Protein Kinase CK2 Mutants Defective in Substrate Recognition

Five mutants of protein kinase CK2 α subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition of the recombinant β subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated α subunits. The kinetic constants and the phosphorylation efficiencies (Vmax/Km) of all the mutants with the parent peptide RRRADDSDDDDD and a series of derivatives, in which individual aspartic acids were replaced by alanines, have been determined. Three mutants, namely K74A,K75A,K76A,K77A; K79A,R80A, K83A; and R191A,R195A,K198A display dramatically lower phosphorylation efficiency and 8-50-fold higher Km values with the parent peptide, symptomatic of reduced attitude to bind the peptide substrate as compared with CK2 wild type. Such differences either disappear or are attenuated if the mutants R191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A,K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type. These data show that one or more of the basic residues clustered in the 191-198, 79-83, and 74-77 sequences are implicated in the recognition of the acidic determinants at positions +1, +3, and +4/+5, respectively, and that if these residues are mutated, the relevance of the other acidic residues surrounding serine is increased. In contrast the other two mutants, namely R228A and R278A,K279A, R280A, display with all the peptides Vmax values higher than CK2 wild type, counterbalanced however by somewhat higher Kmvalues. It can be concluded from these data that all the five mutations performed are compatible with the reconstitution of tetrameric holoenzyme, but all of them influence the enzymatic efficiency of CK2 to different extents. Although the basic residues mutated in the 74-77, 79-83, and 191-198 sequences are clearly implicated in substrate recognition by interacting with acidic determinants at variable positions downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis. Five mutants of protein kinase CK2 α subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition of the recombinant β subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated α subunits. The kinetic constants and the phosphorylation efficiencies (Vmax/Km) of all the mutants with the parent peptide RRRADDSDDDDD and a series of derivatives, in which individual aspartic acids were replaced by alanines, have been determined. Three mutants, namely K74A,K75A,K76A,K77A; K79A,R80A, K83A; and R191A,R195A,K198A display dramatically lower phosphorylation efficiency and 8-50-fold higher Km values with the parent peptide, symptomatic of reduced attitude to bind the peptide substrate as compared with CK2 wild type. Such differences either disappear or are attenuated if the mutants R191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A,K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type. These data show that one or more of the basic residues clustered in the 191-198, 79-83, and 74-77 sequences are implicated in the recognition of the acidic determinants at positions +1, +3, and +4/+5, respectively, and that if these residues are mutated, the relevance of the other acidic residues surrounding serine is increased. In contrast the other two mutants, namely R228A and R278A,K279A, R280A, display with all the peptides Vmax values higher than CK2 wild type, counterbalanced however by somewhat higher Kmvalues. It can be concluded from these data that all the five mutations performed are compatible with the reconstitution of tetrameric holoenzyme, but all of them influence the enzymatic efficiency of CK2 to different extents. Although the basic residues mutated in the 74-77, 79-83, and 191-198 sequences are clearly implicated in substrate recognition by interacting with acidic determinants at variable positions downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis.