CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury

Mark J Ranek,Dong I Lee,Richard C Page,Monte S Willis,Danielle Dillard,Virginia S Hahn ,Mohammed Aslam ,Jonathan C Schisler,Brittany Dunkerly‐Eyring ,Christian U Oeing ,Ronald J Holewinski,Jennifer E Van Eyk,Matthew M Mannion,Cornelia Virus,Masayuki Sasaki,Peter P Rainer,Vineet Agrawal,Rebekah Sanchez‐Hodge ,Sumita Mishra,Huaqun Zhang,Kristen M Kokkonen-Simon,David A Kass

doi:10.1038/s41467-020-18980-x

Abstract

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

Highlights

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases
Exposure to 48 h of simulated ischemia (SI; deoxyglucose + acidosis + hypoxia) increased ubiquitinated proteins (UP) that further rose in cells with PDE5Aoe (Fig. 1a)
In a prior Mass Spec-derived database of proteins in adult rat myocytes subjected to 15 minutes protein kinase G (PKG) activation[25], we found Carboxyl terminus of Hsc70-interacting protein (CHIP) modified at S20 (S20 in rat and mouse, S19 human, Fig. 2b), a highly conserved residue in the tetratricopeptide repeat (TPR) domain (Fig. 2c)

Summary

Introduction

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. We report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc[70], CHIP protein half-life, and consequent clearance of stressinduced ubiquitinated-insoluble proteins. The carboxyl terminus of Hsc70-interacting protein or CHIP (encoded by the gene known as Stub1) is an important member of the UPS system, functioning as both an E3-ligase and co-chaperone, and facilitating protein degradation[5]. CHIP gene upregulation has been protective against such disorders[16,17,18]

Methods

Results

Conclusion