PKC activation induces ubiquitination of the Na-K-2Cl cotransporter 1 in the human colonic epithelial cells T84
Arts and Sciences
Gut clearance (i.e., fluid secretion) is one of the first lines of defense of the intestine against toxins and opportunistic bacteria. In the colon, fluid secretion is coupled to chloride secretion. Previous work has demonstrated that the basolateral Na-K-2Cl cotransporter 1 (NKCC1) represents a site for regulating fluid secretion independently of the apical chloride channels such as the cystic fibrosis transmembrane regulator. In addition, the lab has shown that protein kinase C (PKC) activation causes internalization of NKCC1, which in turn blocks chloride secretion. To date, the post-translational signal responsible for NKCC1 internalization during PKC activation remains unknown. Similarly, the fate of NKCC1 in the endocytic pathway has not been elucidated. In the present study, we investigated the role of ubiquitin as post-translational signal responsible for NKCC1 internalization. Experiments were performed on the human colonic T84 epithelial. Cells were incubated with or without 100 nM phorbol 12-myristate 13-acetate (PMA), a PKC activator, or in presence of 100 µM carbachol, a M3 muscarinic receptor agonist, for 15 or 30 min. NKCC1 ubiquitination was tested by western blot after immunoprecipitating NKCC1. Our preliminary results show that PMA and carbachol induced an increase of NKCC1 ubiquitination compare to control. In addition, blocking the lysosome with 5 µM NH4Cl did not prevent NKCC1 degradation during PKC activation by PMA. Our results suggest that PKC induces NKCC1 internalization through a ubiquitin-dependent pathway and may target NKCC1 for degradation in a lysosomal independent-manner.
Klosa, Payton; Dix, Marie; Bazaldua, Amanda; and Hughes, Emily, "PKC activation induces ubiquitination of the Na-K-2Cl cotransporter 1 in the human colonic epithelial cells T84" (2019). Symposium on Undergraduate Research and Creative Expression (SOURCE). 777.