Abstract
Five mutants of protein kinase CK2 alpha 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 beta 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 alpha subunits. The kinetic constants and the phosphorylation efficiencies (V(max)/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 attentuated 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 V(max) values higher than CK2 wild type, counterbalanced however by somewhat higher Km values. It can be concluded from these data that all 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.
Highlights
Five mutants of protein kinase CK2 ␣ 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  subunit
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
The more specific effect of the mutation occurring in the 79 – 83 stretch as compared with that in the 74 –77 stretch is highlighted if their phosphorylation efficiencies are expressed as percentages of the corresponding phosphorylation efficiency by CK2 w.t. (Fig. 4B). In these histograms the only outstanding efficiency bars with mutants K79A,R80A,K83A and R191A, R195A,K198A are those referring to the peptides RRRADDSDDADD and RRRADDSADDDD, respectively, whereas the outcome with mutant K74A,K75A,K76A,K77A is more promiscuous, with three peptides being represented by bars that surpass that of the parent peptide
Summary
(Received for publication, December 22, 1995, and in revised form, February 14, 1996). 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 order to identify the basic residues responsible for substrate recognition, inhibition, and intrinsic down-regulation, we have applied the “charged-to-alanine” scanning mutagenesis strategy [21] to a number of basic residues of the human ␣ subunit that are conserved across various species but divergent from the homologous residues of other protein kinases Six such mutants in which collectively 16 residues have been singly to quadruply mutated to alanines have been obtained, and three of them have been shown to be seriously defective in catalytic activity [18]. We describe the purification of five of these mutants, either as such or combined with the  subunit to give heterotetrameric holoenzymes, and we analyze their kinetic properties with a set of peptide substrates varying for the replacement of individual aspartyl residues between positions Ϫ2 and ϩ5 within the structure of the reference peptide RRRADDSDDDDD
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