FIGURE 6.

PLK1 phosphorylation regulates MCAK intramolecular interaction. A, MCAK exhibits an intramolecular association in vitro. Recombinant GST-MCAK(1–181) and -(182–586) on glutathione beads were incubated with MCAK-His purified from Sf9 cells as described in Fig. 1B. Anti-MCAK antibody was used in Western blot analysis to determine the intramolecular interaction of MCAK. B, PLK1 phosphorylation promotes the intramolecular association of MCAK in vitro. Recombinant MCAK deletion mutant proteins (GST-MCAK(1–181) and GST-MCAK(182–586)) on glutathione-agarose beads were used as an affinity matrix to isolate MCAK WT-His and its phosphorylation sites mutants from Sf9 cells. Anti-MCAK antibody was used in Western blot analysis to determine the phospho-regulation of the intramolecular interaction of MCAK. C, statistical analysis of the relative binding activity in B. The ordinate indicates the binding ratio of the intensity of MCAK (WT, 6A, or 6E)-His to GST-MCAK (MCAK(1–181) or MCAK(182–586)); the abscissa indicates the corresponding binding assay shown in B.