Probing the Kinesin-Microtubule Interaction*

(Received for publication, December 11, 1996, and in revised form, January 22, 1997)

Carla Tucker Dagger and Lawrence S. B. Goldstein Dagger §

From the § Howard Hughes Medical Institute and Dagger  Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0683

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Kinesin is a mechanoenzyme that couples adenosine triphosphate hydrolysis to the generation of force and movement along microtubules. To gain insight into the interactions of kinesin and microtubules, cross-linking, mapping, and proteolysis experiments were executed. The motor domain of kinesin was consistently cross-linked to both alpha - and beta -tubulin subunits. Initial mapping of the cross-linked kinesin suggested that amino acids within the N- and C-terminal cyanogen bromide fragments of the motor domain formed cross-links to both alpha - and beta -tubulin subunits. Mapping of the cross-linked tubulin suggested that cross-linking to kinesin motors occurred within the negatively charged, C-terminal cyanogen bromide fragments of alpha - and beta -tubulin subunits. Treatment of microtubules with subtilisin, a protease that cleaves C-terminal fragments from alpha - and beta -tubulin, reduced their ability to be cross-linked to kinesin motors supporting the idea that C-terminal sequences of alpha - and beta -tubulin may interact with kinesin motors. Finally, of three synthetic peptides, a peptide consisting of the last 12 C-terminal amino acids of beta -tubulin competitively interfered with the microtubule-stimulated adenosine triphosphatase activity of the kinesin motor, further suggesting that C-terminal sequences of beta -tubulin may be involved in kinesin binding.


INTRODUCTION

Members of the kinesin superfamily of motor proteins harness the energy liberated from ATP hydrolysis to generate a wide variety of intracellular movements (1, 2). To elucidate how kinesin generates force against the microtubule, it is important to understand how kinesin and the microtubule interact. Initial truncation studies of Drosophila kinesin heavy chain revealed that the N-terminal 340 amino acids can hydrolyze ATP and translocate microtubules (3, 4). Consistent with this observation, sequence homology among members of the kinesin superfamily is restricted to this region (2).

Recent work has focused on the interaction of the motor domain with the microtubule, which is composed of repeating dimeric subunits, each consisting of an alpha - and beta -tubulin monomer. The binding stoichiometry of the kinesin motor to tubulin was reported to be one kinesin motor domain/tubulin dimer (5, 6). However, the issue of whether kinesin can be cross-linked only to beta -subunits (6), or to both alpha - and beta -subunits (7), remains controversial. Analysis of the atomic structure of the human kinesin motor domain led to the suggestion that loops 8 and 12 (amino acids 138-173 and 272-280) bind to microtubules, which is consistent with the observation that the C-terminal region of the 340-amino acid kinesin motor may be involved in binding to microtubules (8, 9).

In this paper, the cross-linking reaction between kinesin motors and microtubules was investigated further. Using low concentrations of cross-linker, kinesin motors can be cross-linked consistently to both alpha - and beta -tubulin subunits. Further analysis suggests that cross-linking likely occurs within both the most N- and C-terminal CnBr fragments of the kinesin motor. Finally, to probe the sites in tubulin that interact with the kinesin motor, the sites of cross-linking on alpha - and beta -tubulin were mapped, the behavior of subtilisin-treated tubulin was assessed, and competitive peptide inhibitors were assayed. Together, the data suggest that highly charged C-terminal regions of alpha - and beta -tubulin interact with the kinesin motor.


EXPERIMENTAL PROCEDURES

Constructs

Unless otherwise noted, kinesin motor refers to a protein expressed from the vector pET(23b+) (Novagen); the resulting construct, pET(23b+)K369I, encodes the first 369 amino acids of Drosophila kinesin heavy chain plus one additional isoleucine at the C terminus and is referred to as K369I (11).

Protein Preparation (K369I)

A 20-ml culture of BL21(DE3) containing pET(23b+)K369I was grown for 14 h on a rotary shaker at 37 °C (12, 13). Following 1:100 dilution into LB+ medium, cells were grown at 37 °C to an A600 of 2.0. After induction with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside, the culture was shaken for 3 h at 37 °C. All subsequent steps were performed at 4 °C unless otherwise noted. Cells were collected by centrifugation at 4000 × g, resuspended in 5 ml of lysis buffer (50 mM imidazole, pH 7.9, 50 mM NaCl, 1 mM DTT,1 0.1 mM ADP, 0.1 mM EDTA, 0.5 mM MgCl2, 0.01% Nonidet P-40, 2 µg/ml soybean trypsin inhibitor)/g of cell pellet, and lysed with a French press set at 1000 p.s.i. Following centrifugation at 100,000 g, the supernatant was incubated on a rocker with 2 ml of phosphocellulose resin (Whatman P-11)/10 ml of supernatant. After 30 min, the solution was centrifuged at 3000 × g, and the supernatant carefully removed. This step was repeated two times. The phosphocellulose resin was added to an Econo-Pak column (Bio-Rad, 1.5 × 12 cm) and washed with 10 column bed volumes of lysis buffer. Protein was eluted with lysis buffer containing 500 mM NaCl, dialyzed into lysis buffer, concentrated, and exchanged into 80 mM PIPES, pH 6.9, 2 mM EDTA, 1 mM MgCl2, 1 mM DTT, 1.0 mM ADP using a Centricon-30 (Amicon). Purified protein was snap frozen with liquid N2 in 50-µl aliquots and stored at -80 °C. Before use, each aliquot was thawed on ice and clarified by centrifugation at 100,000 × g. Protein concentration was measured by the Bradford assay (14).

Protein Preparation (Tubulin)

Bovine brain tubulin was purified by cycles of assembly/disassembly followed by phosphocellulose chromatography (15). Purified tubulin was drop frozen into liquid N2 and stored at -80 °C. Before use, tubulin was thawed on ice and clarified by centrifugation at 100,000 × g. Protein concentration was measured by the Bradford assay (14). Microtubules were polymerized by the addition of 7.5% Me2SO, 1 mM GTP, 20 µM Taxol (paclitaxel) and incubation for 15 min at 37 °C (4).

Cross-linking

Kinesin motor (K369I) at 10 µM and microtubules at 10 µM were incubated together in binding buffer (80 mM PIPES, pH 6.9, 2 mM EDTA, 1 mM MgCl2, 50 mM NaCl, 1 mM DTT, 5 mM AMP-PNP, 20 µM paclitaxel) for 30 min at 25 °C. Microtubules and bound K369I were isolated by centrifugation at 50,000 × g. Pellets were resuspended in the original reaction volume with binding buffer. The cross-linking reagent EDC (Pierce) was added to 0.2 mM final concentration, reactions were incubated for 2 h at 25 °C, then terminated by the addition of 5 × sample buffer (25 mM Tris-Cl, pH 6.8, 5% SDS, 5% beta ME, 25% sucrose, 0.05% bromphenol blue). To study the effects of ionic strength, kinesin motors were cross-linked to microtubules under standard conditions with the concentration of NaCl adjusted from 50 to 100, 150, 200, and 250 mM NaCl.

Gel Electrophoresis

Products were diluted 1:5 with 1 × sample buffer (5 mM Tris-Cl, pH 6.8, 1% SDS, 1% beta ME, 5% sucrose, 0.01% bromphenol blue), and separated on 7.5% SDS gels, pH 9.1, or 10% Tris-Tricine gels (16-18). Gels were stained with 0.05% Coomassie Blue R-250 in 40% methanol, 10% acetic acid and destained with 40% methanol, 10% acetic acid.

Western Blot Analysis

Products separated on polyacrylamide gels were electrophoretically transferred to 0.2-µm polyvinylidine difluoride membranes and probed with antibodies specific for kinesin (N18), alpha -tubulin (DM1A), or beta -tubulin (DM1B, 18D6) (19, 20). Bound antibodies were detected using alkaline phosphatase-labeled secondary antibodies and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.

Cyanogen Bromide Digests

Proteins of interest were cut out of polyacrylamide gels with a razor blade. Gel slices were dried under vacuum before the addition of 50 µl/slice of 100 mM CnBr solution in 70% formic acid. The reaction was incubated at 25 °C for 1 h in the dark. Slices were washed with 5 mM Tris-Cl, pH 6.8, 40% methanol, 1% SDS, 1% beta ME, 5% sucrose, 0.01% bromphenol until the dye retained a blue color. Gel slices were then washed with 100% methanol for 15 min and dried under vacuum. Gel slices were rehydrated with 1 × sample buffer (5 mM Tris-Cl, pH 6.8, 1% SDS, 1% beta ME, 5% sucrose, 0.01% bromphenol blue) before loading onto a 10% Tris-Tricine gel (18, 21).

Subtilisin Treatment

Paclitaxel-stabilized microtubules were digested with 1.2% (w/w) subtilisin (Sigma) at 37 °C for 12 h, which has been reported to have no observable effect on microtubule structure as judged by electron microscopy (10, 17, 22). The reaction was terminated by the addition of phenylmethylsulfonyl fluoride to 5 mM. Control, untreated microtubules were incubated in the same manner as microtubules treated with subtilisin. Untreated and subtilisin-treated microtubules were recovered by centrifugation through a 30% sucrose cushion at 50,000 × g and were resuspended in 80 mM PIPES, pH 6.9, 2 mM EDTA, 1 mM DTT, 20 µM paclitaxel.

ATPase Assays

The rate of ATP hydrolysis by the kinesin motor was measured by a molybdate/malachite green colometric assay for Pi release (23). Typically, 10-µl aliquots of a reaction mixture containing 10 µg/ml of the kinesin motor were quenched with 90 µl of ice-cold 0.3 M HClO4, prior to addition of 100 µl of sodium molybdate/malachite green solution in 0.7 M HCl. After 20-30 min at 25 °C, the A650 of the HClO4/malachite green solution was determined using a microtiter plate reader. Rates for ATP hydrolysis were corrected for ATP hydrolysis in the malachite green/HClO4.

Peptide Synthesis

alpha -, beta -, and scrambled peptides were synthesized on a 2-mmol scale using standard solid state methods on a Milligen 9050 peptide synthesizer. Before use, portions of the crude eluted peptides (20 mg) were purified by reverse-phase high performance liquid chromatography using a Waters DeltaPak C18 column and a gradient of 0.1% trichloroacetic acid to 50% acetonitrile to elute peptides. Purified peptides were freeze-dried and stored under vacuum. Before use, peptides were resuspended in 80 mM PIPES, pH 6.9, 2 mM EDTA, 1 mM DTT, 20 µM paclitaxel.

Antibodies

Polyclonal rabbit antibody NK18 was made against a synthetic peptide corresponding to the N-terminal 17 amino acids of Drosophila kinesin heavy chain (MASREREIPAEDSIKVVC) linked through the C-terminal cysteine to bovine serum albumin (Chiron). Polyclonal antibody PAN was generously provided by J. Scholey (University of California, Davis) (24). This antiserum resulted from pooling three separate antibodies raised against the following peptide sequences: LVDLAGSE, SSRSHSVF, and HIPYRNSKLT. Monoclonal antibody His-Tag was purchased from Dianova. Monoclonal antibody 18D6 was generously provided by D. Cleveland (University of California, San Diego) (20). Monoclonal antibodies DM1A and DM1B, which are specific for alpha - and beta -tubulin, respectively, were purchased from Sigma.


RESULTS

Cross-linking Kinesin Motors to Microtubules

To study the interaction between kinesin motors and microtubules, chemical reagents were used to trap these proteins in their bound complexes. To generate these complexes, purified protein consisting of the N-terminal 369 amino acids of Drosophila kinesin heavy chain (K369I) was bound to microtubules using AMP-PNP, a nonhydrolyzable ATP analog that "locks" the motor in the microtubule-bound phase of its mechanochemical cycle (11, 25). After isolating the complexes by centrifugation, the cross-linking reagent EDC was added. To evaluate the optimal concentration of cross-linker, the final concentration of EDC was varied. Increasing the amount of cross-linker from 0.1 to 20 mM increases the prevalence of higher order, oligomeric complexes (Fig. 1). Based on these data, 0.2 mM final concentration of EDC was used in subsequent experiments; this concentration produced a reasonable amount of the 97-kDa product with minimal yield of higher order products. In addition, by using a low concentration of cross-linker, the degree of nonspecific cross-linking was limited.


Fig. 1. Variation of the concentration of EDC in the cross-linking reaction. K369I (10 µM) and microtubules (MT; 10 µM) were cross-linked using EDC at concentrations of 0.1, 0.2, 0.4, 1.0, 3.0, 5.0, 10.0, and 20.0 mM. Products were analyzed by SDS-PAGE; their proposed composition is indicated on the right. K + alpha  indicates K369I cross-linked to the alpha -tubulin subunit; K + beta  indicates K369I cross-linked to the beta -tubulin subunit.
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The effect of increasing the molar ratio of kinesin motors to microtubules on the cross-linking reaction was also evaluated. Cross-linking reactions were carried out with 0.2 mM EDC and 10 µM microtubules, but the concentration of kinesin was varied between 5 and 25 µM. As the molar ratio of kinesin motors to microtubules increased, the yield of the 97-kDa cross-linked product did not obviously change (Fig. 2). Therefore, a "standard" cross-linking reaction was chosen that included 10 µM microtubules, 10 µM kinesin motors, and 0.2 mM EDC in binding buffer.


Fig. 2. Variation of the molar ratio of K to MT in the cross-linking reaction. The final concentration of microtubules was held constant at 10 µM, but the final concentration of K369I was adjusted to 5, 10, 15, 20, or 25 µM, giving molar ratios of K369I to microtubules of 0.5, 1.0, 1.5, 2.0, and 2.5. K + alpha  indicates K369I cross-linked to the alpha -tubulin subunit; K + beta  indicates K369I cross-linked to the beta -tubulin subunit.
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To analyze the composition of the 97-kDa complex, products of a standard cross-linking reaction were separated on a 7.5% SDS-polyacrylamide gel adjusted from pH 8.8 to pH 9.1 to obtain optimal resolution of alpha - and beta -tubulin subunits and tubulin complexes (26). Under these gel conditions, alpha -tubulin has a relative mobility of approx 55 kDa, beta -tubulin has a relative mobility of approx 53 kDa and the 97-kDa complex resolves into a doublet (Fig. 3A). Probing Western blots with subunit-specific antibodies revealed that the upper band of the doublet contains alpha -tubulin, and the lower band contains beta -tubulin (Fig. 3B); both bands react with kinesin-specific antibodies (Fig. 3C). These results support the hypothesis that kinesin motors can be cross-linked to alpha - as well as beta -tubulin subunits.


Fig. 3. Analysis of the 97-kDa products of the cross-linking reaction. K was cross-linked to MT using 0.2 mM EDC. Products were analyzed by SDS-PAGE; however, the pH of the resolving gel was adjusted from 8.8 to 9.1 to obtain optimal resolution of alpha - and beta -tubulin subunits as well as tubulin complexes (26). In control K + K reactions, K369I was cross-linked to K369I, and in control MT + MT reactions, microtubules were cross-linked to microtubules. K + MT indicates reactions in which K369I was cross-linked to microtubules. Products are indicated by asterisks along with their proposed composition; the symbols alpha  and beta  represent alpha - and beta -tubulin subunits, and K represents the K369I. Panel A shows the 97-kDa product resolved into a doublet. Based on their relative mobilities, the components likely resulted from cross-linking of K369I (42 kDa) to either the alpha -subunit (55 kDa) or the beta -subunit (53 kDa). Complexes with relative mobilities slower than 97 kDa may have resulted from cross-linking the alpha - to the beta -subunit (55 kDa + 53 kDa = 108 kDa), the alpha - to the alpha -subunit (55 kDa + 55 kDa = 110 kDa), the beta - to the beta - subunit (53 kDa + 53 kDa = 106 kDa) or from cross-linking K369I to the alpha - and the beta -subunit (42 kDa + 55 kDa + 53 kDa = 150 kDa). Panel B shows the composition of the 97-kDa doublet as determined by antibody analysis. Products were analyzed by SDS-PAGE, pH 9.1, and transferred to nylon membrane. The left half of the membrane was probed with DM1A, an antibody specific for alpha -tubulin, and the right half was probed with DM1B, an antibody specific for beta -tubulin (19). Based on the reactivity of the 97-kDa doublet, the top band is composed of K369I cross-linked to the alpha -subunit (K + alpha ), and the bottom band is composed of K369I cross-linked to the beta -subunit (K + beta ). The complex with the slowest relative mobility may result from cross-linking the alpha - to the beta -subunit (alpha  + beta ), the alpha - to the alpha - subunit (alpha  + alpha ), or the beta - to the beta -subunit (beta  + beta ). Panel C shows that both the upper and lower bands of the 97-kDa doublet react with NK18, a kinesin-specific antibody, lending further evidence that K369I can be cross-linked to both alpha - and beta -tubulin subunits.
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To test whether the cross-linking of K369I to alpha - and beta -tubulin reflects a comparable affinity of interaction, the ionic strength dependence of the reaction was assessed (Fig. 4). The data indicate that as ionic strength increases, the overall efficiency of the cross-linking reaction decreases, which suggests, as reported previously, that the binding reaction between kinesin motors and microtubules contains a significant ionic component (27). However, the relative cross-linking of kinesin motors to alpha - versus beta -tubulin was ionic strength-independent. Comparison of the relative yields of K + alpha  versus K + beta  complexes (where K is kinesin motor) revealed that the ratio of K + alpha  complexes to K + beta  complexes was consistent within a given set of reactions up to 250 mM NaCl. This result argues against the interpretation that cross-links to alpha -tubulin are nonspecific or represent secondary sites of interaction.


Fig. 4. The effect of increasing ionic strength on the cross-linking reaction. K was cross-linked to MT using EDC, and the final concentration of NaCl present in the reaction was adjusted from 50 to 100, 150, 200, or 250 mM. Labeling is as in Fig. 3.
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Mapping Regions of the Kinesin Motor That Interact with the Microtubule

To determine which regions of the kinesin motor make contact with the microtubule, points of cross-linking were localized within the 97-kDa product. Gel slices containing the 97-kDa product (a mixture of K + alpha  and K + beta ) were partially digested with CnBr, a chemical that cleaves proteins specifically at methionine residues (21, 28). Under these conditions, a nested set of kinesin and tubulin fragments resulted. These fragments were run on a second gel, blotted, and probed with antibodies specific for the N- or C-terminal CnBr fragments of the kinesin motor. In the hypothetical case of a kinesin motor cross-linked through its C-terminal fragment to the microtubule, when fragments are probed with an antibody specific for the N terminus of the kinesin motor, only full-length protein should shift out of register as compared with control, uncross-linked kinesin motors (Fig. 5A). If, on the other hand, the motor is probed with a C-terminal kinesin motor antibody, all products should shift out of register (Fig. 5B). When these experiments were carried out, virtually 100% of the full-length protein shifted to a slower relative mobility as compared with control, uncross-linked motor fragments when mapped from the N terminus (Fig. 6A). In addition, there are partial shifts of intermediate-sized fragments as well. Mapping with the C-terminal specific antibody generated comparable data; full-length product shifted completely, while lower fragments shifted partially (Fig. 6B). The simplest interpretation of these data is that cross-links to the microtubule occur within both the N- and C-terminal CnBr fragments of the kinesin motor. Attempts to map the K + alpha  and K + beta  species separately were unsuccessful owing to low yields. Thus, the issue of whether alpha - and beta -tubulin bind to different regions of the kinesin motor could not be resolved.


Fig. 5. Expectations for N- and C-terminal maps of the kinesin motor. If the kinesin motor is digested with CnBr, five fragments are expected. For the purpose of illustration, the motor is assumed to be cross-linked through its C-terminal CnBr fragment to the microtubule (C-terminal cross-link). Panel A diagrams the expected results of an N-terminal map of the kinesin motor cross-linked through its C-terminal fragment to the microtubule (X). In the left control reactions, the motor was not cross-linked to the microtubule before cleavage. On the right, the motor was cross-linked to the microtubule before cleavage. If fragments are separated by SDS-PAGE, blotted, and probed with a kinesin-specific antibody whose site of recognition (*) lies near the N terminus, then only full-length kinesin motor protein should shift to a slower relative mobility as compared with control, uncross-linked motor fragments. Cross-linked products are represented by a large box because they are unlikely to migrate as a well defined band due to partial cleavage of attached tubulin fragments, shown in gray. Panel B diagrams a C-terminal map (using an antibody whose recognition site is at the C terminus (*)) of the kinesin motor cross-linked through its C-terminal CnBr fragment to the kinesin motor. If cross-linking to the microtubule occurs exclusively within the C-terminal fragment, all fragments should shift to a slower relative mobility as compared with control, uncross-linked motor fragments on the left.
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Fig. 6. Antibody mapping of sites on the kinesin motor that interact with the microtubule. Panel A shows an N-terminal map of K369I cross-linked to microtubules and probed with NK18, a kinesin-specific antibody whose site of recognition lies near the N terminus. In the K + MT lane, full-length K369I shifted completely to a slower relative mobility, marked by an arrow, when compared with the K lane. Other fragments partially shifted to slower mobilities, marked by asterisks. Panel B shows a C-terminal map of K369I cross-linked to microtubules. Fragments were probed with PAN, an antibody whose site of recognition lies within the C-terminal CnBr fragment of K369I (24). Comparable to the N-terminal map, in the K + MT lane, full-length protein shifted completely to a slower relative mobility, marked by an arrow, when compared with the K lane. Other fragments partially shifted to slower mobilities, indicated by a bracket.
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Mapping the Sites on the Microtubule That Interact with the Kinesin Motor

To evaluate the regions of alpha - and beta -tubulin that interact with the kinesin motor, partial CnBr digests of the 97-kDa cross-linked product were run on gels, blotted, and probed with tubulin-specific antibodies. If beta -tubulin were linked through its C-terminal CnBr fragment to the kinesin motor, then upon probing with an antibody specific for the N terminus of beta -tubulin, one would have expected that only the largest fragment would shift out of register as compared with control, uncross-linked tubulin. This result was observed, suggesting that contact with the kinesin motor occurs within the most C-terminal CnBr fragment of the beta -subunit (Fig. 7A). To test this result further, maps of the 97-kDa cross-linked product were generated using the antibody specific for the C terminus of beta -tubulin. If cross-linking occurs within the most C-terminal CnBr fragment of beta -tubulin, when fragments are probed with a beta -tubulin-specific antibody whose site of recognition lies near the C terminus, all fragments should shift to slower relative mobilities as compared with control, uncross-linked tubulin fragments. When this experiment was carried out, all fragments appeared to shift, which is consistent with the hypothesis that at least one region of contact between microtubules and the kinesin motor resides within the most C-terminal, CnBr fragment of beta -tubulin (Fig. 7B).


Fig. 7. Antibody mapping of sites on the microtubule that interact with the kinesin motor. Panel A shows an N-terminal map of beta -tubulin cross-linked to K369I. Control microtubules (MT) and microtubules cross-linked to K369I motor (MT + K) were partially digested, seperated, and probed with 18D6, a beta -tubulin-specific antibody whose site of recognition lies near the N terminus (20). In the MT + K lane, only the largest, full-length tubulin fragment shifted to a slower relative mobility as compared with the MT lane. Panel B shows a C-terminal map of beta -tubulin cross-linked to K369I. Control uncross-linked microtubules (MT) and microtubules cross-linked to K369I (MT + K) were partially digested, separated, and probed with DM1B, a beta -tubulin-specific antibody whose site of recognition lies near the C terminus (19). In the MT + K lane, all fragments appeared to shift to slower relative mobilities as compared with the MT lane. Panel C shows a C-terminal map of alpha -tubulin cross-linked to the kinesin motor. Control microtubules and microtubules cross-linked to the kinesin motor were partially digested, separated, and probed with an alpha -tubulin-specific antibody, DM1A, whose site of recognition lies near the C terminus (19). In the MT + K lane, all fragments appeared to shift to slower relative mobilities as compared with the MT lane.
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Contacts between alpha -tubulin and the kinesin motor were analyzed in a similar manner except that an N-terminal alpha -tubulin map was not generated because an appropriate N-terminal alpha -tubulin antibody was not available. If cross-linking to the kinesin motor occurs through the most C-terminal CnBr fragment of alpha -tubulin, then when CnBr fragments from the 97-kDa product are probed with an alpha -tubulin-specific antibody whose site of recognition lies near the C terminus, all fragments should shift to slower relative mobilities as compared with control, uncross-linked tubulin fragments. When this experiment was carried out, all fragments appeared to shift suggesting that at least one region of contact between microtubules and the kinesin motor resides within the most C-terminal CnBr fragment of alpha -tubulin (Fig. 7C).

Further Evidence That the N and C Termini of alpha - and beta -Tubulin Interact with the Kinesin Motor

To confirm whether the C termini of alpha - and beta -tubulin interact with the kinesin motor, paclitaxel-stabilized microtubules were treated with the protease subtilisin under conditions that are reported to remove C-terminal fragments of alpha - and beta -tubulin subunits without obviously affecting microtubule structure (10, 17, 22). These microtubules were then tested for their ability to be cross-linked to the kinesin motor using EDC in the presence of AMP-PNP (25). Cleavage of microtubules with subtilisin changes the relative mobility of microtubules by approx 4 kDa (Fig. 8A); the reaction appears to be complete as there is no obvious uncleaved product (U-MT) remaining in the subtilisin-treated (S-MT) material. In reactions in which S-MT were cross-linked to kinesin motors, very little cross-linked product was detected by Coomassie Blue staining, whereas in those reactions in which U-MT were cross-linked to kinesin motors, cross-linked product is readily observed. Probing a Western blot of these reactions with an anti-kinesin antibody reveals that a comparatively small amount of cross-linked product is present in the reaction with S-MT versus the reaction with U-MT. The observation of a small amount of a S-MT + K product (approx 94 kDa) suggests that treatment of microtubules with subtilisin may not completely remove the kinesin binding site (Fig. 8B). Therefore, additional separate sites of interaction not removed by subtilisin may exist. Nonetheless, these results suggest that treating microtubules with subtilisin markedly reduces their ability to be cross-linked to kinesin motors.


Fig. 8. Cross-linking of subtilisin-treated microtubule to the kinesin motor. S-MT as well as control untreated MT were cross-linked to kinesin motors under standard conditions. K + K indicates control reactions in which K369I was cross-linked to K369I; U-MT + U-MT and S-MT + S-MT indicate control reactions in which the specified microtubules were cross-linked together; U-MT + K indicates reactions in which untreated microtubules were cross-linked to K369I; S-MT + K indicates reactions in which subtilisin-treated microtubules were cross-linked to K369I. Panel A shows that S-MT were less efficiently cross-linked to kinesin motors as determined by SDS-PAGE analysis. Panel B shows that S-MT were less efficiently cross-linked to kinesin motors as determined by Western blot analysis. Products of the cross-linking reactions were separated by SDS-PAGE, blotted, and probed with antibodies directed against the kinesin motor (NK18). The amount of 97-kDa cross-linked product, indicated by arrows, was markedly reduced in the S-MT + K reaction compared with the U-MT + K reaction.
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The functional significance of diminishing kinesin-MT interaction by subtilisin treatment was assessed by examining MT-stimulated ATPase activity. At 50 mM salt, the Km for microtubule-stimulated ATPase activation of K369I was measured to be 1.92 µM, and the Vmax was measured to be 4.4 s-1, both of which are well within published values for comparable constructs (3, 27, 29). When subtilisin-treated microtubules were added to the kinesin motor, the Vmax remained essentially unchanged; however, the Km increased from 1.92 µM to 9.03 µM (Fig. 9). These data also suggest that the interaction of subtilisin-treated microtubules with the kinesin motor is impaired.


Fig. 9. Microtubule activation of the kinesin ATPase with subtilisin-treated microtubules. Panel A shows by Western blot analysis that microtubules were modified by subtilisin. The left half of the blot was probed with DM1A, a monoclonal antibody specific for alpha -tubulin, and the right half was probed with DM1B, a monoclonal antibody specific for beta -tubulin (19). Judging from their faster mobilities, the alpha - and beta -subunits of S-MT both appeared to be fully cleaved by subtilisin. Panel B shows the effect of subtilisin treatment on Km and Vmax for ATPase activation of the kinesin motor. Reciprocal ATPase activity was plotted against reciprocal microtubule concentration for S-MT and control U-MT.
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Since treatment of microtubules with subtilisin affects both alpha - and beta -tubulin, it was important to know whether the modification of alpha -, beta -, or both tubulin subunits resulted in the observed decrease in microtubule-stimulated ATPase activity. To address this question, peptides alpha  (SVEGEGEEEGEE) and beta  (GEFEEEGEEDEA), which correspond to the portions of tubulin removed by subtilisin (30-32), were synthesized and tested for their ability to inhibit microtubule-stimulated ATPase activity. Addition of 1 or 5 mM alpha -peptide had little effect on the Km for ATPase activation of the kinesin motor (Fig. 10A). In contrast, the addition of 1 or 5 mM beta -peptide increased the Km from 1.89 µM to 3.16 µM or 4.15 µM, respectively (Fig. 10B). The addition of a control scrambled peptide (EGEVEESEGEGE) had little effect on the Km (Fig. 10C). In all cases, Vmax remained unchanged. The increase in Km upon the addition of the beta -peptide, but not the alpha - or the scrambled peptide, suggests that the beta -peptide competitively interferes with the binding of microtubules to the kinesin motor.


Fig. 10. Competition of synthetic peptides with microtubules for activation of the kinesin ATPase. The ability of synthetic peptides to compete with microtubules for binding to K369I was assayed. Reported values result from averaging the data from three separate trials. In the control, no peptide reactions, reciprocal microtubule-stimulated ATPase activity of K369I was plotted against reciprocal concentrations of microtubules. Panel A shows the addition of the alpha -peptide, panel B shows the addition of 1 or 5 mM beta -peptide to the reaction, and panel C shows the addition of 1 or 5 mM scrambled peptide.
[View Larger Version of this Image (20K GIF file)]



DISCUSSION

The Kinesin Motor Can Be Cross-linked to Both alpha - and beta -Tubulin Subunits

The data presented here demonstrate that the kinesin motor domain can be reproducibly cross-linked to both alpha - and beta -tubulin subunits of microtubules. These results are in agreement with a recently published study (7), but are in conflict with an earlier report (6). Both previous studies carried out cross-linking reactions with a 10-fold higher concentration of EDC (2.0 mM) than the concentration used in these studies (0.2 mM). If 1 or 3 mM EDC is used in the cross-linking reactions, the resolution between K + alpha  and K + beta  complexes is lower than if 0.2 mM EDC is used; therefore, distinguishing between K + alpha  and K + beta  complexes might be more difficult as the concentration of EDC increases (Fig. 1). An alternative explanation for these discrepancies could be that one study (6) used squid kinesin, while the other study (7), and the present study, used Drosophila kinesin. It is possible that squid kinesin may bind alpha -tubulin, but may not present the EDC-reactive groups in the necessary orientation or proximity to be cross-linked to alpha -tubulin (7). Nevertheless, the comparable ionic strength dependence, and the consistency of cross-linking to alpha - and beta -tubulin observed here, suggests that the cross-links to both subunits are specific, and therefore that both are likely to reflect relevant biological interactions.

The view that kinesin motors make multiple contacts with the microtubule is consistent with other studies of the interactions of kinesin and microtubules. For example, the motor is highly processive and releases the microtubule for only a small fraction of its 50-ms cycle time (25). One way to maintain such a strong hold on the microtubule might be through the use of multiple contacts with alpha - and beta -tubulin monomers as the motor moves over the microtubule lattice. Data indicating an 8-nm step in combination with the data suggesting that kinesin could only be cross-linked to beta -tubulin led to a model in which the kinesin motor steps from beta -subunit to beta -subunit (6, 33). Speculatively, a more complex model may be invoked to explain contacts with both alpha - and beta -tubulin. One possibility is that kinesin takes steps smaller than 8 nm. Indeed, recently improved methodology suggests that the motor might take 5- and 3-nm steps as well as 8-nm steps (34).

The N and C Termini of K369I Interact with the Microtubule

The initial mapping data suggest that both the N- and C-terminal CnBr fragments of K369I interact with the microtubule, which supports models in which the kinesin motor has more than one region capable of interacting with the microtubule, or alternatively, possesses a single, discontinuous microtubule binding site. Analysis of the atomic structure of the kinesin and NCD motors (8, 44) led to the suggestion that two regions, one corresponding to residues 145-180 and a second corresponding to residues 278-287 of K369I, might interact with the microtubule. The latter region is contained within the cross-linked C-terminal CnBr fragment, but the former region is not included within the cross-linked N-terminal CnBr fragment. Although further work will be required to resolve the inconsistencies, both lines of investigation agree that the region defined by the C-terminal CnBr fragment of K369I interacts with the microtubule.

The C Termini of alpha - and beta -Tubulin Interact with the Kinesin Motor

The data presented here suggest that the C termini of alpha - and beta -tubulins may both interact with the kinesin motor domain. Some reports indicated that these regions may also interact with microtubule-associated proteins including kinesin, tau, dynein, and MAP2 (10, 17, 35-37), although other reports contradicted these findings (22, 38, 39). The work reported here is the first attempt to map directly the sites of interaction between microtubules and the kinesin motor; these results point to the C termini of alpha - and/or beta -tubulin as sites where binding may occur. Furthermore, treatment of paclitaxel-stabilized microtubules with the protease subtilisin, which is reported to remove C-terminal fragments from the alpha - and beta -tubulin subunits (30), increased the Km for ATPase activation of the kinesin motor while the Vmax remained unchanged. One interpretation is that subtilisin removes one of the kinesin binding sites on alpha - or beta -tubulin, but that additional sites exist. Perhaps the site removed by subtilisin is a relatively low affinity site whose absence can be overcome by the addition of a greater concentration of microtubules.

Consistent with the idea that the C termini of tubulins interact with K369I is the finding that addition of a C-terminal peptide corresponding to the last 12 amino acids of pig beta -tubulin can increase the Km for microtubule-stimulated ATPase activity of kinesin from 1.89 µM up to 4.15 µM. These data suggest that the beta -peptide competitively interferes with the binding of microtubules to the kinesin motor and may include some portion of a kinesin binding site. The lack of interference by the alpha -peptide is not clear given the cross-linking results, but perhaps the alpha -peptide does not assume a correct conformation. Future work should include the analysis of longer C-terminal tubulin peptides, since it is possible that the peptides used in the current experiments may be too short to encompass fully a C-terminal site of interaction with the kinesin motor. In fact, earlier reports indicate that the sites of subtilisin cleavage are about 20-25 amino acids more N-terminal than the ones chosen for the peptide design (40, 41).

A functional role for the C termini of tubulins has also been suggested previously based on sequence alignments of these regions (17). Although the C termini of tubulins display high phylogenetic variability, the sequences EGEE and EEGEE are well conserved (42, 43). This conservation of sequence and negative charge is consistent with the idea that the C termini of alpha - and beta -tubulin bind to the kinesin motor. The finding that negatively charged amino acids on tubulin make contact with kinesin motors, suggests that positively charged residues on the motor will be important for microtubule binding. Indeed, many conserved positively charged residues are found in the cross-linked N- and C-terminal CnBr fragments.

A Model for the Kinesin-Microtubule Interaction

Combining data contained in this report with what is known about tubulin and kinesin structure, a simple model can be put forth to describe the kinesin-microtubule interaction. Cross-linking data suggest that the C-terminal domain of the tubulin beta -subunit interacts with the N-terminal domain of the tubulin alpha -subunit (45). In addition, a 6-Å resolution structure of tubulin suggests that the monomers are similarly oriented within the filament (46). Therefore, the distance between the C termini of alpha - and beta -tubulin could be as much as 4 nm depending on the flexibility and mobility of the C termini relative to the bulk of the subunits. Accordingly, the motor domain could make contact with the C termini of both alpha - and beta -tubulin, especially if these domains are somewhat flexible.

Given the finding that the highly charged C terminus of beta -tubulin is likely to interact with the kinesin motor, it is tempting to speculate that kinesin binds to its polymeric substrate and generates force using a similar mechanism as myosin. The first step of the myosin-actin interaction is thought to be mediated by ionic interactions that generate weak binding between the two proteins. Subsequent step(s) are thought to be mostly hydrophobic in nature and are proposed to strengthen overall binding before the power stroke occurs (47). One possibility is that the ionic interactions characterized in this report are the counterpart to the initial ionic interactions reported for the myosin-actin system. Other sites of interaction may be important for the final, stereospecific positioning of the motor on the microtubule before the power stroke occurs. It is likely that future studies of how the kinesin motor binds and releases microtubules will address these questions and expand the understanding of how these molecular machines operate.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM35252.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0683. Tel.: 619-534-9700; Fax: 619-534-9701; E-mail: lgoldstein @ucsd.edu.
1   The abbreviations used are: DTT, dithiothreitol; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); AMP-PNP, 5'-adenylylimidodiphosphate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; beta ME, beta -mercaptoethanol; Tricine, N-tris(hydroxymethyl)methylglycine; PAGE, polyacrylamide gel electrophoresis; U-MT, uncleaved microtubule; S-MT, subtilisin-treated microtubule; MT, microtubule; K, K369I.

ACKNOWLEDGEMENTS

We are grateful to D. Woerpel for building the pETK369I construct, S. Farlow for the protocol for purification of the kinesin motor domain, Dr. N. Barton for helpful advice and insight, Dr. D. Cleveland for monoclonal antibody 18D6, Dr. E. Komives for reagents and helpful advice about peptide synthesis, and Dr. J. Scholey for the PAN antibody.


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