Analysis of cellular function of DYRK family protein kinases

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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.