Session: Session 5

Integrative proteomics and phosphoproteomics to investigate new functions of the CHK1 kinase in response to metabolic stress in leukemic cells

Carine FROMENT¹, Lucie FABRIZI², Marie LOCARD-PAULET¹, Stefania MILLEVOI², Stéphane MANENTI², Odile BURLET-SCHILTZ¹

¹Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III - Paul Sabatier (UT3), Toulouse, France
²Centre de Recherches en Cancérologie de Toulouse (CRCT), INSERM UMR1037, CNRS UMR 5071, Université de Toulouse, Toulouse, France

In the context of DNA damage response (DDR) upon different kinds of genotoxic stress, phosphorylation and activation of the serine/threonine checkpoint kinase 1 (CHK1) is known to lead to cell-cycle arrest and DNA repair to preserve genomic integrity. As DDR, autophagy is a biological process also activated in response to different types of stress–including DNA damage– and are both essential for survival and maintenance of cellular homeostasis. However, the cross-talks between DDR and autophagy pathways remain poorly documented. Considering the hypothesis that CHK1 can play a role in autophagy process, the aim of the present study is to identify new substrates of CHK1 that could account for its implication into autophagy triggered by metabolic stress.

For this purpose, we performed a label-free quantitative analysis of the proteome and phosphoproteome of acute myeloid leukemia cells in response to CHK1 inhibition or silencing in two conditions: regular growth medium or amino acid starvation. Here, we present the optimized cell lysis, S-trap tryptic digestion, and TiO2-based phosphopeptide enrichment protocols followed by online nanoLC-MS/MS sample analyses using a FAIMS-equipped Exploris 480 mass spectrometer. In order to identify CHK1-dependent phosphorylation events, we also designed a customized computational data analysis pipeline combining the Proteome DiscovererTM software for identification and quantification, and R scripts for data processing and statistical analysis. The proteins and sites relative quantities were then visualized on STRING networks with Cytoscape to get insights on CHK1 role in autophagy.

Using this approach, we quantified almost 7,000 proteins and more than 14,000 phosphopeptide isoforms. The functional networks that are generated from our (phospho)proteomics data show the down-regulation of Autophagy-related protein LC3 B protein, a key autophagy effector, in response to CHK1 inhibition, and they highlight new potential functions for CHK1.