Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.
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.
Reddy
V S
VS
Department of Biology, Colorado State University, Fort Collins, Colorado 80523, USA. reddy@lamar.colostate.edu
Safadi
F
F
Zielinski
R E
RE
Reddy
A S
AS
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
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