Insert Regions in Domain X of the Casein Kinase II Catalytic Subunit

Casein kinase II, cyclin‐dependent kinases, and glycogen synthase kinase‐3 are members of the protein kinase subfamily with a prominent insert in domain X of their catalytic subunit sequence. The function of the insert sequence in casein kinase II was investigated utilising synthetic peptides corresponding to the insert, cross‐linking experiments, and the generation of casein kinase II insert region mutants. The mutation of basic residues (R276→A, R278→A, R281→A, K277→A) within the major insert sequence (PRFHDILQRHSRKRWERFVHSDNQHL, positions 265–290) did not affect α/β subunit association, enzyme tetramerisation, thermal stability, and peptide (RRRDDDSDDD‐NH₂) phosphorylation. Similarly, replacement of residues 276–290 within the major insert with the corresponding residues from the cell‐cycle kinase cyclin‐dependent kinase 2 (CDK2) (FPKWKPGSLASHVKN) had no significant effect. The mutation of charged residues (H232→A, H234→A, D235→A) within a nearby minor insert sequence (HGHDNY, positions 232–237), or replacement of residues 234–237 with the corresponding residues from CDK2 (DSEI) also did not affect α/β subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated α‐subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent K_m for peptide substrate. The results indicate that the major and minor inserts are not essential for α/β subunit association, but the minor insert region influences substrate binding and thermal stability.