Connexin proteins span the membrane four times, such that the amino terminus, carboxyl terminus and an intracellular loop are formed on the cytoplasmic side of the membrane and two extracellular loops are formed on the outside surface of the cell. Most of the connexin proteins are phosphorylated in vivo, primarily on serine residues with smaller amounts of phosphothreonine or phosphotyrosine reported for some of the connexins. Connexin43 (Cx43) is one of the most ubiquitously expressed connexins and much is known about its phosphorylation. A basal level of serine phosphorylation on at least five sites appears to be required for the assembly and function of Cx43 gap junctions [1
]. An increasing number of studies have implicated growth factors, tumor promoters, oncogene protein kinases, hormones and inflammatory mediators in the regulation of gap junctional communication (GJC) through phosphorylation events occurring on the C-terminal portion of the protein, ~ aa 236–382 [6
]. A number of the serine kinases that target Cx43 in stimulated cells have been identified (reviewed in Ref. [14
]) including PKC (Ser368 and Ser372), MAP kinases (Ser255, Ser279, and Ser282), cdc2/cyclin B (Ser255), and casein kinase I (Ser325, Ser328 and Ser330). v-Src and v-Fps are two of the tyrosine kinases that have been shown to target tyrosine residues in Cx43.
Some of the phosphorylation-induced effects on Cx43 appear to occur directly, such as the tyrosine phosphorylation of Cx43 induced by v-Src and the physical interaction of Cx43 with v-Src that is associated with the disruption of GJC [15
]. These events lead to a chronic and dramatic down-regulation of GJC in v-Src cells [9
]. In other instances, a tyrosine kinase such as activated c-Src has been implicated in intracellular signaling pathways that influence Cx43 function, but tyrosine phosphorylation on Cx43 has not been detected and the disruption of GJC may be transient [18
]. This suggests that the actions of c-Src may be indirect in these cases and dependent on the activation of other kinases such as the Ser/Thr kinases, mitogen-activated protein (MAP) kinase and protein kinase C (PKC) to mediate the effects on GJC. In still other cases, protein phosphorylation does not appear to be directly involved in channel regulation, but rather there is a loss of Cx43 from gap junction plaques in the plasma membrane and this may be associated with a change in the levels of the Cx43 mRNA [22
]. The events that lead to these changes in Cx43 expression are not well understood. Activation of some intracellular signaling pathways has been shown to up-regulate GJC, generally through an increase in Cx43 protein and mRNA. These effects may be more long-term, occurring at 6 or more hours following the activation of intracellular signaling [27
]. The regulation of GJC appears to be complex and has been difficult to dissect, since multiple signaling pathways impinge on Cx43 in different cell types and under different stimulatory conditions. Some of the studies of the regulation of Cx43-mediated GJC by activated tyrosine protein kinases are described in the following sections of this review. They represent some of the spectrum of signaling events that are thought to be involved in the regulation of the Cx43 protein and/or GJC.
This review focuses on the tyrosine kinase-dependent phosphorylation of Cx43, because this represents one of the better-understood mechanisms involved in the regulation of GJC. Many growth factor receptors are tyrosine kinases, as are oncoprotein kinases such as v-Src and v-Fps. In addition, the inhibition of protein tyrosine-phosphatases [29
] has been shown to alter Cx43 regulation. Key intracellular signaling pathways utilize the actions of tyrosine protein kinases/tyrosine protein phosphatases to exquisitely regulate the transmission of molecular signals to downstream effectors and adaptor proteins [30
]. This is particularly important to the regulation of normal cell growth, hyperproliferative diseases such as cancer, gene transcription, the actions of the insulin receptor, and the synchronization of contraction in the heart and the uterus. Protein tyrosine kinases induce protein–protein interactions through the creation of phosphotyrosine binding sites for SH2- or PTB-containing proteins [31
]. This allows transmission of a signal from an activated growth factor receptor to downstream kinases that may mediate increased serine phosphorylation on Cx43. Activation and autophosphorylation of the EGF receptor tyrosine kinase provides phosphotyrosine-docking sites for SH2-containing effector molecules like Grb2 and Shc and leads to the activation of the Ras/Raf/MEK/MAP kinase signaling pathway. In the case of v-Src, the direct phosphorylation of Cx43 on Tyr265 provides a docking site for the SH2 domain of v-Src, leading to an enhanced interaction with Cx43 and the phosphorylation of additional tyrosine sites in Cx43 by the v-Src kinase. Many of the kinases that are important to intracellular signaling pathways induce the phosphorylation of Cx43 and may be involved in the regulation of GJC. The cytoplasmic C-terminal domain of Cx43 is particularly rich in potential kinase phosphorylation target sites and regulatory binding domains that may participate in directing Cx43's interactions with other cellular proteins. Although there is a considerable body of data available on the regulation of Cx43 and GJC by the tyrosine protein kinases, there is some controversy associated with this field and there are a number of areas where additional careful studies are warranted.
This review does not cover some of the other aspects of the regulation of gap junctions by protein phosphorylation. Several recent reviews have addressed some of these other areas of importance to this field, including the role of protein phosphatases in the regulation of gap junctions [14
]. We have provided some background on the subject of Cx43 phosphorylation, but have concentrated on the more recent developments and more-studied examples of the regulation of Cx43 by tyrosine protein kinases. We apologize in advance for the many studies that are not discussed here and that have also provided important contributions to the understanding of the regulation of GJC by connexin protein phosphorylation.