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

Abstract

Casein kinase 11, 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 I1 was investigated utilising synthetic peptides corresponding to the insert, cross-linking experiments, and the generation of casein kinase 11 insert region mutants. The mutation of basic residues (R276+A, R278+A, R281+A, K277-+A) within the major insert sequence (PWHDILQRHSRKRWERFVHSDNQHL, positions 265 -290) did not affect alp subunit association, enzyme tetramerisation, thermal stability, and peptide (RRRDDDSDDD-NH,) phosphorylation. Similarly, replacement of residues 276-290 within the major insert with the corresponding residues from the cellcycle 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 alp subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated a-subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent K,,, for peptide substrate. The results indicate that the major and minor inserts are not essential for dj3 subunit association, but the minor insert region influences substrate binding and thermal stability. Keywords. Casein kinase II; domain X; inserts.Casein kinase I1 is an essential ubiquitous serinehhreonine protein kinase that has been localised in eukaryotes to both the nucleus and the cytoplasm. The enzyme phosphorylates proteins important for cellular growth, differentiation, and metabolism. Casein kinase I1 is a heterotetramer and is typically comprised of two catalytic a (or a') subunits and two non-catalytic j3-subunits (Issinger, 1993 ;Pinna, 1990).Casein kinase I1 belongs to a subfamily of serine/threonine protein kinases including the cyclin-dependent kinases that contain insert sequences in domain X of their catalytic subunits Hanks and Quinn, 1991) (Fig. 1) Hanks and Quinn, 1991) (Fig. 1). The major insert sequence is identical in all known mammalian sequences (Gupta and Singh, 1993;Litchfield et al., 1990;Meisner et al., 1989;Maridor et al., 1991) with only three conservative substitutions in the Drosophila sequence (Saxena et al., 1987). Similarly, the minor insert sequence is also highly conserved between species with 100% similarity existing between the human and the Drosophila sequences (Gupta and Singh, 1993;Litchfield et al., 1990 Enzyme. Protein kinase (EC 2.7.1.37). Meisner et al., 1989; Maridor et al., 1993;Saxena et al., 1987). The crystal structure of cell-cycle kinase (CDK2) (De Bondt et al., 1993) suggests that the major insert may be involved in subunit interactions. In CDK2, the minor insert region forms part of the correspondin...