Identification of a novel phosphorylation site within the KLP10A motor domain. (a) Anti-KLP10A immunoprecipitate from S2 cell lysate separated by SDS-PAGE and stained with ProQ or transferred to nitrocellulose for Western blotting with anti-KLP10A antibodies. (b) Partial sequence alignment of the identified region from the motor domains of different kinesin family members. (c) The cryo-EM map of the Kif1A head–MT complex structure (Protein Data Bank accession no. 2hxh; Kikkawa and Hirokawa, 2006) was used as a model for kinesin motor domain–α-β–tubulin interaction. The motor domain of Kif1A was replaced with the crystal structure of the kinesin 13 Kif2C (Protein Data Bank accession no. 1v8j; Ogawa et al., 2004) using homology-modeling features of Chimera software (University of California, San Francisco, San Francisco, CA). A magnified view of the boxed region is show on the right. (d) Linear map of KLP10A neck/motor construct used for biochemical studies. (e) Coomassie-stained SDS-PAGE gel of purified, bacterially expressed KLP10A proteins used in our assays. (f) Representative KLP10A/MT cosedimentation assay. MT depolymerization assay. n = 18 (wt), 17 (S573A), and 19 (S573E). S, supernatant; P, pellet. MT concentrations are indicated in micromolars. (g) Mean measurements from three independent experiments were fit to rectangular hyperbolas. MT-E, MT-bound KLP10A; E, soluble KLP10A. (h) Apparent dissociation constants of KLP10A constructs for MTs (mean ± SEM). (i and j) Measurement of depolymerase activities and representative MT sedimentation assay, respectively. ***, P < 0.001. Error bars indicate SD.