Importance of phenylalanine residues of yeast calmodulin for target binding and activation.
Recent genetic studies of yeast calmodulin (yCaM) have shown that alterations of different sets of Phe residues result in distinct functional defects (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). To examine the importance of Phe residues for target binding and activation, we purified mutant yCaMs containing single or double Phe to Ala substitutions and determined their ability to bind and activate two target proteins, calcineurin and CaM-dependent protein kinase (CaMK). Binding assays using the gel overlay technique and quantitative analyses using surface plasmon resonance measurements indicated that the binding of yCaM to calcineurin is impaired by either double mutations of F16A/F19A or a single mutation of F140A, while binding to CaMK is impaired by F89A, F92A, or F140A. These same mutant yCaMs fail to activate calcineurin and CaMK, respectively, in vitro. In addition, F19A exhibited a severe defect in activation of both enzymes. F12A activated calcineurin to only 50% of the level achieved by wild-type calmodulin but fully activated CaMK. These results suggest that each target protein requires a specific and distinct subset of Phe residues in yCaM for target binding and activation.
Okano
H
H
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Cyert
M S
MS
Ohya
Y
Y
D002147:Calmodulin / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D000072235:Mating Factor; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D002147:Calmodulin; D010455:Peptides; D011994:Recombinant Proteins; D010649:Phenylalanine; D000072235:Mating Factor