A phage display library displaying random peptides 15 amino acids in length was screened for peptides that interact with soybean (Glycine max L.) CDPKα, an isoform of calcium-dependent protein kinase (EC 220.127.116.11). Interaction of phage displaying the peptide RHPTLTRSPTLRNIQ with CDPKα was confirmed in an independent binding assay. A synthetic peptide corresponding to this sequence plus the surrounding amino acids AERHPTLTRSPTLRNIQPPC was synthesized and found to be a substrate of CDPK isoforms α, β, and γ. A second random peptide phage display library was constructed that displayed the substrate peptide sequence plus an additional 10 random amino acids on its amino-terminal side. Nine new peptides were obtained from the screening, all of which were phosphorylated by CDPKα. Sequence VSPRSFWTTWRHPTLTRSPTLRNIQ appeared twice in the screen. Because it agreed well with the consensus phosphorylation site of CDPKs, its coding sequence was cloned and stably transformed into tobacco cells. The substrate peptide expressed in tobacco was phosphorylated by recombinant CDPKα in vitro and by endogenous CDPK in vivo. Increased phosphorylation of the peptide substrate in response to hydrogen peroxide treatment was observed in transgenic tobacco cells. These results show that the peptide substrate expressed in tobacco cells can be used as a CDPK activity reporter for in vivo studies.
Plant Molecular Biology – Springer Journals
Published: Oct 7, 2004
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