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T. D. Lehman, K. Riese, N. L. Lehman, P. K. Jackson, R. B. Crook; Ubiquitination Is Involved in the Regulation of Na-K-Cl Cotransporter (NKCC) Turnover in Pigmented Ciliary Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5541.
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The Na-K-Cl cotransporter (NKCC), located at the basolateral membrane of pigmented ciliary epithelial cells (CPE cells), facilitates transport of 1 K+, 1 Na + and 2Cl - into the cell via the inwardly directed Na+ gradient from the Na-K ATPase. The NKCC thus plays a major role in aqueous formation by facilitating blood-to-aqueous chloride transport across the ciliary epithelium. Turnover of many proteins is mediated by covalent attachment of the 7.6 Kd protein ubiquitin, which is catalyzed by ubiquitin ligases. Polyubiquitinated proteins undergo proteolysis in the 26S proteasome. Monoubiquitination triggers endocytosis of some membrane proteins, although polyubiquitination is implicated in their degradation in some cases. We examined possible roles for the ubiquitin-proteasomal and endosome-lysosomal systems for NKCC degradation in cultured CPE cells.
Western blotting with an anti-NKCC antibody was used to separately examine NKCC protein within CPE cell membrane and cytosolic compartments over time (0-9 hr) after treatment with proteasomal and lysosomal inhibitors.
Treatment of CPE cells with the proteasomal inhibitor MG132 led to accumulation of the 180 Kd NKCC protein band at the membrane and accumulation of a 62 Kd NKCC fragment in the cytosol, suggesting that NKCC endocytosis and its cytosolic degradation (in part) are mediated by ubiquitin-dependent processes. The lysosomal inhibitor chloroquin had no discernable effect on NKCC protein levels at the membrane, however a small 40 Kd immunoreactive fragment accumulated in the cytosol. This putative NKCC fragment may thus be degraded within the lysosome, likely following proteasomal degradation of larger fragments, since it is not seen with proteasomal inhibition. Next, we immunoprecipitated the NKCC from membrane fractions of cells treated with MG132 and chloroquin. Western blotting with an anti-ubiquitin antibody demonstrated a high molecular weight signal (> 180 Kd) only in the MG132 and chloroquin treated cells and not in control cells. These bands may represent monomers or aggregates of ubiquitinated NKCC proteins.
These data provide evidence that NKCC turnover proceeds, in part, through ubiquitin-dependent proteolysis, and possibly to a lesser extent by the lysosome. Further study of these regulatory mechanisms may reveal novel strategies useful for clinical regulation of aqueous formation and the treatment of glaucoma.
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