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J. Biol. Chem., Vol. 282, Issue 40, 29493-29503, October 5, 2007

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Regulation of Human Cytidine Triphosphate Synthetase 1 by Glycogen Synthase Kinase 3^*

Matthew J. Higgins, Paul R. Graves, and Lee M. Graves¹

From the Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365 and Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298-0058

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3 short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [³²P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.

Received for publication, May 14, 2007 , and in revised form, July 27, 2007.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 116 Manning Dr., 936A Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. Tel.: 919-966-0915; Fax: 919-966-5640; E-mail: lmg{at}med.unc.edu.

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