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PMID: 9049251 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts.

The Journal of cell biology ·Vol. 136 ·No. 4 ·1997-02-24 ·Pages 859-70

Walczak CE, Verma S, Mitchison TJ

Abstract

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.

MeSH Terms
Amino Acid Sequence Animals Cell Extracts/physiology Kinesins/chemistry,isolation & purification,metabolism,physiology Macromolecular Substances Molecular Sequence Data Ovum/cytology,physiology Spindle Apparatus/drug effects,metabolism,physiology Xenopus Proteins Xenopus laevis
Chemicals
Cell Extracts KIFC1 protein, Xenopus Macromolecular Substances Xenopus Proteins Kinesins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Walczak C E
Department of Cellular and Molecular Pharmacology, University of California at San Francisco 94143-0450, USA. walczak@cgl.ucsf.edu
Verma S
Mitchison T J
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.136.4.859
Email
walczak@cgl.ucsf.edu
Published
1997-02-24
Pages
859-70
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2132492
Subset
IM
Grants
NIGMS NIH HHS · F32 GM016100 · United States
Databases
GENBANK
U82809
External Links
PubMed source DOI: 10.1083/jcb.136.4.859 PMC Full Text View journal details
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