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J. Biol. Chem., Vol. 279, Issue 33, 34595-34602, August 13, 2004

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Protein 4.1R, a Microtubule-associated Protein Involved in Microtubule Aster Assembly in Mammalian Mitotic Extract^*

Shu-Ching Huang¶, Ramasamy Jagadeeswaran, Eva S. Liu, and Edward J. Benz, Jr.||**

From the Department of Medical Oncology, Dana Farber Cancer Institute, the Department of Medicine, Harvard Medical School, the ^||Department of Medicine, Brigham and Women's Hospital, the ^**Department of Pediatrics, Children's Hospital of Boston, Boston, and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Non-erythroid protein 4.1R (4.1R) consists of a complex family of isoforms. We have shown that 4.1R isoforms localize at the mitotic spindle/spindle poles and associate in a complex with the mitotic-spindle organization proteins Nuclear Mitotic Apparatus protein (NuMA), dynein, and dynactin. We addressed the mitotic function of 4.1R by investigating its association with microtubules, the main component of the mitotic spindles, and its role in mitotic aster assembly in vitro. 4.1R appears to partially co-localize with microtubules throughout the mitotic stages of the cell cycle. In vitro sedimentation assays showed that 4.1R isoforms directly interact with microtubules. Glutathione S-transferase (GST) pull-down assays using GST-4.1R fusions and mitotic cell extracts further showed that the association of 4.1R with tubulin results from both the membrane-binding domain and C-terminal domain of 4.1R. Moreover, 4.1R, but not actin, is a mitotic microtubule-associated protein; 4.1R associates with microtubules in the microtubule pellet of the mitotic asters assembled in mammalian cell-free mitotic extract. The organization of microtubules into asters depends on 4.1R in that immunodepletion of 4.1R from the extract resulted in randomly dispersed microtubules. Furthermore, adding a 135-kDa recombinant 4.1R reconstituted the mitotic asters. Finally, we demonstrated that a mitotic 4.1R isoform appears to form a complex in vivo with tubulin and NuMA in highly synchronized mitotic HeLa extracts. Our results suggest that a 135-kDa non-erythroid 4.1R is important to cell division, because it participates in the formation of mitotic spindles and spindle poles through its interaction with mitotic microtubules.

Received for publication, April 12, 2004 , and in revised form, May 18, 2004.

^* This work was supported by National Institutes of Health Grants HL61295 (to S.-C. H.) and HL44985 (to E. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Medical Oncology, Dana Farber Cancer Institute, D610, 44 Binney St., Boston, MA 02115. Tel.: 617-632-6965; Fax: 617-632-2662; E-mail: shu-ching_huang{at}dfci.harvard.edu.

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