Unveiling the mechanistic regulation of T148 phosphorylation-mediated neuroprotection by αA-crystallin.

Abstract

α-crystallins (αA- and αB-) are chaperone proteins historically studied in the eye lens as crucial to maintaining lens transparency. More recently, both proteins have garnered increased attention for their protective and chaperone roles in the context of retinal neurodegeneration, and neurodegeneration in the central nervous system more broadly. Critically, for both αA- and αB-crystallin, post-translational modifications (PTMs) and in particular, phosphorylation, regulate these roles. S19, S45, and S59 phosphorylation on αB-crystallin as well as S122 and S/T148 phosphorylation on αA-crystallin have all been implicated in regulating their protective and chaperone abilities. T148 (S148 in rodents) phosphorylation is particularly intriguing, as it is decreased in diabetic retinopathy despite αA-crystallin's upregulation under these conditions. Using phosphomimetic (T148D) and non-phosphorylatable (T148A) αA-crystallin, our lab has shown T148 phosphorylation to be associated with higher protein solubility, greater chaperone function, phosphorylation-dependent protein-protein interactions, greater neuroprotective capabilities in retinal neurons, and regulation of glial function. However, little is known about the mechanistic regulation of T148 phosphorylation, and the involved kinase(s) have yet to be identified. Using the phosphosite-accurate kinase-substrate crosslinking assay (PhAXA) method in R28 retinal neurons and MIO-M1 glial cells, we have identified strong candidate kinases, with the top candidates being mTOR and PI3K-C2α. We also demonstrated significantly decreased mTOR expression in the peripheral retina of patients with diabetic retinopathy in comparison to nondiabetic patients, as well as reduced PI3K activity in an animal model of diabetes, both consistent with the involvement of these kinases. mTOR and PI3K were thus subjected to in-vitro validation by kinase and cell death assays, and T148 phosphorylation was assessed under basal and metabolic stress conditions. These findings shed light on the mechanisms underpinning T148 phosphorylation-mediated neuroprotection and will help further the pursuit of novel therapeutics targeting neurodegenerative retinal diseases.