Highlights d Multiple cell compartments can support accumulation of high levels of polyPs d Over 350 transcripts and 100 proteins show mis-regulation with polyP accumulation d Intracellular polyP results in selective signaling via MAPK effectors d Targets of lysine polyphosphorylation change localization with polyP accumulation

Polyphosphates (PolyP) are composed of long chains of inorganic phosphates linked together by phosphoanhydride bonds. They are found in all kingdoms of life, playing roles in cell growth, infection, and blood coagulation. A resurgence in interest in polyP has shown links to diverse aspects of human disease. However, unlike in bacteria and lower eukaryotes, the mammalian enzymes responsible for polyP metabolism are not known. Many studies have resorted to adding polyP to cell culture media, but it is not clear if externally applied polyP enters the cell to impact signaling events or whether their effect is mediated exclusively by extracellular receptors. For the first time, we use RNA-seq and mass spectrometry to define a broad impact of polyP produced inside of mammalian cells via ectopic expression of the E. coli polyP synthetase Ppk1. RNA-seq demonstrates that Ppk1 expression impacts expression of over 350 genes enriched for processes related to transcription and cell motility. Analysis of proteins via label-free mass spectrometry identified over 100 changes with functional enrichment in cell migration. Follow up work suggests a role for internally-synthesized polyP in promoting activation of mTOR and ERK1/2-EGR1 signaling pathways implicated in cell growth and stress. Finally, fractionation analysis shows that polyP accumulated in multiple cellular compartments and was associated with the relocalization several nuclear/cytoskeleton proteins, including chromatin bound proteins DEK, TAF10, GTF2I and translation initiation factor eIF5b. Our work is the first to demonstrate that internally produced polyP can activate diverse signaling pathways in human cells.Significance StatementFor many years following its discovery in 1890, polyphosphates (polyP) were dismissed as evolutionary fossils. Best understood for its role in bacteria and yeast, our understanding of polyP in mammals remains rudimentary because the enzymes that synthesize and degrade polyP in mammalian systems are currently unknown. In our work, we carried out large-scale transcriptome and proteome approaches on human cells designed to accumulate internally produced polyP via ectopic expression of a bacterial polyP synthetase. Our work is the first to systematically assess the impact of increased intracellular polyP.

The Epidermal Growth Factor (EGF) Receptor (EGFR) is a receptor tyrosine kinase that when deregulated can drive tumor growth and can also contribute to drug resistance. Upon binding its ligand EGF, EGFR triggers the activation of many signaling pathways including phosphatidylinpositol‐3‐kinase (PI3K)/Akt, Ras‐Erk, signal transducer and activator of transcription (STAT), and phospholipase C γ1 (PLCγ1). EGFR may also control DNA repair mechanisms, although this phenomenon this remains poorly understood. Control of DNA repair by EGFR may be particularly relevant in the context of action of and resistance to anti‐cancer drugs that cause DNA damage (e.g. cisplatin). We examined how acute activation (10–30 min) of EGFR by ligand stimulation regulates DNA damage and repair responses induced by chronic (16 h) cisplatin treatment. To do so, we examined various markers of DNA damage and repair such as γH2AX and 53BP1. We observed that as little as 10 min of EGF stimulation is sufficient to elicit remodelling of DNA damage and repair markers such as γH2AX in chronic cisplatin‐treated cells. This indicates that acute EGFR activation triggers signaling pathway(s) that control the DNA damage response and/or DNA repair. Using these methods, we dissected the contribution of various EGFR signaling pathways and membrane traffic phenomena to this EGFR‐dependent control of DNA repair. This work may reveal new ways to enhance the efficacy of existing chemotherapies such as cisplatin for cancer treatment.Support or Funding InformationThis work was supported by a Project Grant and a New Investigator Salary Award from the Canadian Institutes of Health Research (CIHR) to C.N.AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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