Figure 4

Figure 4

Expression and autophosphorylation of chimeric DYRK1A/DYRK1B constructs. (A) Scheme of the chimeric constructs. The designations of the chimeras indicate whether the N-terminal region (NT, first letter), DH box (DH, second letter) or N-terminal and C-terminal part of the catalytic domain (cat, third and fourth letter) originate from DYRK1A (blue) or DYRK1B (red). Information on the amino acid positions of the different sequence parts can be found in the supplementary information. (B–E) Bacterial expression. Tyrosine autophosphorylation of bacterially expressed kinases (37 °C) was detected by Western blot analysis. DYRK1A-D287N served as background control. Panel C shows the expression levels of the different constructs as determined by densitometric evaluation of the Strep-tag signals (means and SD, n = 5, including the experiments presented in Fig. 5C). To allow the quantitative evaluation of tyrosine autophosphorylation of constructs with strongly different expression levels, sample volumes were adjusted to better match the amounts of the recombinant proteins on the Western blot (D). Panel (E) illustrates the results of 5 independent experiments (means and SD). Unless otherwise indicated, statistical significance is given for the comparison with DYRK1A. (F–H) Cell-free expression. Constructs were expressed for 2 h at 37 °C by in vitro translation. The graphs illustrate expression levels (G) and phosphotyrosine signals (H) (means and SD, n = 6; including the experiments presented in Fig. 5F). Relative autophosphorylation was not calculated and results were not statistically evaluated because signal intensities of DYRK1B and BBAA were too close to background levels.