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

Fig. 4. CIP2A displaces PP2A-A from PP2A-B56α trimer and interacts directly with PP2Ac.

Fig. 4

A B56α sites identified in inter-molecular cross-links with CIP2A overlap with B56α contact sites with the scaffolding A subunit (PDB: 6NTS). A subunit is yellow, catalytic subunit is grey, and B56α is wheat. The XL-MS cross-links between CIP2A and B56α are in red. PP2A-A and PP2Ac interaction sites with B56α are shown in magenta, and light purple, respectively (based on refs. 40,41). The overlap between PP2A-A and CIP2A in B56α surface is indicated as transparent oval shape in the right panel. B Coomassie stained SDS-PAGE of PP2A trimer components interacting with CIP2A(1-560) after incubation with the pre-assembled PP2A-B56α heterotrimer (A-B56α-PP2Ac). A = PP2A-A; C = PP2Ac. Representative image from four experiments is shown. C Western blot analysis of the similar experiment as in (B). The different intensities of the Western blot signals between PP2A-A (A), B56α, and PP2Ac in the input (and between B56α, and PP2Ac in the eluates), is due to differential affinities of the antibodies used simultaneously to blot the membranes. N = 3 biological repeats. D GST pull-down assay for PP2A trimer-GST-CIP2A(1-560) interaction. Equal molar amounts of PP2A proteins were used in all the samples. The amount of CIP2A(1-560) protein was titrated against PP2A as indicated. The positions of molecular weight markers are unavailable. E, F Size-exclusion chromatography analysis of protein complexes between GST (F) or GST-CIP2A(1-560) (G) and either pre-assembled PP2A-A-B56α-PP2Ac trimer (ABC) (middle panels) or B56α and PP2Ac (B + C) proteins (lower panels). Proteins eluting from the indicated fractions were analyzed by Western blotting. Approximate molecular weights of protein complexes eluting from each fraction are based on calibration with standard proteins. The positions of molecular weight markers are unavailable. N = 1. G Schematic presentation of CIP2A mediated hijack of the PP2A-B56α complex. In normal cells with low CIP2A expression, the PP2A-B56α holoenzyme (A-B56α-C) remains intact. In cancer with high CIP2A expression CIP2A interacts directly with both B56α and PP2Ac resulting in expel of A-subunit from the trimer. Thus, in cancer cells CIP2A functions as a pseudo-A-subunit stabilizing the trimeric CIP2A-B56α-PP2Ac complex.