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J. Biol. Chem., Vol. 278, Issue 25, 22460-22465, June 20, 2003

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The Second Microtubule-binding Site of Monomeric Kid Enhances the Microtubule Affinity^*,

Katsuyuki Shiroguchi  , Miho Ohsugi ¶, Masaki Edamatsu , Tadashi Yamamoto ¶ and Yoko Y. Toyoshima  ||

From the Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902, Japan and the ^¶Department of Oncology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639, Japan

Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase. To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro. Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric. However, Kid cross-linked microtubules in an ATP-sensitive manner, suggesting that Kid has a second microtubule-binding site in addition to its motor domain. This was ascertained by binding of Kid fragments lacking the motor domain to microtubules. The interaction of the second microtubule-binding site was weak in a nucleotide-insensitive manner. K_mMT of the ATPase activity of Kid was lower than that of the fragments lacking the second microtubule-binding site. Moreover, the velocity of Kid movement in vitro was not affected by the second microtubule-binding site, which is consistent with the weak binding of this site to microtubules. The second microtubule-binding site would be important to enhance the affinity to microtubules for the monomeric motor, Kid. Because the amino acid sequence of this region is highly conserved among species, it seems to have essential roles for the functions of Kid in vivo.

Received for publication, December 3, 2002 , and in revised form, April 9, 2003.

^* This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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.

The on-line version of this article (available at http://www.jbc.org) contains two movies.

Present address: Center for Integrative Bioscience, Okazaki National Research Institutes, Higashiyama 5-1, Myodaiji, Okazaki 444-8585, Japan.

^|| To whom correspondence should be addressed. Tel.: 81-3-5454-6752; Fax: 81-3-5454-6722; E-mail: cyytoyo{at}mail.ecc.u-tokyo.ac.jp.

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