p38-mitogen activated protein kinases (p38-MAPKs) are stress activated serine/threonine kinases that are activated during several different cardiac pathologies. Classically, studies have focused solely on p38α signaling in the heart. However, there is also high cardiac expression of the p38γ isoform but little is known about its cardiac function. The aim of this study was to elucidate the signaling pathway of p38γ, with a particular focus on its role in the progression of pathological cardiac hypertrophy. Comparisons of cardiac function and structure of wild type (WT) and p38γ knock out (KO) mice, in response to abdominal aortic banding, found that KO mice developed less ventricular hypertrophy than their corresponding WT controls, and have preserved cardiac function. Basal p38γ myocardial staining was primarily localised at the membranes and throughout the cytoplasm. Following aortic constriction, nuclear staining of p38γ increased, but no accumulation of p38α was observed. This suggests that the two isoforms play distinct roles in the heart. To elucidate its signaling pathway, we generated an analogue sensitive p38γ, which is mutated at a gatekeeper residue, to specifically track and identify its endogenous substrates in the myocardium. The mutation allows only the mutant kinase, but not WT kinases, to utilise analogues of ATP that are expanded at the N6 position and contain a detectable tag on the γ-phosphate. Transfer of this tag to substrates allows subsequent isolation and identification. Furthermore, unlike other p38-MAPKs, p38γ contains a C-terminal PDZ domain interacting motif. We have utilised this motif in co pull-down assays to identify interacting proteins of p38γ in the heart. Using these techniques we have identified, amongst other substrates, LDB3 and calpastatin as novel substrates of p38γ and we have determined the residues that are targeted for phosphorylation. Lastly we have shown that phosphorylation of calpastatin reduces its efficiency as a calpain inhibitor in vitro, hence proposing a mechanism by which p38γ may mediate its pro-hypertrophic role.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:684459 |
| Date | January 2016 |
| Creators | Loonat, Aminah Ahmed |
| Contributors | Marber, Michael Stephen ; Clark, James |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://kclpure.kcl.ac.uk/portal/en/theses/the-involvement-of-p38gamma-mapk-in-pathological-cardiac-hypertrophy(f00e26a7-dab2-474d-9d3e-a52dfe9e873e).html |
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