A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains.
Recently we isolated a cDNA encoding a tobacco plasma membrane calmodulin-binding channel protein (designated NtCBP4) with a putative cyclic nucleotide-binding domain. Here we analyzed in detail the interaction of NtCBP4 with calmodulin. A full-length recombinant NtCBP4 (81 kDa) expressed in Sf9 insect cells, and the corresponding tobacco membrane protein were solubilized from their respective membrane fractions and partially purified by calmodulin affinity chromatography. NtCBP4 was detected in the eluted fractions using specific antibodies raised against the recombinant protein. By binding [35S]-calmodulin to recombinant NtCBP4 truncations fused to glutathione S-transferase, we identified a single region consisting of 66 amino acids capable of binding calmodulin. A 23 amino acid synthetic peptide from within this region formed a complex with calmodulin in the presence of calcium. We measured the fluorescence of dansyl-calmodulin interacting with this peptide, which revealed a dissociation constant of about 8 nM. The NtCBP4 calmodulin-binding domain was found to perfectly coincide with a phylogenetically conserved alphaC-helix motif of its putative cyclic nucleotide-binding domain. Furthermore, a 23 amino acid region in an equivalent site in the cAMP-binding domain of a mammalian protein kinase regulatory subunit was also found to bind calmodulin. Thus, coinciding calmodulin- and cyclic nucleotide-binding domains may serve as a point of communication between calcium and cyclic nucleotide signal transduction pathways in plants and animals.
Arazi
T
T
Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel.
Kaplan
B
B
Fromm
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D010940:Plant Proteins; D010947:Plants, Toxic; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
C402668:CBP4 protein, Nicotiana tabacum; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008565:Membrane Proteins; D009712:Nucleotides, Cyclic; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium