Interaction of Kinesin Motor Domains with α- and β-Tubulin Subunits at a Tau-independent Binding Site

doi:10.1074/jbc.271.36.22117

Interaction of Kinesin Motor Domains with α- and β-Tubulin Subunits at a Tau-independent Binding Site

REGULATION BY POLYGLUTAMYLATION*

From the Biochimie Cellulaire, CNRS UPR 9065 and the Université P. & M. Curie, Collège de France, 11 place Marcelin Berthelot, 75005 Paris, France

^‡ To whom correspondence should be addressed. Tel.: 33-1-44-27-13-03; Fax: 33-1-44-27-13-09.

Abstract

Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both α- and β-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.

Footnotes

↵* This work was supported by the Centre National de la Recherche Scientifique. 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.
↵¹ The abbreviations used are:

MT

microtubule

PAGE

polyacrylamide gel electrophoresis

MAP

microtubule-associated protein

MES

2-(N-morpholino)ethanesulfonic acid

RK

rat kinesin

ncd

nonclaret disjunctional

PIPES

1,4-piperazinediethanesulfonic acid

MEM

MES-EGTA-MgCl₂ buffer

OV

overlay buffer

HPLC

high pressure liquid chromatography.
- Received March 5, 1996.
- Revision received May 17, 1996.