundation and Instrumentarium Science Foundation (to V. M. P.), Sigrid Juselius Foundation (K. M. D. and T. A. S.) and Tor, Joe, and Pentti Borg's Foundation (T. A. S.), the Swedish Cancer Society (to U. L.), and the National Institutes of Health (CA178974 to R. K.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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