IRBIT a Master Regulator of Cell Physiology

(excerpt) Hormones and neurotransmitters regulate cell functions by binding to their receptors, which activate intracellular signaling and produce the physiological response [1]. There are several intracellular pathways, including but not limited to, leading to the activation of protein kinases, phosphatases and increase in intracellular calcium (Ca2+) [1]

IRBIT a Master Regulator of Cell Physiology

(excerpt) Hormones and neurotransmitters regulate cell functions by binding to their receptors, which activate intracellular signaling and produce the physiological response [1]. There are several intracellular pathways, including but not limited to, leading to the activation of protein kinases, phosphatases and increase in intracellular calcium (Ca2+) [1]

Defining Protein Kinase C Substrates Involved in the Na-K-2Cl Cotransporter 1 Endocytosis in Colonic Epithelial Cells

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

Elucidating protein kinase C substrates involved in internalization of the Na+-K+-2Cl− cotransporter in the human colonic crypt cells T84

Elucidating protein kinase C substrates involved in internalization of the Na+-K+-2Cl− cotransporter in the human colonic crypt cells T84 Jesse Smallwood, Natasa Petreska, Alexander Ahlgrim and Patrice G Bouyer, Valparaiso University Fluid secretion in the lungs and colon lubricates and protects cells lining the cavity of those organs. Dysregulation of fluid secretion is the hallmark of diseases such as: cystic fibrosis (defective secretion) or secretory diarrhea (excessive secretion). In epithelial cells, the driving force for fluid secretion is active transcellular chloride secretion, with the basolateral Na-K-2Cl cotransporter 1 (NKCC1) pumping chloride inside the cell for its secretion by apical chloride channels. Previous studies have highlighted the critical role of NKCC1 in the regulation of chloride secretion in the colon. We have demonstrated that activation of the protein kinase C (PKC) causes a rapid internalization of NKCC1, hence, blunting chloride secretion. Nonetheless, the cellular and molecular details of PKC-mediated NKCC1 internalization remain unclear. Myristoylated, alanine-rich C kinase substrate (MARCKS) and α-adducin are two known PKC substrates participating in PKC-mediated endocytosis in other cells, but their potential role in NKCC1 endocytosis has not been tested. In the present study, we showed by Western blot that α-adducin and MARCKS are expressed in the human colonic crypt cells T84. In addition, using phorbol 12-myristate 13-acetate (PMA), an activator of the conventional and novel PKC caused both α-adducin and MARCKS to be phosphorylated and represent therefore PKC substrates in T84 cells. To test whether α-adducin or MARCKS binds to NKCC1 during PKC activation we performed immunoprecipitation experiments. After PKC activation by PMA, we found that immunoprecipitating NKCC1 pulled down MARCKS and α-adducin. In conclusion, our preliminary data strongly suggest that α-adducin and MARCKS are involved in NKCC1 internalization during PKC activation

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway