Abstract
The HSP90/CDC37 chaperone system not only assists the maturation of many protein kinases but also maintains their structural integrity after folding. The interaction of mature kinases with the HSP90/CDC37 complex is governed by the conformational stability of the catalytic domain, while the initial folding of the protein kinase domain is mechanistically less well characterized. DYRK1A (Dual-specificity tyrosine (Y)-phosphorylation Regulated protein Kinase 1A) and DYRK1B are closely related protein kinases with discordant HSP90 client status. DYRK kinases stoichiometrically autophosphorylate on a tyrosine residue immediately after folding, which served us as a traceable marker of successful maturation. In the present study, we used bacterial expression systems to compare the capacity of autonomous maturation of DYRK1A and DYRK1B in the absence of eukaryotic cofactors or chaperones. Under these conditions, autophosphorylation of human DYRK1B was severely compromised when compared with DYRK1A or DYRK1B orthologs from zebrafish and Xenopus. Maturation of human DYRK1B could be restored by bacterial expression at lower temperatures, suggesting that folding was not absolutely dependent on eukaryotic chaperones. The differential folding properties of DYRK1A and DYRK1B were largely due to divergent sequences of the C-terminal lobes of the catalytic domain. Furthermore, the mature kinase domain of DYRK1B featured lower thermal stability than that of DYRK1A when exposed to heat challenge in vitro or in living cells. In summary, our study enhances the mechanistic understanding of the differential thermodynamic properties of two closely related protein kinases during initial folding and as mature kinases.
Highlights
Abbreviations cellular thermal shift assays (CETSA) Cellular thermal shift assay DYRK homology (DH) box Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) homology box DTT Dithiothreitol glutathione S-transferase (GST) Glutathione S-transferase
Two polyclonal antibodies are commercially marketed for detection of the phosphotyrosine in DYRK1A and DYRK1B or HIPK2, which share closely related sequences in the activation loop (Fig. 1B)
The present results reveal that DYRK1A and DYRK1B differ strikingly regarding their capacity of autonomous folding
Summary
Abbreviations CETSA Cellular thermal shift assay DH box DYRK homology box DTT Dithiothreitol GST Glutathione S-transferase. Tyrosine autophosphorylation (Tyr[321] in DYRK1A, Tyr[273] in DYRK1B) is essential for full catalytic activity of all DYRKs and the related homeodomain-interacting protein kinases (HIPKs)[3,8,10,12,13,14]. DYRK1B harbors three unique residues N-terminal of the catalytic domain that are not found in other class 1 DYRKs (supplementary Fig. S1) These amino acids are located in a conserved sequence designated DYRK homology (DH) b ox[19]. Crystallization of DYRK1A and DYRK2 revealed that the DH box stabilizes the kinase domain through a large network of interactions[22] It is not yet known whether the differences in the DH box sequence are responsible for the differential folding properties and chaperone dependence of DYRK1A and DYRK1B
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