Mapping the residues of protein kinase CK2 implicated in substrate recognition: mutagenesis of conserved basic residues in the alpha-subunit.
Six mutants of protein kinase CK2 alpha subunit in which basic residues have been mutated into alanines were assayed for their capability to phosphorylate the peptide RRRADDSDDDDD. Two mutants (R228A and R278K279R280A) behaved more or less as alpha wild type and one (H160,166A) was nearly inactive, hampering the calculation of kinetic parameters. In contrast 3 mutants (K74-77A, K79R80K83A and R191,195K198A) phosphorylated the peptide with reduced efficiency accounted for by increased Km and decreased Vmax values. By using derivatives of the RRRADDSDDDDD peptide in which individual aspartyl residues were variably replaced by alanine(s) and two peptide substrates derived from I-2 (KYRIREQESSGEEDSDL and RRKDLHDDEEDEEMSETADGE) it was shown that mutations in the 191-198, 74-77 and 79-83 regions were the least detrimental whenever the acidic determinants were lacking at positions +1, +4/+5 and +3, respectively. These data support the conclusion that the basic residues present in the p+1 loop of CK2 alpha specifically recognize the acidic determinant adjacent to the C-terminal side of serine, while the specificity determinants located more down-stream are variably recognized by different residues of the unique basic cluster spanning between Lys74 and Lys83.
Sarno
S
S
Dipartimento di Chimica Biologica, CRIBI, Università di Padova, Italy.
Boldyreff
B
B
Marin
O
O
Guerra
B
B
Meggio
F
F
Issinger
O G
OG
Pinna
L A
LA
D000595:Amino Acid Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D017124:Conserved Sequence; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases