title: Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.
abstract: In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nM, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of (35)S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, Colorado 80523, USA. reddy@lamar.colostate.edu
authors.forename: V S
authors.initials: VS
authors.lastname: Reddy
authors.forename: F
authors.initials: F
authors.lastname: Safadi
authors.forename: R E
authors.initials: RE
authors.lastname: Zielinski
authors.forename: A S
authors.initials: AS
authors.lastname: Reddy
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D016547:Kinesins / Q000378:metabolism*; D008555:Melitten / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; C099551:KCBP protein, Arabidopsis; D010446:Peptide Fragments; D010940:Plant Proteins; D020033:Protein Isoforms; D008555:Melitten; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D016547:Kinesins