Exploring the Mechanisms of a Phosphorylation Induced Inhibition of Microtubule Depolymerization in the Kinesin 13 KLP10A

Abstract

Kinesins 13 are a class of non-motile molecular motors able to regulate microtubule dynamics through their depolymerization activity at microtubule tips. A previous study in our lab on Drosophila Melanogaster KLP10A found a phosphorylation site within the motor domain that reduces microtubule depolymerization activity. This led us to study the inhibition mechanisms related to this phosphorylation site distant from the ATPase site. We have employed an integrated approach to compare the behavior of phosphomutant and wild-type KLP10A constructs with cell biology experiments and a variety of in-vitro biophysical methods. Imaging insect cells having only fluorescently labelled KLP10A variants revealed that the phosphomutant is distributed homogeneously on microtubules (lattice and tips) in contrast with the wild-type or a non-phosphorylatable mutant showing higher kinesin concentration on microtubule tips. Wild-type and phosphomutant neck-motor kinesins constructs have similar properties with isolated structures that mimic these microtubule tips: they can both stabilize curved microtubule protofilaments, an important property for microtubule depolymerization, and have similar affinity and ATPase activity on curved tubulin. However the phosphomutation strongly reduces the microtubule lattice stimulated ATPase activity despite non distinguishable microtubule lattice affinities. Single molecule fluorescence polarization microscopy revealed nucleotide dependent differences in the lattice diffusive behavior between the constructs. EPR and cryo-EM experiments are used to compare the kinesins on the microtubule lattice vs curved protofilaments. The data are compatible with a reduced microtubule depolymerization activity of the phosphomutant caused by a reduction of the lattice stimulated ATPase activity. This reduced activity constrains the kinesin to be in nucleotide states which restrict it from reaching the microtubule ends in comparison to the unphosphorylated kinesin.