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 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.
Tiganis
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T
St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
House
C M
CM
Teh
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Kemp
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BE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D047390:Casein Kinase II; D002417:Cattle; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D004331:Drosophila melanogaster / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000502:physiology; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011817:Rabbits; D013329:Structure-Activity Relationship; D013737:Testis / Q000201:enzymology
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase