Title

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

Primary Submission Contact

Jesse Smallwood

Faculty Sponsor

Patrice Bouyer

Faculty Sponsor Email Address

patrice.bouyer@valpo.edu

College

Arts and Sciences

Department/Program

Biology/Chemistry

Document Type

Poster Presentation

Date

Fall 10-24-2016

Abstract

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.

Biographical Information about Author(s)

Jesse Smallwood and Natasa Petreska have been working on this project for the past four and five semesters respectively,. All of the students involved in this project have heard this project presented at the Biology Colloquium at some point.

Additional Presentation Information

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