Reyland, M. E. | Search bioRxiv

bioRxiv2023

PKCδ regulates chromatin remodeling and DNA repair through SIRT6

Protein kinase C delta (PKC{delta}) is a ubiquitous kinase whose function is defined in part by localization to specific cellular compartments. Nuclear PKC{delta} is both necessary and sufficient for IR-induced apoptosis, while inhibition of PKC{delta} activity provides radioprotection in vivo. How nuclear PKC{delta} regulates DNA-damage induced cell death is poorly understood. Here we show that PKC{delta} regulates histone modification, chromatin accessibility, and double stranded break (DSB) repair through a mechanism that requires SIRT6. Overexpression of PKC{delta} promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKC{delta} increases DNA repair via non-homologous end joining (NHEJ) and homologous recombination (HR) as evidenced by more rapid formation of NHEJ (DNA-PK) and HR (Rad51) DNA damage foci, increased expression of repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKC{delta} depletion is associated with more open chromatin, while overexpression of PKC{delta} reduces chromatin accessibility. Epiproteome analysis revealed that PKC{delta} depletion increases chromatin associated H3K36me2, and reduces ribosylation of KDM2A and chromatin bound KDM2A. We identify SIRT6 as a downstream mediator of PKC{delta}. PKC{delta}-depleted cells have increased expression of SIRT6, and depletion of SIRT6 reverses the changes in chromatin accessibility, histone modification and NHEJ and HR DNA repair seen with PKC{delta}-depletion. Furthermore, depletion of SIRT6 reverses radioprotection in PKC{delta}-depleted cells. Our studies describe a novel pathway whereby PKC{delta} orchestrates SIRT6- dependent changes in chromatin accessibility to increase DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKC{delta}. One Sentence SummaryProtein kinase C delta modifies chromatin structure via SIRT6 to regulate DNA repair.

molecular biology↗

bioRxiv2022

Cleavage of Protein Kinase C δ by Caspase-3 Mediates Pro-inflammatory Cytokine-Induced Apoptosis in the Pancreatic Islet

In type 1 diabetes (T1D), autoreactive immune cells infiltrate the pancreas and secrete pro-inflammatory cytokines that initiate cell death in insulin producing islet {beta}-cells. Protein kinase C {delta} (PKC{delta}) plays a role in mediating cytokine-induced {beta}-cell death; however, the exact mechanisms are not well understood. To address this, we utilized an inducible {beta}-cell specific PKC{delta} KO mouse as well as a small peptide specific inhibitor of PKC{delta}. We identified a role for PKC{delta} in mediating cytokine-induced {beta}-cell death and have shown that inhibiting PKC{delta} protects pancreatic {beta}-cells from cytokine-induced apoptosis in both mouse and human islets. We determined that cytokines induced nuclear translocation and activity of PKC{delta} and that caspase-3 cleavage of PKC{delta} may be required for cytokine-mediated islet apoptosis. Further, cytokine-activated PKC{delta} increases activity both of pro-apoptotic Bax with acute treatment and JNK with prolonged treatment. Overall, our results suggest that PKC{delta} mediates cytokine-induced apoptosis via nuclear translocation, cleavage by caspase-3, and upregulation of pro-apoptotic signaling in pancreatic {beta}-cells. Combined with the protective effects of PKC{delta} inhibition with {delta}V1-1, the results of this study will aid in the development of novel therapies to prevent or delay {beta}-cell death and preserve {beta}-cell function in T1D.

cell biology↗