Actin Protein Phosphorylation And Its Role In The Study

Renal proximal tubular (PT) ceils organize into highly specialized apical membrane domains, constituting microvilli and allowing them to perform the reabsorption of bulk of nutrients and ions in kidney. The establishment and maintenance of microvilli are dependent on the integrity of the actin cytoskeleton because actin polymerizes to form the actin cytoskeleton in the corn of microvilli in renal epithelial cells. Therefore, the disruption of the actin cytoskeleton initiates a cascade of structural and functional alteration in renal epithelial cells and which will lead to the dysfunction of reabsorption in kidney.ATP-depletion is a ideal model for studying various cytoskeletal and functional alternations induced by renal ischemia in renal proximal tubules (PT). Our previous study showed that the actin broken down from the microvillar F-actin filaments was bound to non-actin protein(s) in pellet of renal PT during ATP-depletion. However, the molecular details regarding actin sequestration remained elusive.Phosphorylation is the most common way of regulating protein functions. There is little known about the regulation of actin cytoskeletonal structure and function by actin phosphorylation inrenal epithelial cells.In this report, by using two-dimensional (2D) Western blotting, we separated phosphorylated actin from unphosphorylated actin in ATP-depleted PT, and identified phosphorylation actin by monoclonal anti-actin and which residues of amino acid were phosphorylated by using monoclonal anti-phosphoserine (or anti-phosphotyrosine or anti-phosphothreonine). The purpose of this study was to attempt to demonstrate the correlation between actin sequestration and actin phosphorylation in ATP-depleted PT.The analysis of the sequestered actin indicated that in ATP-depleted PT nearly half of the sequestered actin was phosphorylated on serine residue(s). The further study demonstrated that phosphorylated actin was only found in cytoskletonal fraction rather than in cytoplasmic fractions.In conclusion, the present studies demonstrated that there may be a close correlation between actin sequestration and actin phosphorylation in ATP-depleted PT.