Fig. 5.

Kip2 interacts with Bim1 over its N-terminal extension in a phosphorylation-dependent manner. (A) Alignment of the N-terminal extensions of Kip2 and fission yeast Tea2. The proposed EB1-interacting motifs are shown in green, positively charged residues in yellow. The GSK-3 consensus site comprising S63 is shown in bold. (B,C) Bim1 interacts with the EB1-binding motif at the Kip2 N-terminus, independently of Bik1. Immunoprecipitations of ^HABim1 from kip2Δ or kip2Δ bik1Δ cells expressing depicted plasmid-borne Kip2^13myc variants. Note that only non- (or hypo)-phosphorylated Kip2 isoforms co-precipitate with ^HABim1 (seen in C). P_GAL3HA-BIM1 expression was induced for 3 h prior to immunoprecipitation. Immunoprecipitated proteins (IP) and extracts after the immunoprecipitation (input) were probed in western blots with anti-HA and anti-Myc antibodies. Asterisk indicates antibody heavy chain. SS refers to the Kip2^13myc variant with the SNIP motif mutated to SNSS. (D) Localisation of the Kip2–YFP variants which show decreased interaction with Bim1 (C and D). Representative images of cells expressing CFP–Tub1 and integrated Kip2–YFP variants from the KIP2 promoter as the sole Kip2 source. (E) The two EB1-binding motifs are required for Kip2–Bim1 interaction in vitro. Shown are coomassie stained gels INPUT: amounts of the recombinant proteins used in the pulldown assay, the EB1 domain of Bim1 (^EB1Bim1) as a GST-fusion was bound to beads (bait). S^xIP1 and S^xIP2 denote the recombinant Kip2 variants with respective mutations (SNIP to SNNN) in the two EB1-binding motifs of Kip2 (see main text). Mutation of these motifs does not abrogate but weakens the Kip2-interaction in an additive manner. The Kip2–Bim1 interaction is almost lost upon deletion of the N-terminus (Δ70) or upon mutation of the N-terminal GSK-3 clusters to aspartates (allD, refer also Fig. 1A).