Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Joshua Dawson - University of Washington
Co-Author(s): Joell Solan, Paul Lampe
Gap Junctions (GJ) are specialized portions of cell membranes that connect adjacent cells and are critical for synchronous electrical signaling in the heart. Therefore, better understanding of GJs can lead to more effective treatment for individuals with arrhythmia. GJs are a collection of channels that pass small molecules and metabolites between cells. They are composed of connexin proteins, the most common being Connexin 43 (Cx43). Kinases phosphorylate Cx43 to control the assembly and disassembly of GJs. Specifically, Protein Kinase C (PKC) and Extracellular Signal-Regulated Kinase (ERK1/2) phosphorylate Cx43 during assembly and disassembly and are activated during cardiac ischemia. PKC phosphorylates amino acid S368 on Cx43, which changes channel permeability, while residues S279/282 can be phosphorylated by ERK1/2 which results in channel closure. Lastly, it is known that S262 is phosphorylated under conditions which lead to GJs disassembly. However, both PKC and ERK1/2 have been implicated as the S262 kinase, in part because typical reagents that activate PKC also result in activation of ERK1/2. Our study is designed to clarify which of these kinases phosphorylates S262 by quantifying phosphorylation of Cx43 at S368, and S262 in the presence of PKC and ERK1/2 kinase activators and inhibitors. By examining S262 phosphorylation using different combinations of these reagents, we should be able to discern which kinase phosphorylates S262 or, at least, whether this event correlates with phosphorylation on S368. Our data indicates that ERK1/2 is the kinase involved in S262 phosphorylation due to the greatly diminished phosphorylation activity observed when ERK1/2 inhibitors were introduced; and that the PKC activator, TPA, in reality plays a role in stimulating ERK1/2. I now want to find out which phosphorylation event is the most important aspect for gap junction disassembly between MAPK and PKC and perhaps if the two kinases work together. To investigate, I will make a S368A mutant that cannot be phosphorylated and monitor how the ability to phosphorylate S368 affects the disassembly process of gap junctions.
Funder Acknowledgement(s): Richard C. Goldstein Private Foundation
Faculty Advisor: Joell Solan, email@example.com
Role: I ran an SDS Page on B5 MDCK cell lines which were previously treated in cell culture with various kinase activators and inhibitors. I then used primary and secondary antibodies (conjugated to fluorescent markers) to quantified the ratio of phosphorylation on Connexin 43 using an Odyssey scanner to indicate which kinase was involved in phosphorylation of our target serine residues.