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. 2023 Feb 28;14:1143. doi: 10.1038/s41467-023-36693-9

Fig. 6. Validation of hijack and mute model of B56α inhibition by CIP2A in cellulo.

Fig. 6

A NIH3T3 cells, stably expressing human CIP2A(1-905) V5, or empty vector control (see Supplementary Fig. 8D), were transiently transfected with YFP-B56α WT, followed by immunoprecipitation using GFPTrap and analysis by mass spectrometry. B Proteins differentially interacting with B56α in CIP2A(1-905)V5 over-expressing cells (N = 3) vs. control (N = 3). Indicated proteins exceed the threshold of 2-fold difference with p < 0.05 (two-sided t-test using log2 of protein abundances, normalized to the protein abundance of B56α in the CTRL_1 sample, as an input). C Pie-chart of share of proteins displaying either decreased or increased binding to B56α upon CIP2A over-expression (excluding PPP2R1A and CIP2A). Bar graph indicates percentual share of proteins with putative LxxIxE motif among the proteins which interaction with B56α was decreased by CIP2A. D Pie-chart presentation of percentual share of proteins dephosphorylated (FDR < 0.05) in CIP2A K21A clone1 as compared to control clone in relation to putative LxxIxE motif found from the dephosphorylated protein (in orange), or whether dephosphorylated protein is an interactor of a protein with validated LxxIxE motif (in red). E Significantly enriched kinase motifs based on phosphopeptides dephosphorylated in CIP2A K21A clone1. Background indicates the number of motifs found from all phosphopeptides identified by MS from the same samples. F Reactome analysis image capture of the process Senescence enriched in targets dephosphorylated in CIP2A K21A clone1 as compared to control clone. Schematic presentation of CIP2A-mediated PP2A-B56α inhibition by the identified hijack and mute mechanism. In normal cells with low CIP2A expression, PP2A-B56α binds to its substrate protein (light blue) via LxxIxE groove and the adjacent acidic patch (orange) resulting in substrate dephosphorylation. In cancer cells with high CIP2A gene transcription, CIP2A binding to PP2A-B56α trimer results in expel of A and the formation of CIP2A-B56α-PP2Ac trimer. In this alternative trimer CIP2A shields the LxxIxE groove and the adjacent acidic patch from B56α substrates and thereby the substrate remains phosphorylated (dark blue). Binding of CIP2A to B56α via its head domain further stabilizes CIP2A protein.