Phosphoproteomics analyses reveal that phosphorylation of heat shock protein 70 (hsp70) by protein kinase B (Akt1) on ser631 controls activity of mitochondrial superoxide‐2 (SOD2).
Authors: AFOLAYAN Adeleye James , Zemanovic Sara M. , Konduri Girija Ganesh 
Published: June 2021

BackgroundSOD2 is a nuclear encoded gene whose product must be imported into the mitochondria for activation. Previously, we showed that hsp70 chaperones SOD2 to the mitochondria. The chaperone activity of hsp70 is negatively regulated by the C‐terminus of hsp70‐interacting protein (CHIP). However, the mechanism that controls SOD2 activity proportional to activating signals to maintain mitochondrial redox balance is unknown.MethodsTo identify the signaling protein that regulates activity of hsp70, HEK‐293T cells were transfected with GST‐tagged‐hsp70. The GST epitope was immuno‐precipitated and the differential band was sent for mass spectrometry and Akt1 was identified. To examine the contributions of Akt1 to SOD2 induction in response to stimuli, we used PI3K inhibitor, Ly294002 treatment as well as Akt1‐siRNA knockdown methods. Pulmonary artery endothelial cells (PAECs) were transfected with Akt1‐siRNA and we re‐expressed myristoylated Akt1 or dominant negative Akt1. We then quantified SOD2 protein and activity in isolated mitochondria using Western blots and colorimetric assays. To identify the amino acid residue (s) in hsp70 that are phosphorylated by Akt1, we incubated recombinant Akt1 proteins with purified hsp70 in an in‐vitro kinase reaction and the band containing phosphorylated hsp70 was sent for phosphoproteomics analysis and site identification using LC‐MS/MS. We characterized the newly identified residue by point mutation and assessed the effect of mutations on SOD2 localization to the mitochondria by hsp70 using immuno‐fluorescent staining. To examined the effect of hsp70 phosphorylation on SOD2 activity in response to stimuli, we knockeddown Akt1 using siRNA and generated global akt1 null mice and measured SOD2 import, and activity and rates of superoxide and hydrogen peroxide levels in cells and lung lysates following stimulation using LC‐MS/MS following mitoSOX/mito‐B stainings. We determined the effect of hsp70 phosphorylation on the interaction between CHIP and hsp70, and the functional relevance of CHIP interaction on SOD2 function using CHIP overexpression, ubiquitin assays and immunoprecipitation methods.ResultsInduction of Akt1 activity enhanced SOD2 activity in PAECs within minutes following PDGF stimulation, reaching a peak at 20 minutes and is then downregulated. Conversely, inhibition of Akt1 activity robustly decreased SOD2 protein and activity in isolated mitochondria and increased mitochondrial O2− formation compared to controls. Re‐expression of myr‐Akt1 restored SOD2 activity whereas DN‐Akt1 failed to improve SOD2 activity. Mechanistically, we demonstrated that Akt1 binds to and phosphorylates hsp70 at ser‐631. Substitution of ser for Ala at position 631 in hsp70 blocked the PDGF mediated induction of SOD2 accumulation in the mitochondria. Phosphorylated hsp70 inhibited the chaperone interaction with CHIP, and conversely, dephosphorylated hsp70 preferentially recruited CHIP and facilitated SOD2 degradation. Treatment with mito‐TEMPO induced PP2A/2C protein expression, inactivated Akt1 and dephosphorylated hsp70‐ser‐631, leading to the eventual termination of SOD2 import.ConclusionAkt1 integrates activation and inhibition signals, links them to hsp70 to regulates SOD2 activity.Support or Funding InformationSupported by NIHKO8 (AJA), Children Research Institute (CRI) and Department of Pediatrics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.