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

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.

The structure, binding and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3

“…The structure shows that consistent with our current and previous binding studies ( 22 ) L3MBTL3 binds entirely to the BTD of RBPJ (Figure 3A ), threading through a narrow groove and down the front face of the BTD (Figure 3B – E ). L3MBTL3 residues 56–69 (-KKATATTTWMVPTA-) were built into the electron density; however, the N-terminal lysines ( 56–57 ), which contribute to binding ( see below ), have poorly resolved sidechain density (Figure 3B ). L3MBTL3 residues T63, W64, V66, and P67 bury their sidechains into an exposed hydrophobic pocket on the surface of the BTD, which anchors complex formation, and are flanked N- and C-terminally by an extensive polar network between L3MBTL3 and RBPJ (Figure 3D , E).…”

Identification of senescence-related subtypes, establishment of a prognosis model, and characterization of a tumor microenvironment infiltration in breast cancer

“…For instance, it was shown that Notch3 accelerated the development of prostate cancer-induced bone lesions through MMP-3 ( 35 ). Meanwhile, previous research proved that breast cancer tumorigenesis could be regulated by phosphorylation of Notch3, which might be affected by PTEN transactivation ( 36 – 38 ). Furthermore, many molecules can also affect tumorigenesis by influencing the Notch3 pathway.…”

Roles of Notch Signaling in the Tumor Microenvironment

“…Notch ICD modifications include phosphorylation, hydroxylation, methylation, sumoylation and acetylation [ 11 ]. Among the kinases phosphorylating Notch are Nemo-like kinases, AKT, CDKs, aPKC, GSK3β and PIM kinases [ 12 , 13 , 14 , 15 , 16 , 17 ]. Phosphorylation by CDK8 in the PEST domain located in the C-terminal part of Notch ICD converts the PEST domain to a phosphodegron [ 18 ], which becomes ubiquitylated by the FBXW7 E3 ubiquitin ligase, inducing rapid proteasome-mediated degradation of Notch ICD [ 19 , 20 , 21 , 22 ].…”

“…Phosphorylation by CDK8 in the PEST domain located in the C-terminal part of Notch ICD converts the PEST domain to a phosphodegron [ 18 ], which becomes ubiquitylated by the FBXW7 E3 ubiquitin ligase, inducing rapid proteasome-mediated degradation of Notch ICD [ 19 , 20 , 21 , 22 ]. Phosphorylation by PIM kinases is interesting because it is paralog-specific, i.e., the PIM phosphorylation sites differ between the Notch1 and Notch3 receptors, and PIM-mediated phosphorylation leads to distinct functional consequences for the two receptor paralogs [ 16 , 17 ]. One phosphatase, Eya1, has been identified for Notch ICD, and Eya1-induced removal of a phosphate group from a threonine residue in the transcriptional activation domain of Notch1 ICD increased the stability of Notch1 ICD [ 23 ].…”