Kinesin and Tau Bind to Distinct Sites on Microtubules
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Journal of Cell Science 107, 339-344 (1994) 339 Printed in Great Britain © The Company of Biologists Limited 1994 JCS8430 Kinesin and tau bind to distinct sites on microtubules Pankaj K. Marya, Zarrin Syed, Paul E. Fraylich and Peter A. M. Eagles* Department of Molecular Biology and Biophysics, The Randall Institute, King’s College, University of London, 26-29 Drury Lane, London WC2B 5RL, UK *Author for correspondence SUMMARY We have used a fluorescent derivative of kinesin, AF- β-tubulin subunits. These treated microtubules can no kinesin (kinesin conjugated with 5-(iodoacetamido)fluores- longer bind tau, but are able to bind AF-kinesin in the cein), to investigate the binding site of kinesin on micro- presence of AdoPP[NH]P. Finally, AF-kinesin will support tubules and to compare this site with that to which tau the gliding of subtilisin-digested microtubules in the binds. Microtubules saturated with tau will bind AF- presence of ATP at rates comparable to those obtained with kinesin in the presence of the ATP analogue, 5′-[β,γ- non-digested microtubules. These results show directly that imino]triphosphate (AdoPP[NH]P). This shows that there the binding site for kinesin is outside the C-terminal region are distinct binding sites for the two proteins. Further of tubulin that is removed by subtilisin and is distinct from evidence comes from digestion studies where taxol-sta- the binding site of tau. bilised microtubules were treated with subtilisin, resulting in the cleavage of C-terminal residues from both the α- and Key words: kinesin, tau, microtubule INTRODUCTION al. (1990) showed that microtubules that have been digested with subtilisin, so that the MAP2 binding site was lost, were Microtubules have on their surface a number of binding sites still capable of binding kinesin. Using the approach of in vitro for associated proteins, and much work is currently aimed at motility assays (von Massow et al., 1989; Heins et al., 1991), identifying domains that might bind microtubule motors like it was found that the binding of MAP2 to microtubules does kinesin or dynein, and domains that might bind other micro- not prevent microtubule gliding due to kinesin. Thus from a tubule-associated proteins (MAPs) like MAP2 and tau. It is variety of evidence, the two sites for binding are distinct. important to elucidate these issues in order to find out, for Regarding the binding site for tau, the cloning of the genes example, whether those microtubules that do not act as tracks for tau has enabled the microtubule binding domain of the for vesicle transport in neurones (Miller et al., 1987) do so protein to be determined (Himmler et al., 1989; Butner and because their binding sites for a motor are sterically blocked Kirschner, 1991). The domain, in the basic C-terminal part of by other molecules. the molecule, consists of four repeat regions together with a With MAP2, it has been well demonstrated that the C proline-rich sequence (Kanai et al., 1992; Aizawa et al., 1991). termini of α- and β-tubulin are required for its binding to There is considerable similarity in the repeat regions with those microtubules (Serrano et al., 1984; Littauer et al., 1986), and found in MAP2, which has lead to the proposal that on micro- there is evidence that cytoplasmic dynein also utilizes these tubules MAP2 and tau probably occupy a similar site. The regions for interaction (Paschal et al., 1989). For example, sub- corollary of this is that the tau site is distinct from the kinesin tilisin-digested microtubules, which lack the C-terminal binding site; however, the idea has not been proved by direct peptides of tubulin (Serrano et al., 1984; Littauer et al., 1986; experiment. This would be important to test because there are Maccioni et al., 1986) failed to stimulate the ATPase activity good grounds for believing that the line of argument that of cytoplasmic dynein (Paschal et al., 1989). However, the assumes that what is true for the binding of MAP2 is also true issue is complex because other workers, who also used subtil- for the binding of tau may not be entirely valid. isin-digested tubules, showed that such tubules were in fact Firstly, although MAP2 and tau share similarity in their capable of interacting with dynein (Rodionov et al., 1990). repeat regions they have low homology in the sequence of the The region on α- and β-tubulin to which MAP2 binds is proline-rich region, which is also important for microtubule probably different from the site on β-tubulin to which kinesin binding (Kanai et al., 1992; Aizawa et al., 1991), thus tau and binds (Song and Mandelkow, 1993). This follows from a MAP2 may differ in their binding details. Secondly, the pro- variety of studies. Those of Cowan and colleagues (Lewis et jection domains on tau and MAP2 differ considerably not only al., 1988, 1989; Noble et al., 1989) indicate that the micro- in sequence but also in length (Lewis et al., 1988; Lee et al., tubule binding domain of MAP2 shares no obvious sequence 1988). The steric influence from these regions on the binding homology with kinesin (Yang et al., 1989), and Rodionov et of other molecules in the vicinity has not been clearly 340 P. K. Marya and others addressed. So again we see that conclusions concerning the Preparation of microtubules with saturating amounts of attachment of kinesin and MAP2 to microtubules may not be tau valid if transposed to the situation regarding the attachment of HPLC-purified tau (300 µg/ml) was incubated (20 minutes, 25°C) kinesin and tau. with microtubules (up to 60 µg/ml) in Pipes buffer supplemented with In view of this, we have examined, by direct experiment, the 10 µM taxol. The mixture was centrifuged on an airfuge (0.17 MPa, issue of whether there is overlap between the binding sites for 30 minutes) and the pellet, containing microtubule-bound tau, was kinesin and tau by using a novel fluorescent derivative of removed and used for analysis. kinesin, AF-kinesin (Marya et al., 1990a,b, 1991; Löffler et al., Binding assay 1992). In summary, we show by experiment that the binding A mixture of HPLC-purified tau and taxol-stabilized microtubules of tau to microtubules does not interfere with their interaction was made up and incubated as above. A 10 µl sample was allowed involving kinesin. to adsorb to a polylysine-coated, number 1, glass coverslip for 10 minutes. A 20 µl sample of 1% BSA was added, left for 30 minutes and then removed. A 10 µl sample of AF-kinesin (10 µg/ml) con- taining 0.1% BSA and 2.5 mM AdoPP[NH]P was added. The kinesin MATERIALS AND METHODS was left to bind for 1 hour, after which unbound protein was washed away with two additions of Pipes buffer plus taxol (10 µM), and the Materials coverslip was fixed in cold methanol. The coverslip was then blocked DEAE-cellulose 52 was from Whatman. Coverslips were purchased with 1% BSA and stained for the presence of tau using an anti-tau 1 from Clay Adams (Gold Seal). Taxol was a gift from Dr M. Suffness monoclonal antibody (1:500) and an anti-mouse (donkey) Texas (National Cancer Institute). Horseradish peroxidase-conjugated and Red-linked secondary antibody (1:50). Several washes with PBS Texas Red-linked secondary antibodies were purchased from were carried out subsequent to each antibody incubation. After the Amersham. All other chemicals were from Sigma. Kinesin was final wash, 20 µl PBS with anti-photobleaching reagent (ascorbic isolated from ox spinal nerve roots, conjugated with 5-(iodoac- acid, pH 7.0, 100 mg/ml) was added to the sample, which was then etamido)fluorescein and purified by high-performance liquid chro- covered with a number 0 glass coverslip and observed under matography (Marya et al., 1990a). immunofluorescence microscopy. The procedure was repeated using subtilisin-digested microtubules in place of the non-digested micro- Preparation of tubulin tubules. Tubulin, prepared by recycling (Murphy and Heibsch, 1979), was purified by DEAE-cellulose column chromatography, and polymer- Electrophoresis ized in Pipes buffer (25 mM Pipes, 0.5 mM EGTA, 0.5 mM EDTA, SDS/PAGE was performed by the method of Laemmli (1970) with 5 mM magnesium chloride, 1 mM dithiothreitol, pH 7.0) by the 10% acrylamide gels. The gels were stained with PAGE blue 83 addition of taxol to 10 µM and GTP to 1 mM. Microtubules (0.3 (BDH). Proteins were immunoblotted onto nitrocellulose, which was mg/ml) prepared in this way were stored frozen in liquid N2, and were subsequently blocked in 1% BSA and then reacted with DM1A, then thawed as required. DM1B YL 1/2, or YOL 1/34. Secondary antibodies were conjugated with horseradish peroxidase (Amersham). Microtubule gliding assay This was based on the assay developed by Vale et al. (1985) and Fluorescence microscopy performed as in the method of Marya et al. (1990a). Samples were viewed using a Zeiss Axiomat microscope equipped with epifluorescence optics. Illumination was from an HBO 100 W Preparation of subtilisin-digested microtubules mercury arc via a heat filter. Sets of standard Zeiss fluorescence filters Taxol-stabilized microtubules (200 µg/ml) in Pipes buffer (supple- (BP 450-490, LP 515-565; BP 546, LP 590) were used. A ×50 mented with 10 µM taxol), generated from DEAE-cellulose 52 objective (numerical aperture, 1.0) together with a ×4 intermediate purified tubulin, were digested with subtilisin (60%, w/w) at 37°C for lens were employed for magnification. Areas were viewed under illu- 60 minutes. Proteolysis was halted by the addition of phenylmethyl- mination appropriate for Texas Red or fluorescein fluorophores, and sulfonyl fluoride (10 mM), after which, digested microtubules showed images were photographed using 35 mm Kodak 3200 ASA film at 30 no changes in their appearance, when viewed by video microscopy second exposure times.