Fig 2: Phosphorylation of Notch1 ICD (N1ICD) and Notch3 ICD (N3ICD) by proviral integration site for Moloney murine leukemia virus (PIM) kinases occurs in different domains and results in differential Notch signaling outputs.A, a schematic model of mouse Notch3 with the PIM target site at S1673. B, comparison of mouse N1ICD and N3ICD phosphorylation sites and their outcomes. PIM-mediated phosphorylation inhibits CSL-dependent activity of N3ICD but promotes that of N1ICD (7). In both cases, phosphorylation leads to increased tumor growth, albeit via different mechanisms. ANK, ankyrin repeat domain; CSL, C promoter–binding factor 1, Suppressor of Hairless, Lag-1; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin12-Notch repeat; NECD, Notch extracellular domain; NLS, nuclear localization sequence; NRR, negative regulatory region; PEST, proline-, glutamic acid-, serine-, and threonine-rich domain; RAM, RBPJ-associated molecule domain; S2, ADAM family metalloprotease cleavage site; S3, γ-secretase cleavage site; TAD, transcription activation domain; TM, transmembrane domain.

Fig 3: PIM1 colocalizes and interacts with Notch3 ICD (N3ICD). RFP-tagged PIM1 and/or GFP-tagged WT, phosphodeficient (SA), or phosphomimicking (SE) N3ICD were transiently overexpressed in MCF-7 cells. Empty vectors were used as controls. A, the localization of the fluorescent proteins was imaged by confocal microscopy. The figure shows representative single channel or merged images. B, equivalent expression levels of overexpressed N3ICDs were confirmed by Western blotting, and signal intensities were normalized to actin beta (ACTB) staining. C, the physical interactions between PIM1 and WT or mutant N3ICD were analyzed by fluorescence-lifetime imaging. The figure shows the results from three independent experiments along with sample numbers inside the black bars and representative images. D, proximity ligation assays were performed with one PIM1 and two different N3ICD antibodies (AB) to visualize the presence of endogenous proteins in the proximity of each other in MCF-7 cells. Representative images along with average results from three independent experiments are shown. ∗p < 0.05 was used as a limit for significant difference. Error bars represent standard deviation. SA, serine to alanine; SE, serine to glutamic acid; GFP, green fluorescent protein; RFP, red fluorescent protein.

Fig 4: PIM kinases phosphorylate mouse Notch3 ICD (N3ICD) at S1673.A, radioactive in vitro kinase assays were performed with GST-tagged PIM kinases and WT or serine to alanine (SA) mutants of mouse N3ICD. Phosphorylated proteins were separated by electrophoresis and detected by autoradiography. Protein loading was visualized by PageBlue staining. Shown are representative images from one of two experiments. B and C, MCF-7 cells were transiently transfected with plasmids encoding FLAG-tagged N3ICD, His-tagged PIM1, and/or their controls. About 24 h later, cells were treated with DMSO (−) or 10 μM DHPCC-9 (+). The ectopically expressed proteins were immunoprecipitated with the anti-FLAG affinity agarose gel or the HisLink resin. N3ICD phosphorylation was detected with the phosphospecific RXXpS/pT antibody and protein levels by anti-FLAG (N3ICD) or anti-PIM1 antibodies. Cell lysates and actin beta (ACTB) stainings were used as controls. Average signal intensities for phosphorylation or coimmunoprecipitation (CO-IP) were determined relative to overexpression levels. Shown are representative examples from three independent experiments. D, endogenous NOTCH3 was immunoprecipitated from MCF-7 cells cultured in the absence or the presence of DHPCC-9. N3ICD phosphorylation was detected with the phospho-AKT substrate antibody. Average signal intensities of phosphorylation were determined relative to overexpression levels. Shown are representative examples from three independent experiments.

Fig 5: Structure and binding properties of CSL and the NOTCH3 RAM (N3RAM) domain.A, CSL, shown with a transparent white surface, consists of three major domains: the N-terminal domain (NTD) in cyan, the beta-trefoil domain (BTD) in green, and the C-terminal domain (CTD) in orange. The N3RAM peptide corresponding to residues 1665 to 1682 is shown as a stick representation in purple. B, close-up view of the N3RAM bound to the BTD of CSL. Residues of CSL colored green directly contact RAM, as determined by the PISA server (15). C, close-up view of S1672 colored in yellow. The dashed line represents the hydrogen bond between the carbonyl group of the peptide backbone on E259 in CSL and the hydroxyl group of S1672 in N3RAM. Select CSL side chains that make up the hydrophobic pocket accommodating RAM S1672 are shown as stick representations in green. D, isothermal titration calorimetry experiments measuring CSL binding to RAM peptides. Representative thermograms from triplicate experiments of the binding of CSL to the NOTCH1 RAM residues 1754 to 1781 (left), nonphosphorylated N3RAM residues 1665 to 1682 (middle), and N3RAM residues 1665 to 1682 with a phosphorylation at S1672 (right). NBD signifies no binding detected.