Molecular identification of phenylalanine ammonia-lyase as a substrate of a specific constitutively active Arabidopsis CDPK expressed in maize protoplasts.
Phenylalanine ammonia-lyase (PAL) is a key enzyme in pathogen defence, stress response and secondary metabolism and is subject to post-translational phosphorylation. In order to address the significance of this phenomenon it is necessary to identify the protein kinase (PK) responsible and place it in its regulatory circuit. Using protoplast transient expression of Arabidopsis kinase genes coupled to immunocomplex kinase assay, it has been possible to screen for specific PAL kinase. We show here that AtCPK1 (calcium dependent PK), but not other closely related PKs could phosphorylate both a recombinant PAL protein and a peptide (SRVAKTRTLTTA) that is a site phosphorylated in vivo. Identification of the specific CDPK as a PAL kinase now opens up the possibility of exploring the calcium link in biotic stress signalling, salicylate and phytoalexin production as well as the significance of PAL phosphorylation. The protoplast transient expression system is a potentially powerful method to determine and screen for plant gene functions utilising genomic and proteomic data.
Cheng
S H
SH
Department of Molecular Biology, Harvard Medical School, Massachusetts General Hospital, Boston 02114, USA.
Sheen
J
J
Gerrish
C
C
Bolwell
G P
GP
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
D009842:Oligopeptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D010650:Phenylalanine Ammonia-Lyase