Figure 2. Serine 2152 in Notch1 is phosphorylated by Pim kinases.

A. GST-tagged Pim kinases were treated with 0.1% DMSO or 10 μM DHPCC-9 prior to in vitro kinase assays with GST-tagged NICDs or GST control protein. Pim (P) autophosphorylation and NICD (N) phosphorylation signals were analysed by autoradiography (above), while protein loading was detected by Page Blue™ staining (below). B. N1ICD was immunoprecipitated from MCF-7 cells that stably expressed the doxycycline-inducible N1ΔE protein and that were treated with 10 μM DHPCC-9 and/or 1 μg/ml of doxycycline for 24 h, after which the phosphorylation status of N1ICD was analysed by Western blotting with antibodies targeting phosphorylated S/T residues or N1ICD. C. Phosphorylation of wild-type (WT) N1ICD or phosphodeficient (SA) mutants by Pim family members were analysed by in vitro kinase assays. At least two independent experiments were performed and shown are representative results of autoradiography (above) and protein staining (below) in one experiment. D. A schematic model shows Pim target sites within the Notch1 protein. Abbreviations: NECD = The Notch extracellular domain, EGF = Epidermal Growth Factor, NRR = negative regulatory region, LNR = the Lin12-Notch repeat, HD = heterodimerization domain, S2 = ADAM family metalloprotease cleavage site, TM = the transmembrane domain, S3 = γ-secretase cleavage site, RAM = Rbp-associated molecule domain, ANK = ankyrin repeat domain, PPD = potential phosphorylated domain, NLS = nuclear localization signal, TAD = transcription activation domain, PEST = domain rich in proline, glutamic acid, serine and threonine.