Insert regions in domain X of the casein kinase II catalytic subunit.
Tiganis, T, et al.
Eur. J. Biochem., 229: 703-9 (1995)
1994
Afficher le résumé
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 alpha/beta 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 alpha/beta subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated alpha-subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent Km for peptide substrate. The results indicate that the major and minor inserts are not essential for alpha/beta subunit association, but the minor insert region influences substrate binding and thermal stability. | | 7758466
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Casein kinase II beta-subunit inhibits the activity of the catalytic alpha-subunit in the absence of salt.
Tiganis, T, et al.
Biochim. Biophys. Acta, 1203: 282-9 (1993)
1992
Afficher le résumé
Casein kinase II (CKII) has a subunit structure of alpha 2 beta 2 where alpha is the catalytic subunit. Recombinant Drosophila casein kinase II alpha-subunit expressed in insect cells is inhibited by NaCl, thermally labile and inactivated by binding to plastic. In the presence of detergent (Tween 80) recombinant alpha-subunit has a kcat of 249 min-1 (Km 170 microM) for the peptide substrate RRRDDDSDDD-NH2, compared to recombinant Drosophila CKII with a kcat of 71 min-1 (per mol alpha) (Km 42 microM) and bovine CKII with a kcat of 123 min-1 (per mol alpha) (Km 45 microM) when measured in the absence of NaCl. The kcat values of bovine CKII and recombinant Drosophila CKII measured in the presence of 150 mM NaCl were 429 min-1 (per mol alpha) (Km 82 microM) and 204 min-1 (per mol alpha) (Km 51 microM), respectively. Since the kcat for the Drosophila alpha-subunit is approx. 3-fold greater than the Drosophila CKII measured in the absence of added salt these results indicate that the beta-subunit acts primarily as an inhibitory subunit. | | 8268212
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