C-terminus of mitotic centromere-associated kinesin (MCAK) inhibits its lattice-stimulated ATPase activity

Abstract

Mitotic centromere-associated kinesin (MCAK) is a microtubule (MT)-destabilizing molecular motor. In the present study we show that the final 8 amino acids of the C-terminus of MCAK inhibit lattice-stimulated ATPase activity of the motor. Surprisingly, loss of this C-terminal 'tail' (MCAK-Q710) leads to more rapid depolymerization of MTs relative to full-length MCAK (wt-MCAK). Biochemical and microscopic assays revealed that MCAK-Q710 bound to the MT lattice with higher apparent affinity as compared with wt-MCAK. End-stimulated depolymerization was similar for both enzymes. These data suggest that lattice-bound MCAK can increase the rate of MT depolymerization, but at an energy cost. The function of the C-terminus of MCAK may be to selectively inhibit lattice-stimulated ATPase activity, resulting in limited interactions of the motor with the MT lattice. This increases the coupling between ATP hydrolysis and tubulin dimer release, but it also limits MT depolymerization.

Figure 1. C-terminal truncations increase the depolymerization activity of MCAK

The MT depolymerization in CHO cells transfected with wt-MCAK and MCAK-Q710 is shown. (A, C, E) show MTs, with antibody against α-tubulin. (B, D, F) show fluorescent expression of GFP-tagged motor protein. (A) and (B) show a cell transfected with GFP-tagged wt-MCAK. (C) and (D) show a cell transfected with GFP-tagged wt-MCAK, cultured in the presence of 15 μM paclitaxel. (E) and (F) show a cell transfected with GFP-tagged MCAK-Q710 in the presence of 15 μM paclitaxel. (G) shows the C-terminal truncations of GFP-tagged MCAK in conjunction with their amino acid truncation sites. Each construct was transiently transfected into CHO cells cultured in the presence of 15 μM paclitaxel. Cells were fixed 16 h post transfection and antibody labelled against α-tubulin. The extent of MT depolymerization was scored for each of the constructs. + was the maximum observed depolymerization represented by the shortest bundles; ++++ was the minimum represented by longer MT bundles. AA, amino acid.

Figure 2. MCAK-Q710 shows enhanced MT depolymerization in vivo

(A) shows the difference in tubulin polymer levels for the same populations of cells. (B) is a comparison of the cytoplasmic MCAK expression levels between wt-MCAK and MCAK-Q710. Fluorescent intensity levels are measured over 256 grey-scale values. Error bars are shown for each (S.E.M., n=44).

Figure 3. MCAK-Q710 depolymerizes paclitaxel-stabilized MTs faster than wt-MCAK in vitro and exhibits increased apparent affinity for the MT

SDS/PAGE gel shows the difference in localization and depolymerization activity between wt-MCAK and MCAK-Q710. Supernatant lanes (S) show the amount of free tubulin dimer and unbound protein left in solution after the reaction was centrifuged. Pellet lanes (P) show the amount of larger polymer and any bound motor protein after centrifugation. The upper bands in the gel are MCAK motor and the lower bands are tubulin. (A) In each experiment, 50 nM of active motor was added to 2200 nM paclitaxel-stabilized MTs and 1 mM MgATP. MTs were sheared to a length of 15 μm on average. The depolymerization reactions were carried out for 16 min at room temperature (25±1 °C). (B) shows the binding of motor to the MT over a range of motor concentrations. Saturating experiments demonstrate that in the presence of 50 μM MgATP and 8.8 μM MT polymer, MCAK-Q710 saturates the MT polymer at 527 μM, whereas wt-MCAK saturates at 453 μM, a 16% increase. Curves were fitted over data points from 3 independent experiments.

Figure 4. Longer MTs increase MT-stimulated ATPase activity of MCAK

Active motor (50 nM) was incubated in the presence of 11 μM tubulin, 250 μM [γ-³²P]ATP (6000 Ci/mmol) and 250 μM unlabelled MgATP. All Figures display P_i release (μM) over time. Linear curve fits (A, B) were generated by Microsoft Excel. All others (C–E) were generated with MATLAB (The Mathworks). The curves fitted to the data in (C–E) are exponential association curves, fitted via non-linear least-squares regression. (A) shows free tubulin-stimulated ATPase with wt-MCAK and (B) is with MCAK-Q710. (C) shows ATPase stimulation in the presence of unsheared MTs. (D) shows ATPase stimulation in the presence of paclitaxel-stabilized MTs, 5 μm in length. (E) shows ATPase stimulation in the presence of MTs, 15 μm in length. Curves were fitted over data points from 4 independent experiments.

Figure 5. wt-MCAK exhibits tighter 1:1 coupling between MT depolymerization and ATP hydrolysis in comparison with MCAK-Q710

Depolymerization and ATP hydrolysis were compared for MTs 15 μm in length. (A) is wt-MCAK and (B) is MCAK-Q710. The stoichiometry of ATP hydrolysis and MT depolymerization quickly diverges with MCAK-710. However, this activity is suppressed with wt-MCAK.

Figure 6. wt-MCAK shows more significant aggregation than MCAK-Q710 and differential MT binding in the absence of nucleotide

Images of EGFP–wt-MCAK (A) and EGFP–MCAK-Q710 (B) bound to pp[CH₂]pG-stabilized MTs with varying nucleotide conditions are presented. Motor only-labelled rows show the interactions of the motor alone in the absence of MTs. wt-MCAK displays more significant aggregation than MCAK-Q710. p[NH]ppA-labelled (AMP-PNP) rows illustrate the motor in the presence of MTs and p[NH]ppA. Both motors bind the ends of MTs in the presence of p[NH]ppA. Again, wt-MCAK displays larger aggregate on the MT ends in comparison with MCAK-Q710. ADP-labelled rows show the motor with MTs in the presence of ADP. Neither motor appears to bind the MT in the presence of ADP. No nucleotide-labelled rows show the motors with MTs, but in the absence of nucleotide. Both motors bundle MTs and decorate the MT lattice. However, wt-MCAK decorates the lattice in a more punctate fashion, whereas MCAK-Q710 appears to more uniformly decorate the MT lattice with increased apparent-affinity. All binding experiments were done in 75 mM KCl. Incubations were for 5 min at room temperature (25±1 °C).

Figure 7. Proposed model of the difference between wt-MCAK and MCAK-Q710

(A) is a detailed illustration of the proposed ATP hydrolysis cycle of wt-MCAK based on the EGFP–wt-MCAK binding assays. The model proposes that the binding of MCAK to the MT lattice promotes the dissociation of ADP from the motor and the subsequent binding of ATP. MCAK oscillates along the lattice in the ATP bound state until it finds a MT end. Association with the high-affinity-end binding sites triggers ATP hydrolysis and terminal tubulin dimer dissociation. P_i release then liberates the motor from the cleaved tubulin dimer. This model does not differentiate between processivity and non-processivity. Thus both scenarios are illustrated. (B) illustrates how wt-MCAK oscillates back and forth along the MT in a weak binding state until it finds an end, at which time the C-terminal inhibition is relieved. ATPase activity is uninhibited by this interaction and the ATP-binding state then allows the motor to bind tightly to the high-affinity binding site on the terminal tubulin dimer. (C) illustrates how MCAK-Q710 may interact with the MT. While it may primarily oscillate in the loose binding state, similar to wt-MCAK, it may also be able to hydrolyse ATP sporadically along the MT lattice. This ATP hydrolysis along the MT may allow the motor to bind tightly to the lattice. As the MT end approaches these tightly bound motors, they may be able to facilitate more efficient MT depolymerization. This may happen as a result of more efficient end targeting via a power stroke (in comparison with the diffusion-based targeting of wt-MCAK) or it may result from a cooperative interaction between the lattice-bound motors and those bound to the MT end.