Phosphorylation by p34cdc2 Protein Kinase Regulates Binding of the Kinesin-related Motor HsEg5 to the Dynactin Subunit p150Glued

doi:10.1074/jbc.272.31.19418

Phosphorylation by p34^cdc2 Protein Kinase Regulates Binding of the Kinesin-related Motor HsEg5 to the Dynactin Subunit p150^Glued*

From the ^‡Swiss Institute for Experimental Cancer Research (ISREC), 155, Chemin des Boveresses, CH-1066 Epalinges, Switzerland and the ^¶Department of Molecular Biology, Sciences II, University of Geneva, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

Abstract

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34^cdc2 protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, α-helical region of human p150^Glued, a prominent component of the dynactin complex. The interaction between HsEg5 and p150^Glued was enhanced upon activation of p34^CDC28, the budding yeast homolog of p34^cdc2, provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150^Glued to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150^Glued during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150^Glued may interact in mammalian cells in vivo and that p34^cdc2 may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.

Footnotes

↵* This work was supported by Swiss National Science Foundation Grant 31-33615.92/2, Swiss Cancer League Grant DKL 267-1-1996, and by the Canton of Geneva.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U90445.
↵§ Recipient of postdoctoral fellowships from European Molecular Biology Organization and the Roche Foundation. Present address: CRBM-CNRS, 1919, route de Mende, F-34033 Montpellier Cedex, France.
↵‖ To whom correspondence should be addressed. Tel.: 41 22 702 6127; Fax: 41 22 702 6868; E-mail:nigg{at}sc2a.unige.ch.
↵1 The abbreviations used are: KRP, kinesin-related protein; DBD, DNA binding domain; AD, activation domain; GST, glutathione S-transferase; PBS, phosphate-buffered saline.
- Received January 14, 1997.
- Revision received April 28, 1997.