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J Biol Chem, Vol. 273, Issue 41, 26375-26382, October 9, 1998
,¶
From the 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.
Department of Biological Sciences, Graduate
School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo
113-0033, Japan, the § Department of Biological Sciences,
Stanford University, Stanford, California 94305, and the ¶ Unit
Process and Combined Circuit, PRESTO, Japan Science and Technology
Corporation, Graduate School of Science, University of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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