Spatial alterations of De Novo purine biosynthetic enzymes by Akt-independent PDK1 signaling pathways.

Danielle L Schmitt,Bao Tran Luu,Miji Jeon,Anand Sundaram,Songon An,Marco Falasca

doi:10.1371/journal.pone.0195989

Abstract

A macromolecular complex of the enzymes involved in human de novo purine biosynthesis, the purinosome, has been shown to consist of a core assembly to regulate the metabolic activity of the pathway. However, it remains elusive whether the core assembly itself can be selectively controlled in the cytoplasm without promoting the purinosome. Here, we reveal that pharmacological inhibition of the cytoplasmic activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) selectively promotes the formation of the core assembly, but not the purinosome, in cancer cells. However, alternative signaling cascades that are associated with the plasma membrane-bound PDK1 activity, including Akt-mediated cascades, regulate neither the core assembly nor the purinosome in our conditions. Along with immunofluorescence microscopy and a knock-down study against PDK1 using small interfering RNAs, we reveal that cytoplasmic PDK1-associated signaling pathways regulate subcellular colocalization of three enzymes that form the core assembly of the purinosome in an Akt-independent manner. Collectively, this study reveals a new mode of compartmentalization of purine biosynthetic enzymes controlled by spatially resolved signaling pathways.

Highlights

Purines are associated with a variety of cellular processes as secondary messengers, cofactors and nucleic acids [1]
Using a series of pharmacological inhibitors and time-lapse live-cell imaging approaches, we have found that the inhibition of phosphatidylinositol-3-kinase (PI3K), Akt, or mammalian target of rapamycin (mTOR), whose cascade starts at the plasma membrane, have no impact on the subcellular distribution of de novo purine biosynthetic enzymes in human cancer cells
Both cancer cell lines were maintained in nutrient-rich conditions (Materials and Methods), where all the enzymes in de novo purine biosynthesis diffusively stain the cytoplasm in the absence of exogenous stimuli [7,8]

Summary

Introduction

Purines are associated with a variety of cellular processes as secondary messengers, cofactors and nucleic acids [1]. Inosine monophosphate (IMP), a metabolic product of purine biosynthesis, is synthesized in one step by a purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), which conjugates hypoxanthine with phosphoribosyl pyrophosphate (PRPP). The salvage pathway appears to be sufficient to meet the cellular demand for purines in normal healthy cells [2]. De novo purine biosynthesis contributes to cellular purine levels by converting PRPP to IMP in ten chemical reactions (Fig 1A). Six enzymes, three of which are multifunctional enzymes, are responsible for the sequential reactions; i.e. PRPP amidotransferase (PPAT; step 1), a trifunctional enzyme (TrifGART; steps 2, 3 and 5) with glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase and aminoimidazole ribonucleotide synthetase activities, formylglycinamidine ribonucleotide synthase (FGAMS; step 4), a bifunctional enzyme (PAICS; steps 6 and 7) with carboxyaminoimidazole ribonucleotide synthetase and succinylaminoimidazolecarboxamide ribonucleotide.

Methods

Results

Conclusion