Analysis of cellular function of DYRK family protein kinases

Dyrk(双特异性酪氨酸磷酸化调节蛋白激酶)家族由DYRK1A、DYRK1B、DYRK2、DYRK3和DYRK4几个相关蛋白激酶组成。DYRKs与果蝇小脑有显著的同源性，人类21号染色体上的DYRK1a被证明与人类唐氏综合症有关。在这里，我们报告了与DYRKs特定成员相关的细胞蛋白的鉴定。分子量为90、70和50 kDa的细胞蛋白，与DYRK1B和DYRK4特异结合，但不与其他DYRKs结合。这些蛋白分别被鉴定为分子伴侣Hsp90、Hsp70和CDC37。GFP-DYRKs的显微分析表明，DYRK1A和DYRK1B为细胞核，而DYRK2、DYRK3和DYRK4主要为细胞质。用特定的Hsp90抑制剂格尔达那霉素处理细胞，可以消除Hsp90和CDC37与DYRK1B和DYRK4的关联，但不能消除Hsp70的关联。抑制Hsp90分子伴侣活性影响DYRK1B和DYRK4的细胞内动力学，但不影响其他DYRKs。DYRK1B和DYRK4在用格尔达霉素处理细胞后迅速形成胞质斑点，提示Hsp90的伴侣功能是阻止靶蛋白的蛋白聚集所必需的。长时间抑制Hsp90会降低DYRK1B和DYRK4的细胞水平，但不会降低其他DYRKs的水平。最后，DYRK1B和DYRK4在细胞中被特异性泛素化，而泛素化的DYRK1B和DYRK4被蛋白酶体抑制进一步增加。综上所述，这些结果表明，Hsp90和CDC37区分Dyrk激酶家族的特定成员，并且是这些客户激酶在细胞中的溶解性和稳定性所必需的。

The DYRK (Dual-specificity tYrosine-phosphorylation Regulated protein Kinase) family consists of several related protein kinases, DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4. DYRKs show significant homology to Drosophila Minibrain, and DYRK1A in human chromosome 21 has been shown to be responsible for the human Down's syndrome. Here we report identification of cellular proteins that associate with specific members of DYRKs. Cellular proteins with molecular masses of 90, 70, and 50-kDa associated specifically with DYRK1B and DYRK4, but not with other DYRKs. These proteins were identified as molecular chaperones Hsp90, Hsp70, and Cdc37, respectively. Microscopic analysis of GFP-DYRKs showed that DYRK1A and DYRK1B were nuclear, while DYRK2, DYRK3, and DYRK4 were mostly cytoplasmic. Treatment of cells with the specific Hsp90 inhibitor, geldanamycin, abolished the association of Hsp90 and Cdc37 with DYRK1B and DYRK4, but not of Hsp70. Inhibition of Hsp90 molecular chaperone activity affected the intracellular dynamics of DYRK1B and DYRK4, but not other DYRKs. DYRK1B and DYRK4 underwent rapid formation of cytoplasmic punctate dots after treatment of cells with geldanamycin, suggesting that the chaperone function of Hsp90 is required for the prevention of protein aggregation of target kinases. Prolonged inhibition of Hsp90 decreased the cellular levels of DYRK1B and DYRK4, but not other DYRKs. Finally, DYRK1B and DYRK4 were specifically ubiquitinated in cells, and the ubiquitinated DYRK1B and DYRK4 further increased by proteasome inhibition. Take together, these results indicate that Hsp90 and Cdc37 discriminate specific members of the DYRK kinase family and are required for the solubility and stability of these client kinases in cells.