The p38 Pathway Regulates Oxidative Stress Tolerance by Phosphorylation of Mitochondrial Protein IscU
In this paper, the authors examined the role of the P38-mitogen activated protein kinase pathway (MAPK), an eukaryotic evolutionary conserved pathway which mediates responses to abiotic stress stimuli. The specific role of oxidative stress tolerance is conserved within this pathway, and was the authors predominant target of inquiry.
The role of Iron-Sulfur Clusters (ISC) within the pathway, cofactors which play major roles in promoting electron transport and redox reactions as well as acting as an upstream protein in the formation of Aconitase, was further examined with the understanding that such cofactor formation takes place via a highly complex and catalyzed process. The protein component IscU was specifically analyzed, as it acts as a scaffolding protein for overall ISC biosynthesis and eventual transfer to acceptor proteins. While there exist two isoforms of IscU within human populations, Drosophilla melanogaster possesses a single isoform which makes it an attractive experimental population for analysis of ISC biosynthesis, specifically as it pertains to function.
The role of MAPK activated protein kinase 2 (MK2) was another element of significant interest within the research, with the understanding that it is known to regulate gene transcription, cytoskeleton architecture formation, and play a role in inflammatory response within mammalian populations. The Drosophila specific variant, dMK2, had a corresponding lack of detailed information regarding its function. To resolve this, analytical techniques including RT-PCR, In-vitro kinase and activity assays, oxidative stress assays, and immunoblotting were utilized. The authors plan was to perform a wide-spectrum analysis within likely parameters ; The impact upon oxidative stress as resulting from successful ISC formation, the mechanistic details thereof, and the ramifications felt on aconitase activity in-vitro.
From these disparate analytical techniques, several functional factors were identified. It was determined that dMK2 knockout flies as generated by p-mediated genomic excisions, a method which reduces disruption outside the excision cite, have shorter lifespans under oxidative stressor conditions as effected by H2O2 or Paraquat (a redox-active heterocyclic compound) addition. This effect was maintained across both genders, and proper genomic modification verified by PCR analysis.
In order to examine downstream effects of the dIscU gene product as mediated by dMK2, in-vitro kinase assays and co-immunoprecipitation techniques were utilized using E. coli generated dMK2 recombinant proteins as a generative source. The likely possible phosphorylation sites on dIscU were analyzed by alignment with human and mouse sequences of IscU, and individual mutation of the residues performed in order to discern any effect upon phosphorylation by dMK2. The Ser-20 site mutation was found to be the only site which fully inhibited phosphorylation, painting it as the most likely target of dMK2. This was borne out by kinase assay.
In order to perform analysis regarding in-vivo effects, isoelectric focusing followed by western blotting was performed upon tissue harvested from dIscU-overexpressed eyes of the transgenic population. From this analysis, there was evidenced a decrease in phosphorylation of dIscU which while significant, did not wholly exclude the possibility of other kinases acting to phosphorylate dIscU. This was enough data however to demonstrate that dMK2 phosphorylates dIscU at Ser20 both in vitro and in vivo within the fly population. Measurements of aconitase activities in vivo revealed that there was no significant control exerted by Ser-20 phosphorylation. Analysis of dMK2 further revealed a lack of control over aconitase activities. However, mitochondrial respiratory complex 1 was determined to be negatively regulated by phosphorylation of Ser-20, as mediated by dMK2.
Oxidative stress within the fly population was the subject of further analysis, as the transgenic flies were exposed to oxidative stressors and their lifespans measured. The researchers were able to determine a quantitative decrease in both the homozygous and heterozygous transgenic populations, and thus helping to confirm the role of the p38 pathway (as mediated by dMK2 phosphorylation) in managing oxidative stress.
The point which likely makes this paper so significant as to be a hallmark in JBC lies in the final experimentation, where the phosphorylation of IscU by MK2 was evidenced to occur in mammalian cells. The authors embarked on this line of reasoning with the knowledge that IscU (and the phosphorylation site on Serine) are conserved in mammals, and subsequently demonstrated in HeLa cells hMK2 (a human isoform of MK2)phosphorylates IscU. As a result of this analysis, the authors were able to determine that phosphorylation of IscU by way of MK2 mediates key elements of the TCA cycle, specifically complex 1 activity. While the increased activity thereof possibly harbors responsibility for reduced tolerance to oxidative stress (as seen with fly populations) more investigation is admitted by the authors as necessary to solidify any claims thereof. The authors likely aim to carry on research to elucidate the precise cause for oxidative stress tolerance within this system, and how it carries over to human populations, as oxidative stress management is directly pertinent in regards to cancer treatments.