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,Cecilia Sahlgren

doi:10.1016/j.jbc.2021.100593

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.

Highlights

The Notch signaling pathway orchestrates tissue development and homeostasis, but when dysregulated, it can promote tumorigenesis and support cancer progression [1,2,3,4]
We showed that PIM kinases phosphorylate Notch1 to promote tumorigenicity of estrogen receptor (ER)–positive breast cancer cells [7]
The parental MCF-7 cells efficiently formed tumors as expected, but the growth of tumors derived from the NOTCH1 or NOTCH3 KO cells was significantly decreased (Fig. 1C), indicating that both genes are essential for estrogendependent mammary tumorigenesis

Summary

Results

We showed that PIM kinases phosphorylate Notch to promote tumorigenicity of estrogen receptor (ER)–positive breast cancer cells [7]. Analysis with the PAS antibody showed that PIM inhibition reduces the phosphorylation status of N3ICD (Fig. 2D), indicating that NOTCH3 is an endogenous PIM kinase target. Minimization of the N3ICD complex phosphorylated at S1672 (corresponding to mouse S1673) twisted the conformation of the NOTCH3 RAM (N3RAM) peptide, causing the phosphate group to point toward the solvent instead of interacting with CSL. This resulted in a slight displacement (root-mean-square deviation of 1.5 Å for Cα-atoms) of the phosphorylated N3RAM peptide as compared with the nonphosphorylated N3RAM. The binding site for S1672 resides in a small and hydrophobic cavity on CSL, suggesting that both the size and the

Number 1 at risk

25 Phosphorylation

Discussion

N3 SAKI n

Experimental procedures