In vivo phosphorylation of a recombinant peptide substrate of CDPK suggests involvement of CDPK in plant stress responses.
A phage display library displaying random peptides 15 amino acids in length was screened for peptides that interact with soybean (Glycine max L.) CDPKalpha, an isoform of calcium-dependent protein kinase (EC 2.7.1.37). Interaction of phage displaying the peptide RHPTLTRSPTLRNIQ with CDPKalpha 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 alpha, beta, and gamma. 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 CDPKalpha. 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 CDPKalpha 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.
Shao
Jiahong
J
Department of Botany, University of Florida, Gainesville, FL 32611-8526, USA.
Harmon
Alice C
AC
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D030821:Plants, Genetically Modified; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology; D013379:Substrate Specificity; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D006861:Hydrogen Peroxide; D019151:Peptide Library; D010455:Peptides; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases