Novel Insights into the Biochemical Mechanism of CK1epsilon and its Functional Interplay with DDX3X

Casein Kinase 1 epsilon (CK1epsilon) is a member of the serine (Ser)/threonine (Thr) CK1 family, known to have crucial roles in several biological scenarios and, ever more frequently, in pathological contexts, such as cancer. Recently, the human DEAD-box RNA helicase 3 X-linked (DDX3X), involved in cancer proliferation and viral infections, has been identified as one of CK1epsilon substrates and its positive regulator in the Wnt/beta-catenin network. However, the way by which these two proteins influence each other has not been fully clarified. In order to further investigate their interplay, we defined the kinetic parameters of CK1epsilon towards its substrates: ATP, casein, Dvl2 and DDX3X. CK1epsilon affinity for ATP depends on the nature of the substrate: increasing of casein concentrations led to an increase of KmATP, while increasing DDX3X reduced it. In literature, DDX3X is described to act as an allosteric activator of CK1epsilon. However, when we performed kinase reactions combining DDX3X and casein, we did not find a positive effect of DDX3X on casein phosphorylation by CK1epsilon, while both substrates were phosphorylated in a competitive manner. Moreover, CK1epsilon positively stimulates DDX3X ATPase activity. Our data provide a more detailed kinetic characterization on the functional interplay of these two proteins.