Figure 1

Figure 1

Detection of tyrosine autophosphorylation in DYRK1A, DYRK1B and MNB. (A) Domain structure of selected class 1 DYRKs and design of expression constructs. Conserved sequence regions include the catalytic domain (cat), the DH box and the N-terminal region (NT), which harbors a binding site for the adaptor protein DCAF7 and a bipartite nuclear localization signal (NLS). Expression constructs employed in the present study (designated ΔC) were fused to a Strep-tag (S), glutathione S-transferase (GST), or the FLAG (F) and HiBiT (H) tags. The shorter constructs (ΔNΔC) have been described previously¹¹. The exact boundaries of the different subdomains are listed in the supplementary information. (B) Sequence surrounding the autophosphorylated tyrosine in the activation loop. The alignment illustrates the structural basis for the cross-reaction of the HIPK2(pY361) antibody with class 1 DYRKs. (C) Detection of tyrosine autophosphorylation in E. coli. The indicated deletion constructs of rat DYRK1A, human DYRK1B and Drosophila MNB were expressed in E. coli for 3 h at 37 °C. Total cell lysates were subjected to immunoblot analysis with different commercial antibodies that were directed against the tyrosine in the activation loop of HIPK2 (pY361) or DYRK1A/B (pY321/273), or with an antibody that detects phosphotyrosine (pTyr) independent of the sequence context (PY99). Total recombinant protein was visualized by detection of the N-terminal Strep-tag. Point mutants used as controls include a catalytically inactive point mutant of DYRK1A (DN, D287N) and mutants of the autophosphorylation site in the activation loop (YF, Y321F in DYRK1A and Y326F in MNB). (D) Comparison of DYRK1A and DYRK1B. DYRK1-ΔC constructs were expressed in E. coli for 3 h at 37 °C or for 20 h at room temperature (20–25 °C). Tyrosine autophosphorylation was detected by immunoblot analysis of total bacterial lysates with the pY361(HIPK2) antibody. Relative phosphorylation was densitometrically quantitated (ratio of pTyr to Strep-tag signal). The graphs illustrate the results of n = 4 experiments (means and SD; One sample t test).