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© The Rockefeller University Press, 0021-9525/1997//859 $5.00
The Journal of Cell Biology, Volume 136, Number 4, , 1997 859-870

Article

XCTK2: A Kinesin-related Protein That Promotes Mitotic Spindle Assembly in Xenopus laevis Egg Extracts



Claire E. Walczak, Suzie Verma, and Timothy J. Mitchison

Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California 94143-0450

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central {alpha}-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor- arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.

Abbreviations used in this paper: CSF, cytostatic factor-arrested; CTK, carboxy-terminal kinesin; KRP, kinesin-related protein.

Please address all correspondence to Claire Walczak, Department of Cell and Molecular Pharmacology, University of California at San Francisco, 513 Parnassus Box 0450, San Francisco, CA 94143-0450. Tel.: (415) 4760836; Fax.: (415) 476-5233; E-mail: walczak{at}cg1.ucsf.edu


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