Fig. 4.

Phosphorylation regulates association of Kip2 with MTs. (A) Mck1-dependent phosphorylation regulates Kip2 load on aMTs. Fluorescence intensity of Kip2–YFP, Kip2-AT–YFP and allD-Kip2–YFP in wild-type cells and of Kip2–YFP in ^HAMck1-overexpressing or Mck1-depleted cells. Examples of quantified images are given above each graph. Numbers of counted aMTs in brackets, arbitrary intensity units ±s.e.m. are shown. Scale bars: 3 µm. (B) Cycloheximide chase of plasmid-borne Kip2^13mycAA under control of the KIP2 promoter in cells expressing Kip2^TAP from the KIP2 locus as an internal control. Kip2 phosphoisoforms were not resolved here in order to facilitate comparison between different Kip2 variants. (C) A variant of Kip2 with a phosphorylation-mimicking N-terminus (allD-^MBPKip2ΔC^GFP) binds less efficiently to MTs in vitro, compared to the unchanged variant. Shown are Coomassie-Blue-stained gels of microtubule sedimentation assays, after incubation of in vitro polymerised, taxol-stabilized MTs with recombinant and ^MBPKip2ΔC^GFP or allD-^MBPKip2ΔC^GFP. S, supernatant; P, pellet. Refer also to Fig. S2D.