Fig. 6
From: A novel cardiomyopathy phenotype linked to a CHD7 missense variant
Kinases phosphorylate human CHD7 at T730 and S734, pivotal sites in a regulatory kinase pathway for CHD7. (a) Schematic diagram of the T730 and S734 sites along with the key protein domains in CHD7. The consensus sequence of the GSK3β substrate, ‘Ser/Thr-X-X–X-Ser/Thr-P’, is highlighted in a red box and aligned with the amino acid sequence near T730. The WT T730 peptide used for the in vitro kinase assays is underlined. (b) List of synthesized peptides surrounding T730 used in the in vitro kinase assays, including a peptide with pre-priming phosphorylation at S734 (pS734) and several mutant peptides. (c) In vitro kinase assays using DYRK2 or HIPK1 kinases on the indicated peptides near CHD7 T730 and its mutant variants. Relative kinase activities were normalized to the luminescence signal of WT controls, measured using a GloMax plate reader. (d) In vitro kinase assay using GSK3α or GSK3β on the indicated peptides near CHD7 T730. Enzyme activity was measured by luminescence as shown in (c). (e) Autoradiography and Coomassie blue staining of an SDS-PAGE gel showing the results of in vitro kinase reactions using GSK3β on human full-length CHD7 protein in the presence of radioactive [γ−32P]ATP. (f) Model for the regulatory phosphorylation of CHD7 by kinases and the effects of nearby P737L mutation. Statistical significance is indicated as follows: *, **, ***; P < 0.05, P < 0.01, and P < 0.001, respectively (Student t-test).
