IDENTIFICATION OF PHOSPHORYLATION SITES IN HUMAN CTPS1

Abstract

Cytidine triphosphate synthetase (CTPS) is an essential enzyme in the de novo synthesis of CTP, which is important in phospholipid metabolism and DNA synthesis. CTPS catalyzes the rate-limiting step in the formation of cytidine nucleotides by transferring the amine from glutamine to uridine triphosphate (UTP) to form CTP. Although CTPS phosphorylation has been well characterized in yeast, relatively little is known regarding the phosphorylation of mammalian CTPS. In this study, we characterized the phosphorylation of human CTPS1 in human embryonic kidney (HEK) 293 cells and examined possible kinases responsible for CTPS1 phosphorylation. In yeast, CTPS1 has been shown to be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC) on multiple residues. We investigated phosphorylation of human CTPS1 by immunoprecipitating endogenous human CTPS1 from lysates from 32P labeled HEK293 cells, separating tryptic peptides by two-dimensional phosphopeptide mapping and identifying phosphorylation sites by mass spectrometry. We identified the two major phosphorylation sites in human CTPS1 and have begun characterizing the significance of these phosphorylation sites on CTPS1 function using Flag tagged CTPS1 bearing an alanine mutation for the phosphoamino acid. Alanine mutants expressed in HEK293 cells implicate CTPS1 phosphorylation is regulating CTPS1 expression. Additionally, we have also have preliminary evidence implicating CDKs and CK2 in phosphorylating CTPS1. Funding to LMG from NIH