Unraveling the role of DDX RNA helicases in protein kinase regulation

DEAD-box (DDX) RNA helicases are a large family of ATPases, many of which have unknown functions. We previously identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-β-catenin network in embryonic development, where it acts as a regulator of Casein kinase 1 epsilon (CK1e). DDX RNA helicases commonly promote RNA processing, but intriguingly, the DDX3X oncogene is as we showed also an activator of Casein kinase 1e (CK1e) in Wnt signaling.

We demonstrated that kinase stimulation is a latent property of many DDX proteins. Stimulation resides in the helicase core domain and engages RNA binding- but not catalytic motifs. In DDX3X, the C-terminus provides specificity for DVL2 condensates in Wnt/CK1esignaling. For Casein kinase 2 (CK2a2), we identified DDX1, -24, -41, and -54 as physiological activators. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy converge on the conclusion that DDX proteins function as nucleotide exchange factors to stimulate CK2a2 activity and overcome substrate inhibition. Our studies reveal protein kinase stimulation by nucleotide exchange as a new principle in kinase regulation and an evolved function of DDX proteins.

We conducted several large-scale screens to investigate if protein kinase-DDX interaction is a more widespread phenomenon. In these screens, we retrieved Ser/Thr protein kinases as prominent interactors of DDX/DHX proteins and found hundreds of binary interactions. We extracted members of eleven protein kinase families, which bind to- and are stimulated by DDX proteins, including CAMK, CDK, CK1, CK2, DYRK, MARK, NEK, PRKC, SRPK, STE7/MAP2K, and STE20/PAK family members. MARK1 was identified in all screens and enzyme kinetic validation confirmed DDX proteins as V-type activators of MARK1. The findings indicate pervasive interactions between protein kinases and DEAD box RNA helicases and they will provide a rich resource to explore their regulatory relationships.

Our working hypothesis is that the interaction between DDX proteins and protein kinases will be particularly important in phase separation where protein concentrations are very high. We therefore investigate the role of DDX3X in biomolecular condensates during Wnt signaling.