The Xenopus laevis Aurora-related Protein Kinase pEg2 Associates with and Phosphorylates the Kinesin-related Protein XlEg5

doi:10.1074/jbc.274.21.15005

The Xenopus laevis Aurora-related Protein Kinase pEg2 Associates with and Phosphorylates the Kinesin-related Protein XlEg5*

From the ^‡Groupe Cycle Cellulaire and ^¶Groupe Structure Dynamique de la Chromatine, Faculté de Médecine, Laboratoire de Biologie et Génétique du Développement, CNRS UPR 41, 35043 Rennes Cedex, France

Abstract

We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from thebimC family (Le Guellec, R., Paris, J., Couturier, A., Roghi, C., and Philippe, M. (1991) Mol. Cell. Biol. 11, 3395–3408) as well as pEg2, an Aurora-related serine/threonine kinase (Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Dorée, M., Philippe, M., and Prigent, C. (1998) J. Cell Sci. 111, 557–572). Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation. Here, we report that inXenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interactin vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active when bound to the beads and phosphorylate XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylatedin vivo first confirmed that XlEg5 was phosphorylated by p34^cdc2 and next revealed that in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain serine residue that was phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is to our knowledge the first in vivo substrate ever reported for an Aurora-related kinase.

Footnotes

↵* This work was supported by European Economic Community Grant CHRX-CT94-0568, the French government (CNRS and ACC-SV4), the Association pour la Recherche sur le Cancer, and the Ligue Nationale contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: Cell Cycle Group, Div. of Electron Microscopy, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia.
↵‖ To whom correspondence should be addressed. Tel.: 33-2-99-33-62-16; Fax: 33-2-99-33-62-00; E-mail:claude.prigent{at}univ-rennes1.fr.
Abbreviations:

XlEg5

X. laevis kinesin-like protein Eg5

BSA

bovine serum albumin

CENP-E

centromere protein E

GST

glutathioneS-transferase

IgG

immunoglobulin G

Ipl1

increase in ploidy 1

KLP61F

kinesin-like protein 61F

NuMA

nuclear mitotic apparatus protein

PP1 and PP2A

protein phosphatases 1 and 2A, respectively

TLC

thin layer cellulose

XKCM1

Xenopuskinesin central motor 1

XCTK2

Xenopus C-terminal kinesin 2

Xklp1

Xenopus kinesin-like protein 1

XlEg5HS

head and stalk domain of XlEg5

XlEg5ST

stalk and tail domain of XlEg5

XlEg5T

tail domain of XlEg5

PBS

phosphate-buffered saline

NTA

nitrilotriacetic acid

IP

immunoprecipitation

TBS

Tris-buffered saline

TBST

Tris-buffered saline with Tween 20

IB

interaction buffer

PCR

polymerase chain reaction
- Received December 28, 1998.
- Revision received February 22, 1999.
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