Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli.
The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.
Jakobi
R
R
Department of Biochemistry, University of California, Riverside 92521.
Traugh
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D047390:Casein Kinase II; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010770:Phosphotransferases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases