Fig. 3: Substrate phosphorylation assay and autophosphorylation assay for CDK19 variants. | Genetics in Medicine

Fig. 3: Substrate phosphorylation assay and autophosphorylation assay for CDK19 variants.

From: CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

Fig. 3

(a,b) Substrate phosphorylation assays were performed using the equal amounts of each purified recombinant CDK19 kinase module (wild-type [wt], Gly28Arg [G28R], or Tyr32His [Y32H]) and either recombinant Pol II-CTD (a) or STAT1 (b) as a substrate (30 °C, 3 hours). The reaction products were analyzed by immunoblotting using indicated phospho-specific antibodies for evaluating phosphorylation levels and anti-GST antibody (Pol II-CTD) or anti-STAT1 antibody (STAT1) for evaluating the total amount of the substrates (top row). Each assay was performed multiple times and one representative result is shown. Phosphorylation degree (bar charts on the bottom) was evaluated by quantification of the intensity of immunoblotting signal using ImageJ software. Error bars indicate the standard error of the mean (SEM). The asterisks represent statistically significant differences between wt and the indicated variant (Fisher’s exact t-test, **p < 0.01, ***p < 0.001). Note that the Gly28Arg mutant almost completely loses the ability to phosphorylate both the CTD (a) and STAT1 (b), although this mutant still can form the complex with other subunits (cyclin C, MED12, and MED13) as seen in Fig. S1A. The Tyr32His variant, on the other hand, tends to show higher kinase activity than wild-type CDK19 (a,b). (c) Autophosphorylation activity rates of wild-type CDK19, Gly28Arg (G28R)-mutated CDK19, and Tyr32His (Y32H)-mutated CDK19 using HEK293 cells. Note that kinase activity of Gly28Arg (G28R) mutant tended to be lower than that of wild-type CDK19. Kinase activity of Tyr32His (Y32H) mutant, was (significantly) higher than that of wild-type CDK19. The values shown are the average of the values observed in one representative experiment in which each transfection was performed in triplicate. Error bars indicate the standard error of the mean (SEM). The imunoprecipitated CDK19 proteins were analyzed by immunoblotting (bottom panel) showing stable levels.

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