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J. Biol. Chem., Vol. 282, Issue 20, 15258-15270, May 18, 2007

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Regulation of the Rab5 GTPase-activating Protein RN-tre by the Dual Specificity Phosphatase Cdc14A in Human Cells^*

Letizia Lanzetti¹, Valentina Margaria¹², Fredrik Melander, Laura Virgili, Myung-Hee Lee, Jiri Bartek, and Sanne Jensen³

From the Dipartimento di Scienze Oncologiche, Università degli Studi di Torino, Istituto per la Ricerca e la Cura del Cancro, Strada Provinciale 142, 10060 Candiolo, Torino, Italy and the Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark

The Cdc14 family of dual specificity phosphatases regulates key mitotic events in the eukaryotic cell cycle. Although extensively characterized in yeast, little is known about the function of mammalian Cdc14 family members. Here we report a genetic substrate-trapping system designed to identify substrates of the human Cdc14A (hCdc14A) phosphatase. Using this approach, we identify RN-tre, a GTPase-activating protein for the Rab5 GTPase, as a novel physiological target of hCdc14A. As a Rab5 GTPase-activating protein, RN-tre has previously been implicated in control of intracellular membrane trafficking. We find that RN-tre forms a stable complex with the catalytically inactive hCdc14A C278S mutant but not with the wild type protein in human cells, indicative of a substrate/enzyme interaction. In support, we show that RN-tre is regulated by cell cycle-dependent phosphorylation peaking at mitosis, which can be antagonized by hCdc14A activity in vitro as well as in vivo. Furthermore, we show that RN-tre phosphorylation is critical for efficient hCdc14A association and that RN-tre binding can be displaced by tungstate, a competitive inhibitor that binds to the active site of hCdc14A. Consistent with the preference of hCdc14A for phosphorylations mediated by proline-directed kinases, we find that RN-tre is a direct substrate of cyclin-dependent kinase. Finally, phosphorylation of RN-tre appears to finely modulate its catalytic activity. Our findings reveal a novel connection between the cell cycle machinery and the endocytic pathway.

Received for publication, January 31, 2007 , and in revised form, March 16, 2007.

^* This work was supported by the Danish National Research Foundation and the Danish Cancer Society (to J. B.); the Danish Cancer Society and Novo Nordisk Foundation (to S. J.); Svenska Sällskapet för Medicinsk Forskning, Stockholm (to F. M.); and Associazione Italiana per la Ricerca sul Cancro, the European Community (VI Framework), and Association for International Cancer Research (AICR) (to L. L.). 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.

¹ These authors contributed equally to this work.

² Supported by a fellowship from AICR.

³ To whom correspondence should be addressed: Evolva A/S, Bülowsuej 25, DK-1870 Frederiksberg, Denmark. Tel.: 4532502028; Fax: 4535283885; E-mail: sannej{at}evolva.com.

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