Project Summary/Abstract Intracellular bacteria often secrete proteins (effectors) that hijack and rewire cellular pathways to establish their replicative niche. Studying the mechanisms by which these bacterial proteins function have frequently led to the identification of key cellular processes. Given that protein synthesis is essential to sustain cellular life and function, several bacteria manipulate host cell protein synthesis. We recently discovered that the intracellular bacterium, Legionella pneumophila (L.p.) secretes a eukaryotic-serine-threonine-protein-kinase (eSTPK) effector called LegK4, that translocates into the host cell cytosol and phosphorylates the chaperone HSC70 on T495 (pHSC70). Interestingly, this single phosphorylation causes a global block in protein synthesis. Often, bacterial proteins mimic the activities of host proteins to usurp their cellular function. Given that HSC70 plays diverse roles in the cell that includes the regulation of critical cellular processes such as protein homeostasis, we studied whether a host kinase was capable of phosphorylating HSC70 on T495. Indeed, we discovered that treatment of cells with methyl methanesulfonate, an alkylating agent that causes DNA damage, results in the accumulation of pHSC70. Global mRNA translation is blocked during the DNA damage response (DDR) and it is known that the type of stressor determines the mechanism of the block. For example, ionizing radiation and topoisomerase II inhibition leads to an inhibition of mTOR signaling, while UV exposure leads to the phosphorylation of eukaryotic initiation factor 2 by the kinase GCN2. However, the role of pHSC70 in inhibiting protein synthesis during the DDR has never been studied. Here, we propose to investigate the role of pHSC70 in response to DNA damage and elucidate the mechanisms by which pHSC70 leads to protein synthesis attenuation. In the first aim, we will use biochemical and fluorescence-based assays to determine how phosphorylation of HSC70 affects its chaperone function. In the second aim, we will perform polysome runoff assays to determine which step of protein synthesis is blocked when HSC70 is phosphorylated. In addition, we propose to identify the proteins that escape the protein synthesis block mediated by pHSC70. Finally, in the third aim, we will identify the kinases that phosphorylate HSC70 during the DNA damage response and the spatio-temporal regulation of signaling events that link the stress induced DNA damage response to pHSC70 and protein synthesis inhibition. Overall, the aims of this proposal will help elucidate the role of a multifunctional chaperone in mediating protein synthesis inhibition during DDR. The results of this study will not only be beneficial for the investigators studying HSC70, but also those that study the DNA damage response and host-pathogen interactions.