Defining Protein Kinase C Substrates Involved in the Na-K-2Cl Cotransporter 1 Endocytosis in Colonic Epithelial Cells
Arts and Sciences
Fluid secretion in the lungs and intestine lubricates and protects cell lining in these organs’ cavities and defect in secretion is observed in situations such as cystic fibrosis or Sjögren’s syndrome. In colonic epithelial cells, the driving force for fluid secretion is an active transcellular chloride secretion, and the basolateral Na-K-2Cl cotransporter 1 (NKCC1) represents the main transporter pumping chloride inside the cell for its secretion by apical chloride channels. Human colonic T84 cells are used to characterize cellular and molecular mechanisms regulating chloride secretion. Previously it was shown that activated protein kinase C (PKC) epsilon and delta, cause internalization of NKCC1, thus blunting chloride secretion. Today, PKC-mediated internalization of NKCC1 is not fully understood. Myristoylated, alanine-rich C kinase substrate (MARCKS) and α-adducin are known PKC substrates participating in PKC-mediated endocytosis in other cells, but their potential role in NKCC1 endocytosis has not been tested. Here, we show by western blot and immunocytochemistry that T84 cells express both MARCKS and α-adducin and display cytosolic and some plasma membrane localization. Also, activation of PKCs by phorbol 12-myristate 13-acetate (PMA) induced α-adducin and MARCKS phosphorylation in T84 cells. We have performed co-immunoprecipitation experiment to determine whether MARCKS and/or α-adducin participate in NKCC1 internalization during PKC activation. T84 cells exposed to PMA revealed that immunoprecipitating NKCC1 pulled down α-adducin and MARCKS. Finally, using T84 cells and Mardin Darby Canine Kidney cells expressing eGFP-NKCC1 we found that PMA causes co-localization of α-adducin with NKCC1. In conclusion, our data support the hypothesis that α-adducin and MARCKS may participate in NKCC1 endocytosis during PKC activation.
Petreska, Natasa and Smallwood, Jesse, "Defining Protein Kinase C Substrates Involved in the Na-K-2Cl Cotransporter 1 Endocytosis in Colonic Epithelial Cells" (2016). Symposium on Undergraduate Research and Creative Expression (SOURCE). 532.