Is Ubiquitination Responsible for the Internalization of NKCC1 During PKC Activation?

Faculty Sponsor

Patrice Bouyer

College

Arts and Sciences

Department/Program

Biology Department

Presentation Type

Poster Presentation

Symposium Date

Summer 7-24-2024

Abstract

In the colon, the Cl-driven fluid secretion is dependent on the basolateral Na+ K+ Cl- cotransporter 1 (NKCC1). Activation of protein kinase C (PKC) causes the internalization of NKCC1. However, the PKC-dependent signal causing NKCC1 internalization is not known. We hypothesize that ubiquitin may be the signal responsible. We used Madin-Darby Canine Kidney (MDCK) cells expressing eGFP-tagged NKCC1 to monitor its internalization using fluorescent microscopy. We used phorbol 12-myristate 13-acetate (PMA) which activates PKC and PYR-41 (dissolved in DMSO) to inhibit ubiquitin ligase. Finally, we combined both PYR+PMA to test whether inhibiting ubiquitin ligase will reduce the effect of PKC on NKCC1 internalization. We used FIJI to count the number of internalized NKCC1 vesicles and the number of nuclei. Our results, calculated as vesicles per cell, were as follows: control (3.6±0.6 vesicles/cell, n=18), DMSO (2.4±0.5, n=18), PMA (12.6±3.7 vesicles/cell, n=12), PYR (5.6±0.8 vesicles/cell, n=30), and PYR+PMA (4.5±0.4 vesicles/cell, n=18). A one-way ANOVA found a significant difference between our conditions (p=<0.001). A Tukey’s post-hoc test showed that PMA significantly increased internalization compared to control (p=<0.001), PYR+PMA had significantly less internalization than PMA alone (p=<0.001), and control vs PYR was not significant (p=0.7). Our results suggest that ubiquitin may be the signal responsible for the internalization of NKCC1 during PKC activation.

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