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J. Biol. Chem., Vol. 281, Issue 8, 4887-4893, February 24, 2006

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Identification of in Vivo Phosphorylation Sites on Human Deoxycytidine Kinase

ROLE OF SER-74 IN THE CONTROL OF ENZYME ACTIVITY^*

Caroline Smal¹, Didier Vertommen, Luc Bertrand^¶2, Sandrine Ntamashimikiro, Mark H. Rider, Eric Van Den Neste^||, and Françoise Bontemps³

From the Laboratory of Physiological Chemistry and Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, the ^¶Division of Cardiology and the ^||Department of Hematology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, 1200 Brussels, Belgium

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [³²P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of ³²P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.

Received for publication, November 10, 2005 , and in revised form, December 16, 2005.

^* This work was supported in part by grants from the Belgian Fonds National de la Recherche Scientifique (Télévie, FRSM, Crédit au Chercheur), the Interuniversity Attraction Poles Program-Belgian Science Policy (P5/05), The Bauchau Foundation, The Maisin Foundation, The Goor Foundation, and The Salus Sanguinis Foundation. 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 Télévie Fellow.

² Research Associate of the Belgian Fonds National de la Recherche Scientifique.

³ To whom correspondence should be addressed: Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, UCL-ICP 7539, 75 Av. Hippocrate, 1200 Brussels, Belgium. Tel.: 32-2-7647539; Fax: 32-2-7647598; E-mail: bontemps{at}bchm.ucl.ac.be.

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