Mapping Post-Translational Modifications of de Novo Purine Biosynthetic Enzymes: Implications for Pathway Regulation

Chunliang Liu, Giselle M. Knudsen, Anthony M. Pedley, Jingxuan He, Jared L. Johnson, Tomer M. Yaron, Lewis C. Cantley, Stephen J. Benkovic

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14 Scopus citations


Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.

Original languageEnglish
Pages (from-to)2078-2087
Number of pages10
JournalJournal of Proteome Research
Issue number5
StatePublished - 3 May 2019
Externally publishedYes

Bibliographical note

Publisher Copyright:
© Copyright 2019 American Chemical Society.


*E-mail: [email protected]; phone: 1-814-865-2882. ORCID Anthony M. Pedley: 0000-0003-1034-5072 Stephen J. Benkovic: 0000-0003-3680-3481 Present Addresses ○Boragen, Inc., Durham, North Carolina 27709, United States ∇Alaunus Biosciences, Inc., San Francisco, California 94107, United States Author Contributions C.L., G.M.K., and A.M.P. contributed equally to this work. C.L., G.M.K., A.M.P., and S.J.B. contributed to the design of the experiments for this study. C.L., A.M.P., and J.H. conducted all the biochemical experiments. G.M.K. and A.M.P. performed the mass spectrometry (PTM analysis: G.M.K., metabolite analysis: A.M.P.). J.L.J., T.Y., and L.C.C. performed the kinase prediction. C.L., G.M.K., A.M.P., and J.H. analyzed the data. All authors contributed to the writing of the manuscript, reviewed the results, and approved the final version of the manuscript. Notes The authors declare the following competing financial interest(s): L.C.C. is a founder and member of the BOD of Agios Pharmaceuticals; he is also a co-founder, member of the SAB, and shareholder of Petra Pharmaceuticals. These companies are developing novel therapies for cancer. The L.C.C. laboratory receives some funding support from Petra Pharmaceuticals. The authors thank members of the Benkovic Laboratory for critical analysis of the manuscript, the University of California San Francisco Mass Spectrometry Facility directed by A.L. Burlingame (A.L.B.), and the Pennsylvanie State Metabolo-mics Core Facility at University Park, PA directed by P. Smith for processing and technical assistance in the analysis of metabolite measurements. Financial support for this study was provided by The National Institutes of Health (R01GM024129 to S.J.B., P41GM103481 to A.L.B., and R35CA197588 to L.C.C.) and the Adelson Family Foundation (A.L.B.).

FundersFunder number
Adelson Family Foundation
Petra Pharmaceuticals
National Institutes of HealthR35CA197588, P41GM103481, R01GM024129
National Cancer InstituteU54CA210184


    • AKT
    • PTM
    • de novo purine biosynthesis
    • metabolism
    • phosphorylation
    • post-translational modification


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