Imaging kinase: connexin43 interactions using phosphorylation activity biosensors and phospho‐specific antibodies (802.2)

Abstract

Connexin43 (Cx43) is a ubiquitous gap junction protein with widespread tissue expression, serving important functions in cell homeostasis and signaling. The C‐terminus of Cx43 contains multiple phosphorylation sites and protein binding domains that are involved in regulation of connexin trafficking and channel gating. Several kinases phosphorylate Cx43 (Protein Kinase A, Protein Kinase C, Akt (also known as Protein Kinase B), Src, CK1, MAPK) at specific serines or tyrosines. We used kinase biosensors,for dynamic readouts of function, and combined it with image analysis to obtain spatial information. We fused a genetically encoded fluorescence resonance energy transfer‐based reporter for PKC delta activity, delta C kinase activity reporter (dCKAR) to the C‐terminus of Cx43 (Cx43‐dCKAR) and expressed it in Cos7 cells. Similarly, we genetically appended an activity reporter for Protein Kinase A (AKAR‐CR) to Cx43 and expressed it in Cos7 cells. Time lapse imaging of stimulated PKC resulted in phosphorylation of Ser368 Cx43‐dCKAR channels and discrete domains in the center of the gap junction, followed by internalization and degradation. Phosphorylated Cx43‐AKAR‐CR was predominantly found in the Golgi apparatus and to a lesser extent around the plaque edges. Thus, the spatio‐temporal localizations of PKC and PKA phosphorylated Cx43 represent degradative and synthetic pathways, respectively.Grant Funding Source: Supported by NIH grants GM072881, GM043154, GM103412