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. 2018 Dec 6;294(7):2543–2554. doi: 10.1074/jbc.RA118.005465

Figure 1.

Figure 1.

Recruitment of active calcineurin to mAKAPβ signalosomes in differentiating C2C12 skeletal myoblasts. A, mAKAPβ in striated myocyte is identical to aa residues 245–2314 of the neuronal mAKAPα (14). The three spectrin repeats (SR) required for nuclear envelope targeting are indicated (40). Binding sites are shown for those mAKAP binding partners for which there is evidence of direct binding: 3-phosphoinositide-dependent kinase-1 (PDK1) (14); adenylyl cyclase 5 (AC5) (41); MEF2 (11); phospholipase Cϵ (PLCϵ) (21); nesprin-1α (23); ryanodine receptor (RyR) (42); CaN (13); phosphodiesterase 4D3 (PDE4D3) (38); p90 ribosomal S6 kinase 3 (RSK3) (43); protein kinase A (PKA) (40); protein phosphatase 2A (PP2A) (44). HDAC5 binding to mAKAPβ has been mapped to the MEF2D site (12). MEF2D (aa 301–500) and CaN (aa 1285–1345) binding domain peptides are designated MBD and CBD, respectively. B, C2C12 cells transfected with an expression plasmid for mCherry- and FLAG-tagged CaN were cultured in GM or DM in the absence or presence of cyclosporin A (500 nm) for 3 h before immunoprecipitation using FLAG antibodies and detection of associated mAKAPβ. p (one-way ANOVA) < 0.0001. C, same as in B except using cells co-expressing CBD-mCherry or mCherry control. Note that CaN-mCherry-FLAG and the smaller CBD-mCherry (37 kDa) and mCherry (29 kDa) were readily separated by SDS-PAGE. p (two-way ANOVA for both factors and interaction) < 0.02. D, C2C12 cells cultured as in B were fractionated into cytosolic and nuclear fractions. GAPDH and lamin A antibodies were used to show the efficiency of fractionation. Detection of endogenous CaNAβ in each fraction was determined by Western blotting. p (two-way ANOVA for culture conditions and interaction) < 0.01. E, C2C12 cells were transfected with mAKAP or control siRNA before fractionation and analysis as in D. F, C2C12 cells were transfected with mCherry or CBD-mCherry expression plasmids before fractionation and analysis as in D. p (two-way ANOVA for both factors and interaction) < 0.02 for both cytosolic and nuclear fractions for E and F. n = 3 independent experiments for all panels. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Error bars, S.E.