Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean.
Calmodulin-like domain protein kinases (CDPKs) are a family of calcium- but not calmodulin-dependent protein kinases found in a wide variety of plants and in protists. CDPKs are encoded by large multigene families, and to assess whether family members play distinct or redundant roles in vivo, we characterized soybean CDPK isoforms alpha, beta, and gamma, which share 60-80% identity in amino acid sequence. RNA blot analysis showed that the three CDPKs were expressed in most plant tissues examined and in suspension-cultured soybean cells. Recombinant CDPKalpha, -beta, and -gamma phosphorylated peptide substrates containing the four-residue motif R/K-X-X-S/T, but CDPKalpha was the most selective for residues outside of the motif. The CDPKs were inhibited by the general protein kinase inhibitors K252a and staurosporine and by calphostin C, which is an inhibitor of protein kinase C. The calcium-binding properties of each CDPK were distinct. The Kd's for Ca2+ determined by flow dialysis in the absence of substrates were 51, 1.4, and 1.6 micro M for CDPKalpha, -beta, and -gamma, respectively. In the presence of the peptide substrate syntide-2 the Kd of CDPKalpha decreased to 0.6 microM. Also, the sensitivity of this isoenzyme's activity to calcium varied with protein substrate. The concentrations of Ca2+ required for half-maximal activity (K0.5) for each CDPK with syntide-2 as substrate were 0.06, 0.4, and 1 micro M, respectively. These results show that members of the CDPK family differ in biochemical properties and support the hypothesis that each isoform may have a distinct role in calcium signal transduction.
Lee
J Y
JY
Program in Plant Molecular and Cellular Biology, Department of Botany, University of Florida, Gainesville 32611-8526, USA.
Yoo
B C
BC
Harmon
A C
AC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018749:RNA, Plant / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D000255:Adenosine Triphosphate; D002118:Calcium; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases