Interaction of a Kinesin-like Protein with Calmodulin Isoforms from Arabidopsis

Vaka S Reddy,Farida Safadi,Raymond E Zielinski,Anireddy S.N Reddy

doi:10.1074/jbc.274.44.31727

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.

Highlights

In Arabidopsis and other plants there are multiple calmodulin isoforms
Binding of kinesin-like calmodulin-binding protein (KCBP) Synthetic Peptide to AtCaM Isoforms—In a protein-protein interaction-based screening with animal CaM as a probe, we isolated a novel CaM-binding domain containing kinesin motor protein from Arabidopsis [24]
In Arabidopsis, molecular and biochemical analyses led to the identification of at least four distinct groups of CaM isoforms that differ in their deduced amino acid sequences [5]

Summary

The abbreviations used are

CaM, calmodulin; AtCaM, Arabidopsis CaM; KCBP, kinesin-like calmodulin-binding protein; PDE, cyclic nucleotide phosphodiesterase; BSA, bovine serum albumin; DTT, dithiothreitol. Cam-related genes encode TCH2, TCH3 [21], CaBP-22 protein [22], and a putative Ca2ϩ-binding protein [23] which show 44, 70, 65, and 34.8% amino acid sequence identity, respectively, with CaM2. The divergent group contains two isoforms CaM4 and 5 that differ from the conserved group in 32 amino acids These are the most divergent group of CaM isoforms identified so far in plants [14]. Plants, in contrast to animals that produce an identical CaM protein, possess multiple CaM isoforms [5] These findings suggest that different CaM isoforms may perform different functions in Ca2ϩ-signaling pathways. The interaction of Arabidopsis KCBP with plant CaM, especially with different isoforms of CaMs from the same system, has not been tested. The binding of AtCaM isoforms to KCBP was tested using 35S-labeled AtCaM isoforms

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION