Institute of Pharmacology and Toxicology, RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany.
Institute of Molecular Pharmacology, RWTH Aachen University, 52074, Aachen, Germany.
Sci Rep. 2022 Feb 14;12(1):2393. doi: 10.1038/s41598-022-06423-0.
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.
HSP90/CDC37 伴侣系统不仅协助许多蛋白激酶的成熟，而且在折叠后还维持其结构完整性。成熟激酶与 HSP90/CDC37 复合物的相互作用受催化结构域构象稳定性的控制，而蛋白激酶结构域的初始折叠在机制上的特征描述较少。DYRK1A（双特异性酪氨酸（Y）磷酸化调节蛋白激酶 1A）和 DYRK1B 是密切相关的蛋白激酶，但其 HSP90 客户状态不同。DYRK 激酶在折叠后立即在一个酪氨酸残基上进行同工型自磷酸化，这是成熟成功的可追踪标记。在本研究中，我们使用细菌表达系统比较了在没有真核辅助因子或伴侣的情况下 DYRK1A 和 DYRK1B 的自主成熟能力。在这些条件下，与 DYRK1A 或斑马鱼和非洲爪蟾的 DYRK1B 同源物相比，人 DYRK1B 的自磷酸化严重受损。通过在较低温度下进行细菌表达，可以恢复人 DYRK1B 的成熟，这表明折叠不一定完全依赖于真核伴侣。DYRK1A 和 DYRK1B 的不同折叠特性主要归因于催化结构域 C 末端叶的不同序列。此外，当在体外或活细胞中受到热应激时，DYRK1B 的成熟激酶结构域的热稳定性低于 DYRK1A。总之，我们的研究增强了对两种密切相关的蛋白激酶在初始折叠和成熟激酶时的差异热力学性质的机制理解。
