br Conflict of interest statement br Introduction

Conflict of interest statement
Introduction
Gap junction assembly
Induced disassembly of gap junctions The regulation of connexin and gap junction turnover are still not well understood. This is, in part, due to the fact that while the overall turnover of connexin molecules is fairly consistent and rapid across most cell types and conditions, the stability of individual gap junction plaques can be highly variable [43]. This is presumably due to altered kinetics of connexin assembly into and removal from a given gap junction. However, rapid restructuring followed by internalization of gap junctions occurs in most cell types in response to a variety of stimuli, including treatment with phorbol esters or growth factors [44], [45], oncogene activation [46], [47], ischemia [48], [49], [50], [51] and epidermal wounding [52]. These events are coincident with increased phosphorylation on specific serines representing a kinase program which coordinates induced gap junction disassembly [16]. Fig. 3, Fig. 4 show examples of a time course of acute disassembly, where waves of phosphorylation occur within minutes. This program appears to be initiated by phosphorylation of Cx43 on S373 [53]. Fig. 3 shows immunofluorescence of NRK cells that have been treated with TPA and immunolabeled with erbb2 inhibitor to total Cx43 (green) and Cx43 phosphorylated on S373 (red); after 5min of treatment (middle panel), we see a dramatic increase in S373 phosphorylation visible as yellow. This is coincident with rapid gap junction growth providing a temporary increase in channel open probability. Under control conditions we see a variety of sizes of gap junctions, while after 5min of TPA treatment most have taken on a larger more elongated appearance. By 30min, gap junctions are diminished and in various states of disassembly and internalization. Over this time course, the western blot in Fig. 4 shows sustained phosphorylation on S262 and S279/282 as channels close and gap junctions are broken down in response to EGF or TPA (see Fig. 3). However, there are differences in phosphorylation patterns where TPA mediated activation of PKC also results in phosphorylation at S368. We discuss these events in a stepwise (i.e., waves) fashion below.
Src and gap junction disassembly Activation of Src has long been shown to result in downregulation of gap junction communication and decreased Cx43 in gap junction plaques [46], [47]. Gap junction channel closure can occur relatively rapidly and there is still debate as to whether this is due to a direct effect of Src phosphorylation on Cx43 [73], [74] or due to Src activation of ERK1/2 and subsequent phosphorylation at ERK sites [75]. While Src is able to directly phosphorylate Cx43 on Y247 and Y265 to help mediate these effects there appears to be both cooperativity and redundancy via activation of other kinases by Src including ERK1/2 and PKC that could affect channel gating and gap junction stability [76]. During chronic expression of v-src, we find that while S262, S279/282 and S368 are phosphorylated on Cx43, Akt activity and consequent S373 phosphorylation are diminished [16]. While this seems distinct from the pathways described above where disassembly is initiated by phosphorylation on S373 and inhibition of Cx43:ZO-1 interaction, Src itself can displace ZO-1 binding from Cx43 [77], [78] perhaps bypassing the need for S373 phosphorylation under these conditions. Additionally, while Src activity is clearly associated with diminished gap junctions, a population of (closed) gap junctions often remains [75], [76]. Interestingly, we have shown by immunostaining that these gap junctions contain variable levels of pY247 [16] and these data suggest that pY247 may serve to mark specific areas, or subdomains, of a gap junction for disassembly, similar to what was observed with phosphorylated S368 and the PKC kinase sensor [67] (discussed in Section 3.3). The combination of diminished and non-functional gap junctions with a Cx43 phosphorylation pattern consistent with disassembly may indicate that Src activation destabilizes gap junctions through increased disassembly kinetics.