Calcium and lipid regulation of an Arabidopsis protein kinase expressed in Escherichia coli.
Calcium-dependent protein kinases (CDPKs) represent a new family of protein kinases which are proposed to contain, in a single polypeptide, both a kinase domain and an adjoining calmodulin-like domain with four calcium-binding EF-hand motifs [Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H., & Harmon, A.C. (1991) Science 252, 951-954]. DNA cloning and Western blot analysis indicate that multiple CDPK isoforms are present in the model plant system Arabidopsis thaliana. One CDPK gene called AK1 was isolated from Arabidopsis as a full-length cDNA. The predicted AK1 protein has a M(r) of 72,645 and is 116 amino acid residues longer at the amino terminus than the prototype CDPK alpha gene previously identified in soybean. The most highly conserved region between these two CDPKs is a region of 31 amino acids that joins the kinase and calmodulin-like domains. To verify the kinase activity of the enzyme encoded by AK1, a fusion of an amino-terminally truncated AK1 to the C-terminus of glutathione S-transferase was expressed in Escherichia coli. The fusion protein was purified and displayed a maximum kinase activity of 40 nmol of phosphate/(min.mg), using histone IIIs as a substrate. The enzyme activity was stimulated 3-6-fold by calcium and 2-5-fold by crude lipid. However, a synergistic stimulation of 16-30-fold was observed by the addition of both calcium and crude lipid. Lipid stimulation was specific for lysophosphatidylcholine and phosphatidylinositol and did not occur with the addition of phosphatidylserine or phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)
Harper
J F
JF
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Binder
B M
BM
Sussman
M R
MR
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli; D017343:Genes, Plant; D008055:Lipids / Q000494:pharmacology; D008969:Molecular Sequence Data; D005810:Multigene Family; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D004247:DNA; D008055:Lipids; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases