Regulation of CDPKs, including identification of PAL kinase, in biotically stressed cells of French bean.
Changes in protein kinase activity have been investigated during the early response of suspension cultured cells of French bean to fungal elicitor. One of the kinases activated has a known target, phenylalanine ammonia-lyase (PAL), which has an important role in plant defence responses, and was purified. Kinase acivity during purification was monitored for both the PAL-derived peptide and syntide-2, which it also phosphorylated. The kinase had an Mr of 55,000 on the basis of gel migration, 45Ca2+ binding, autophosphorylation and phosphorylation of various substrates using in-gel assays. The kinase has been characterised with respect to kinetics and other properties in vitro and appears to be a CDPK. In-gel assays were also used to show that this kinase and a number of other CDPKs of similar Mr showed complex changes in elicitor-treated suspension-cultured cells of French bean. An activation was observed within 10 min and was maintained for up to 4 h. The time course of activation was different from MAP kinase and casein kinase assayed in the same extracts. However, at 5 min after addition of elicitor there is a transient inactivation of the CDPKs before activation. This inactivation can be mimicked by adding forskolin to the cells 30 min before elicitation, which brings about changes in the cellular pH. Forskolin potentiates the oxidative burst when elicitor is subsequently added while the CDPK cannot be activated by elicitor upon forskolin treatment. In contrast, intracellular acidification brought about by forskolin brings about slight activation of MAPkinase.
Allwood
Ellen G
EG
Division of Biochemistry, School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Egham, Surrey, UK.
Davies
Dewi R
DR
Gerrish
Chris
C
Bolwell
G Paul
GP
D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008970:Molecular Weight; D027805:Phaseolus / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D004791:Enzyme Inhibitors; D005947:Glucose; D007546:Isoquinolines; D010650:Phenylalanine Ammonia-Lyase; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides; C017967:W 7; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide