The calmodulin-binding domain from a plant kinesin functions as a modular domain in conferring Ca2+-calmodulin regulation to animal plus- and minus-end kinesins.
Plant kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that interacts with calmodulin (CaM) via its CaM-binding domain (CBD). Activated CaM (Ca(2+)-CaM) has been shown to inhibit KCBP interaction with microtubules (MTs) thereby abolishing its motor- and MT-dependent ATPase activities. To test whether the fusion of CBD to non-CaM-binding kinesins confers Ca(2+)-CaM regulation, we fused the CBD of KCBP to the N or C terminus of a minus-end (non-claret disjunction) or C terminus of a plus-end (Drosophila kinesin) motor. Purified chimeric kinesins bound CaM in a Ca(2+)-dependent manner whereas non-claret disjunction, Drosophila kinesin, and KCBP that lack a CBD did not. As in the case of KCBP with CBD, the interaction of chimeric motors with MTs, as well as their MT-stimulated ATPase activity, was inhibited by Ca(2+)-CaM. The presence of a spacer between the motor and CBD did not alter Ca(2+)-CaM regulation. However, KCBP interaction with MTs and its MT-stimulated ATPase activity were not inhibited when the motor domain and CBD were added separately, suggesting that Ca(2+)-CaM regulation of CaM-binding motors occurs only when the CBD is attached to the motor domain. These results show that the fusion of the CBD to animal motors confers Ca(2+)-CaM regulation and suggest that the CBD functions as a modular domain in disrupting motor-MT interaction. Our data also support the hypothesis that CaM-binding kinesins may have evolved by addition of a CBD to a kinesin motor domain.
Reddy
Vaka S
VS
Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins 80523, USA. reddy@lamar.colostate.edu
Reddy
Anireddy S N
AS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004330:Drosophila; D016547:Kinesins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*
D002147:Calmodulin; D017931:DNA Primers; D016547:Kinesins; D002118:Calcium