Critical role of the CMGC insert sequence for tyrosine autophosphorylation in the protein kinase DYRK1B

Many CMGC group kinases require phosphorylation of a conserved tyrosine residue in the activation loop to achieve catalytic activity. DYRK family members use a distinctive mechanism involving constitutive cis-autophosphorylation of this tyrosine. The structural basis of this process has remained unclear, as it occurs while the kinase is still in an inactive conformation, and the tyrosine does not match the known substrate consensus of DYRKs. Here, we exploited the different autophosphorylation capacities of the paralogs DYRK1A and DYRK1B to define structural determinants of this process. DYRK1A efficiently autophosphorylates even in cell-free systems, whereas DYRK1B does not. Using domain swaps and point mutations, we identify the CMGC insert in the C-terminal lobe and two adjacent proline residues (P332/P333 in DYRK1B) as critical for proper folding and activation. Mutation of either proline impaired DYRK1B autophosphorylation and nuclear localization but had no effect in DYRK1A. Substitution of the DYRK1B CMGC insert with that of DYRK1A rescued the maturation defect, demonstrating functional interplay between the insert and flanking prolines. Furthermore, the pathogenic R349W mutation in DYRK1B, associated with monogenic obesity and type 2 diabetes, also disrupted autophosphorylation. These findings highlight the role of the CMGC insert and adjacent prolines in DYRK kinase maturation and autoactivation.