Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. Selective binding of kinesin to detyrosinated tubulin and vimentin.
We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995) J. Cell Biol. 131, 1275-1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an approximately 2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of approximately 120 kDa and a light chain of approximately 64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.
Liao
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Department of Anatomy and Cell Biology, Columbia University, New York, New York 10032, USA.
Gundersen
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GG
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D002417:Cattle; D005973:Glutamine; D006367:HeLa Cells; D006801:Humans; D007382:Intermediate Filaments / Q000502:physiology*; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000502:physiology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014404:Tubulin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine; D014746:Vimentin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D011994:Recombinant Proteins; D014404:Tubulin; D014746:Vimentin; D005973:Glutamine; D014443:Tyrosine; D016547:Kinesins