FIGURE 4.

Identification of critical residues for the interaction of AtMKP1 with CaM. A, CaM binding analysis of WT and mutated CaMBD constructs of the D5 and D7 domains shown in Fig. 1A. CaMBDI WT, W453R, and L456R indicate wild-type CaMBDI (D5 domain), and CaMBDI mutants containing single amino acid substitutions, respectively. CaMBDII WT, W678R, and I684R represent wild-type CaMBDII (D7 domain), and CaMBDII mutants containing single amino acid substitutions, respectively. B, interaction of WT and CaMBD mutants of full-length AtMKP1 (D0 described in Fig. 1A) with CaM. Wild-type and CaMBD mutants were fused to the C terminus of GST and expressed in E. coli. Expressed recombinant proteins were analyzed by Western blotting with an anti-GST antibody (Western). CaM binding was analyzed using a CaM: HRP overlay assay in the presence of 1 mm CaCl₂ or 5 mm EGTA.