Investigation of Protein Kinase C-mediated internalization of the Na+-K+-2Cl– cotransporter 1 in Madin-Darby Canine Kidney cells

Faculty Sponsor

Patrice Bouyer

Department/Program

Biology Department

Presentation Type

Poster Presentation

Symposium Date

Summer 7-23-2025

Abstract

In the colon, the Cl- driven fluid secretion is dependent on the Na+-K+-2Cl– cotransporter 1 (NKCC1). The effect of prolonged activation of PKC on NKCC1 expression is unknown; we used immunoblotting to test this effect. In preliminary experiments, prolonged activations of PKC on NKCC1 resulted in a decrease in expression. While ubiquitination is a known signal for degradation, we tested whether ubiquitination is a signal for NKCC1 internalization using fluorescence microscopy in Madin-Darby Canine Kidney cells expressing eGFP-NKCC1. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC, and PYR-41 to inhibit the ubiquitin ligase E1. We quantified the number of endocytosed vesicles using ImageJ software. One-way ANOVA shows a significant difference among our conditions (P < 0.001). A Tukey’s post-hoc test shows no significant difference between control (0.9 ± 0.2 vesicles/cell, n = 89) and DMSO (1.4 ± 0.4, n=41, p=0.2). PMA (8.3 ± 1.1, n = 27) significantly increases the number of vesicles/cells compared to control (P < 0.001). Unexpectedly, PYR (7.0 ± 0.7, n = 77) significantly increased the number of vesicle counts (P < 0.001), and PYR + PMA (8.7 ± 1.5, n = 83) was not statistically different from PYR or PMA (P = 0.3 and 0.8, respectively). We can infer that inhibiting ubiquitination reveals a ubiquitin-independent mechanism of NKCC1 internalization. While degradation is present during PKC activation, it cannot be linked to ubiquitination due to inconclusive microscopy results.

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