Figure 8.

Ser244 phosphorylation inhibits microtubule bundling but not microtubule binding or ATPase activity. (A) Western blot (WB) of pellets from microtubule (MT) pelleting assays performed in 25 mM KCl. Ser244 mutation to Ala or Glu does not affect the ability of Mklp2 to bind microtubules. (B) Mklp2 WT, S244A, or S244E was incubated with or without Nek6 followed by addition of taxol-stabilized microtubules and centrifugation onto coverslips. Microtubule bundling was observed by immunofluorescence. Nek6 inhibits bundling of Mklp2 WT, but Mklp2 S244A is refractory to Nek6 inhibition, whereas Mklp2 S244E is significantly attenuated in bundling activity in both conditions. Representative images of three independent experiments are shown. Bar, 20 µm. (C) Ser244 mutation to Ala or Glu does not affect the microtubule-dependent ATPase activity of Mklp2. Recombinant Mklp2 WT, S244A, and S244E were incubated with or without taxol-stabilized microtubules for 30 min at 28°C, and then ATPase activity was measured. Graphs are normalized to mean WT ATPase activity from three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD.