Fig. 5.

Site-directed mutagenesis of the hosphorylation site on peptide P5-2. In order to identify the phosphorylation residue in P5-2, site-directed mutagenesis was performed. P5T-3 is a peptide which has a nine amino acid sequence overlapping with P5-2. P5-2 S→A is a peptide with a mutation of serine (Ser299) to alanine in P5-2; P5-2 T→A is a threonine (Thr296) to alanine mutation peptide; P5-2 TS→AA is a double mutation peptide of serine and threonine to alanine. The results indicated that both serine and threonine in P5-2 can be phosphorylated by G-CDPK16-6His in vitro. Peptide phosphorylation signals are marked with arrowheads.