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Sci Rep
2022 Feb 14;121:2393. doi: 10.1038/s41598-022-06423-0.
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Differential maturation and chaperone dependence of the paralogous protein kinases DYRK1A and DYRK1B.
Papenfuss M
,
Lützow S
,
Wilms G
,
Babendreyer A
,
Flaßhoff M
,
Kunick C
,
Becker W
.
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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.
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).
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