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. 2020 Aug 26;9:e57617. doi: 10.7554/eLife.57617

Figure 2. PRMT7 methylated SHANK2 at R240.

(A) The purified SHANK2 from MDA-MB-231 cells transfected with HA-SHANK2 was analyzed for methylation by mass spectrometry. The SHANK2 R240 residue in fragmentation of KAARMRN was methylated. The Mascot score was 27.28, and the expectation value was 3.74E-04. (B) Alignment of the consensus SHANK2 sequences between different species near the R240 methylation site. (C) MDA-MB-231 cells with or without expressing SHANK2 shRNA and with or without reconstituted expression of WT SHANK2 or SHANK2 R240K mutant. Immunoprecipitation of HA with anti-HA antibody was performed. (D) Flag-tagged SHANK2 WT or SHANK2 R240K was transfected into MDA-MB-231 cells. HA-purified SHANK2 WT or SHANK2 R240K proteins were incubated with purified bacterially expressed PRMT7 and SAM. Methylation of SHANK2 protein was determined by western blot. (E) HEK293T cells were co-transfected with increasing amounts of Flag-PRMT7 and SHANK2 WT or SHANK2 R240K expression plasmids. Immunoprecipitation of SHANK2 with anti-SHANK2 antibody was performed.

Figure 2.

Figure 2—figure supplement 1. The role of PRMT5 in PRMT7 mediated SHANK2 methylation.

Figure 2—figure supplement 1.

Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (A) HEK293T cells were co-transfected with control pcDNA3.1 or PRMT5 plasmids. Immunoprecipitation of SHANK2 with anti-SHANK2 antibody was performed. (B) HEK293T cells were co-transfected with increasing amounts of Flag-PRMT5 and SHANK2 WT or SHANK2 R240K expression plasmids. Immunoprecipitation of SHANK2 with anti-SHANK2 antibody was performed. (C) HEK293T cells with increasing amounts of Flag-PRMT7, PRMT5 and SHANK2 were treated with GSK591 (5 µM) or DMSO as control. Immunoprecipitation of HA with anti-HA antibody was performed. Western blot was performed with the indicated antibodies. (D) MDA-MB-231 cells with increasing amounts of Flag-PRMT7 were treated with GSK591 (5 µM) or DMSO as control. Immunoprecipitation of SHANK2 with anti-SHANK2 antibody was performed. Western blot was performed with the indicated antibodies.