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M. Narayan, S. P. Mirza, S. S. Twining; Differential Phosphorylation of Intracellular and Secreted Corneal Epithelial Cell Maspin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1972.
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Maspin, a 42 kDa non-classical serpin (serine protease inhibitor), expressed and secreted by corneal epithelial cells regulates migration and adhesion of corneal stromal fibroblasts through its RSL and exhibits anti-angiogenic activity in the cornea. The function of maspin in the corneal epithelium is unclear at present. Our approach to understanding the role of maspin in the corneal epithelium is to characterize the modification of maspin by phosphorylation, which may help link maspin to pathways involved in maintenance of epithelial homeostasis.
Immortalized human corneal epithelial cells (HCEC) and primary epithelial cells were lysed directly in iso-electric focusing (IEF) buffer. The proteins in the cell lysates were separated by IEF and then by SDS-electrophoresis in the second dimension (2D-PAGE). Tyrosine phosphorylation of maspin was detected by western blotting. Maspin was also immunoprecipitated (IP) from lysates and conditioned medium of primary and immortalized corneal epithelial cells in culture, and analyzed by tandem mass spectrometry for phosphorylation. Maspin was localized by immunofluorescence.
We previously reported that maspin secreted by human corneal epithelial cells exists in multiple forms as evidenced by IEF-2D-PAGE analysis. Tandem mass spectrometry analysis indicated secreted maspin is phosphorylated at multiple serine and threonine residues (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310 and Ser316). Intracellular corneal epithelial maspin, which localizes to the cytosol and nucleus, also exhibits similar differences in charge as evidenced by IEF-2D-PAGE analysis with isoelectric points (pIs) lower than the predicted value of 5.6. Interestingly, IP and western blotting analysis indicates the presence of two intracellular forms of maspin, one that is not phosphorylated and one that is phosphorylated on one or more tyrosine residues. The absence of phosphorylation on one form has been confirmed by mass spectrometry.
Corneal epithelial cells differentially phosphorylate intracellular and secreted maspin suggesting distinct roles for this molecule based on its location. Understanding the pathway for phosphorylation of maspin may be important for resolving the role of maspin in the corneal epithelium.
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