LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization.
The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 microM). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 microM, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting.
Rutschmann
Frank
F
Institute of Plant Sciences, Plant Biochemistry and Physiology Group, Swiss Federal Institute of Technology, Universitätstrasse 2, CH-8092 Zürich, Switzerland.
Stalder
Urs
U
Piotrowski
Markus
M
Oecking
Claudia
C
Schaller
Andreas
A
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D018551:Solanum lycopersicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics
D002118:Calcium; D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D011494:Protein Kinases; D006180:Proton-Translocating ATPases