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T. J. Lukas, H. Miao, A. Crabb, M. R. Hernandez; Hydrostatic Pressure Sensitive Protein Phosphorylation in Optic Nerve Head Astrocytes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):885.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve head (ONH) astrocytes are involved in the pathology found in glaucoma. ONH astrocytes exhibit changes in signal transduction induced by elevated hydrostatic pressure (HP) resulting in altered cell morphology and migration properties. However, the molecular mechanisms leading to these changes are unknown. To investigate the origins of these effects, we analyzed changes in phosphoprotein patterns in normal Caucasian ONH astrocytes subjected to elevated HP.
Soluble protein extracts from HP-treated astrocytes were subjected to reduction/alkylation and digestion with trypsin. Phosphopeptides were enriched using Gallium-chelate chromatography. Phosphopeptides were separated and detected by high sensitivity HPLC-Mass spectrometry. Database searches with the peptide mass spectrometry data identified the source proteins. Phosphoproteins were curated based upon spectral quality and number of identifications. Proteins from each set (Control, 3hr, 6hr, 24hr, 48hr) were further grouped based upon Gene Ontology (GO) analysis of function and localization (GoMiner and Profcom).
Three different primary cultures of ONH astrocytes were used in the analysis. For each time series approximately 150 curated phosphopeptides were found. GO analysis indicated that nuclear phosphoproteins were the most significant enriched species in the controls (25 proteins, p=0.009) followed by proteins associated with the cell cycle (7 proteins, p=0.045). However, a time-dependent decrease in nuclear phosphoproteins (25->12) was observed after HP-treatment. This was complemented by an increase in phosphoproteins associated with the cytoskeleton (2->6). One of the newly detected phosphoproteins was ELMO, a component of the DOCK180-p130Cas complex that is associated with focal adhesions and RAC1 GTPase activation. Phosphorylation of p130Cas is known to be triggered by mechanical force (Sawada, et al., Cell 127:1015 2006) and we found that phosphorylation of p130CAS (Y165) was also increased in HP-treated ONH astrocytes.
HP-treatment increases phosphorylation of proteins associated with focal adhesion complexes that are linked to the cytoskeleton. These signaling events may be partly responsible for changes in the cell adhesion and migration properties of ONH astrocytes that lead to the reactive phenotype in glaucoma.
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