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Critical role for the EB1 and APC interaction in the regulation of microtubule polymerization

2001; Elsevier BV; Volume: 11; Issue: 13 Linguagem: Inglês

10.1016/s0960-9822(01)00297-4

1879-0445

Masafumi Nakamura, Xiao Zhen Zhou, Kun Ping Lu,

Genetic factors in colorectal cancer

Human EB1 was originally cloned as a protein that interacts with the COOH terminus of adenomatous polyposis coli (APC) [1Su L.K. Burrell M. Hill D.E. Gyuris J. Brent R. Wiltshire R. et al.APC binds to the novel protein EB1.Cancer Res. 1995; 55: 2972-2977PubMed Google Scholar]. Interestingly, this interaction is often disrupted in colon cancer, due to mutations in APC. EB1 also interacts with the plus-ends of microtubules and targets APC to microtubule tips [2Morrison E.E. Wardleworth B.N. Askham J.M. Markham A.F. Meredith D.M. EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.Oncogene. 1998; 17: 3471-3477Crossref PubMed Scopus (194) Google Scholar, 3Berrueta L. Kraeft S.K. Tirnauer J.S. Schuyler S.C. Chen L.B. Hill D.E. et al.The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules.Proc Natl Acad Sci USA. 1998; 95: 10596-10601Crossref PubMed Scopus (154) Google Scholar, 4Mimori-Kiyosue Y. Shiina N. Tsukita S. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.Curr Biol. 2000; 10: 865-868Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 5McCartney B.M. Peifer M. Teaching tumour suppressors new tricks.Nat Cell Biol. 2000; 2: E58-E60Crossref PubMed Scopus (20) Google Scholar, 6Tirnauer J.S. O'Toole E. Berrueta L. Bierer B.E. Pellman D. Yeast Bim1p promotes the G1-specific dynamics of microtubules.J Cell Biol. 1999; 145: 993-1007Crossref PubMed Scopus (207) Google Scholar]. Since APC is detected on the kinetochores of chromosomes, it has been hypothesized that the EB1-APC interaction connects microtubule spindles to the kinetochores and regulates microtubule stability [7Fodde R. Kuipers J. Rosenberg C. Smits R. Kielman M. Gaspar C. et al.Mutations in the APC tumour suppressor gene cause chromosomal instability.Nat Cell Biol. 2001; 3: 433-438Crossref PubMed Scopus (591) Google Scholar, 8Pellman D. Cancer. A CINtillating new job for the APC tumor suppressor.Science. 2001; 291: 2555-2556Crossref PubMed Scopus (20) Google Scholar, 9Kaplan K.B. Burds A.A. Swedlow J.R. Bekir S.S. Sorger P.K. Nathke I.S. A role for the Adenomatous Polyposis Coli protein in chromosome segregation.Nat Cell Biol. 2001; 3: 429-432Crossref PubMed Scopus (471) Google Scholar]. In yeast, EB1 regulates microtubule dynamics [6Tirnauer J.S. O'Toole E. Berrueta L. Bierer B.E. Pellman D. Yeast Bim1p promotes the G1-specific dynamics of microtubules.J Cell Biol. 1999; 145: 993-1007Crossref PubMed Scopus (207) Google Scholar, 10Beinhauer J.D. Hagan I.M. Hegemann J.H. Fleig U. Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form.J Cell Biol. 1997; 139: 717-728Crossref PubMed Scopus (188) Google Scholar, 11Schwartz K. Richards K. Botstein D. BIM1 encodes a microtubule-binding protein in yeast.Mol Biol Cell. 1997; 8: 2677-2691Crossref PubMed Scopus (168) Google Scholar], and its binding domain in APC may be conserved in Kar9, an EB1 binding protein involved in the microtubule-capturing mechanism [12Bienz M. Spindles cotton on to junctions, APC and EB1.Nat Cell Biol. 2001; 3: E67-E68Crossref PubMed Scopus (65) Google Scholar, 13Korinek W.S. Copeland M.J. Chaudhuri A. Chant J. Molecular linkage underlying microtubule orientation toward cortical sites in yeast.Science. 2000; 287: 2257-2259Crossref PubMed Scopus (181) Google Scholar, 14Lee L. Tirnauer J.S. Li J. Schuyler S.C. Liu J.Y. Pellman D. Positioning of the mitotic spindle by a cortical-microtubule capture mechanism.Science. 2000; 287: 2260-2262Crossref PubMed Scopus (240) Google Scholar]. These results suggest that the interaction of EB1 and APC is important and may be conserved. However, it is largely unknown whether the EB1-APC interaction affects microtubule dynamics. Here, we show that EB1 potently promotes microtubule polymerization in vitro and in permeabilized cells, but, surprisingly, only in the presence of the COOH-terminal EB1 binding domain of APC (C-APC). Significantly, this C-APC activity is abolished by phosphorylation, which also disrupts its ability to bind to EB1. Furthermore, yeast EB1 protein effectively substitutes for the human protein but also requires C-APC in promoting microtubule polymerization. Finally, C-APC is able to promote microtubule polymerization when stably expressed in APC mutant cells, demonstrating the ability of C-APC to promote microtubule assembly in vivo. Thus, the interaction between EB1 and APC plays an essential role in the regulation of microtubule polymerization, and a similar mechanism may be conserved in yeast.