Figure 3

Figure 3

Tyrosine autophosphorylation of different vertebrate class 1 DYRKs. (A) Dendrogram of selected class 1 DYRKs. Invertebrates harbor one class I DYRK while most vertebrates contain the paralogous DYRK1A (blue) and DYRK1B (red). Genome duplications resulted in the presence of two DYRK1A genes in Zebrafish (Danio rerio) and Xenopus laevis. Sequence similarity is indicated by branch lengths. The DYRK1 proteins characterized below are underlined. The rat DYRK1A construct differs by a single amino acid from the human sequence. (B,C) Cell free expression. The indicated class 1 DYRKs from rat (r), human (h), Xenopus (x), zebrafish (z) or Drosophila (MNB) were expressed by coupled in vitro transcription and translation for 2 h at 37 °C. The catalytically inactive mutant of DYRK1A (DYRK1A-D287N) was included as a technical control to monitor the background signal of the unphosphorylated kinase. Tyrosine autophosphorylation was detected by immunoblot analysis of total cellular lysates and relative phosphorylation was quantitated by densitometric evaluation (ratio of p-Tyr to Strep-tag signal). hDYRK1B values are not shown because signal intensities were too close to background levels. The scatter plot shows the means and SD of n = 4 replicate experiments (except for zDYRK1Aa, n = 3). (D,E) Expression in E. coli. Expression of the indicated class 1 DYRKs proceeded for 3 h at 37 °C. Tyrosine autophosphorylation of the recombinant kinases was assessed by immunoblot analysis of total bacterial lysates (D). Panel E illustrates the results of n = 3 replicate experiments (means and SD).