Lin R, Warn-Cramer BJ, Kurata WE, Lau AF.. v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication. J Cell Biol 154: 815-827

Molecular Carcinogenesis Section, Cancer Research Center of Hawaii, Honolulu, HI 96813, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2001; 154(4):815-27. DOI: 10.1083/jcb.200102027
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ABSTRACT The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.

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Available from: Alan F Lau, Sep 28, 2015
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    • " , a proline - rich region ( amino acids 274 – 284 ) and then phosphorylates tyrosine 265 providing an SH2 - binding domain with the subse - quent phosphorylation at tyrosine 247 ( Fig . 1C ) ( Kanemitsu et al . , 1997 ) . As a consequence of these phosphorylations gap junctional intercellular communica - tion is reduced ( Swenson et al . , 1990 ; Lin et al . , 2001 ; Giepmans et al . , 2001a ) and Cx43 turnover is initiated ( Solan and Lampe , 2014 ) . Interestingly , NMR studies show that although binding of Src is confined to the SH3 - binding domain of Cx43CT , conformational changes resulting from this binding extend for large distances along the Cx43CT ( Sorgen et al . , 2004 ) affecting the "
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    ABSTRACT: Connexin43 (Cx43) as a building block of gap junction channels and hemichannels exerts important functions in astrocytes. When these cells acquire a malignant phenotype Cx43 protein but not mRNA levels are downregulated, being negligible in high-grade astrocytoma or glioblastoma multiforme, the most common and deadliest of malignant primary brain tumours in adults. Some microRNAs associated to glioma target Cx43 and could explain the lack of correlation between mRNA and protein levels of Cx43 found in some high-grade astrocytomas. More importantly, these microRNAs could be a promising therapeutic target. A great number of studies have confirmed the relationship between cancer and connexins that was proposed by Loewenstein more than 40 years ago, but these studies have also revealed that this is a very complex relationship. Indeed, restoring Cx43 to glioma cells reduces their rate of proliferation and their tumorigenicity but this tumour suppressor effect could be counterbalanced by its effects on invasiveness, adhesion and migration. The mechanisms underlying these effects suggest the participation of a great variety of proteins that bind to different regions of Cx43. The present review focuses on an intrinsically disordered region of the C-terminal domain of Cx43 in which converges the interaction of several proteins, including the proto-oncogene Src. We summarize data that indicate that Cx43-Src interaction inhibits the oncogenic activity of Src and promotes a conformational change in the structure of Cx43 that allosterically modifies the binding to other important signalling proteins. As a consequence, crucial cell functions, such as proliferation or migration, could be strongly affected. We propose that the knowledge of the structural basis of the antitumorigenic effect of Cx43 on astrocytomas could help to design new therapies against this incurable disease. Copyright © 2015. Published by Elsevier Ltd.
    Neuroscience 02/2015; DOI:10.1016/j.neuroscience.2015.02.029 · 3.36 Impact Factor
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    • "A study on Cx43 mutants has implied that phosphorylation of Tyr265 alone is not sufficient for complete gap junction closure. Instead, phosphorylation of both Tyr265 and Tyr247 are required to disrupt metabolic coupling through Cx43 gap junction channels (Lin et al., 2001). Together, these results support a model for v-Src induced Cx43 tyrosine phosphorylation, where interaction is initiated by binding of the v-Src SH3 domain to the Pro274-Pro284 region of Cx43. "
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    ABSTRACT: Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins.
    Frontiers in Pharmacology 10/2013; 4:130. DOI:10.3389/fphar.2013.00130 · 3.80 Impact Factor
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    • "However, contrary results have been published by Swenson, et al. (1990) and Lin et al. (2001), who showed that Y265 and Y247 are required for v-src induced disruption of coupling in both Xenopus oocytes (Swenson et al. 1990) and in a mouse Cx43 knockout cell line transfected with different Cx43 mutants (Lin et al. 2001). In the latter study, the ERK1/2 phosphorylation sites (S255, 279, and 282) were found not to be required for closure. "
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    ABSTRACT: Attenuation in gap junctional coupling has consistently been associated with induction of rapid or synchronous cell division in normal and pathological conditions. In the case of the v-src oncogene, gating of Cx43 gap junction channels has been linked to both direct phosphorylation of tyrosines (Y247 and 265) and phosphorylation of the serine targets of Erk1/2 (S255, 279 and 282) on the cytoplasmic C-terminal domain of Cx43. However, only the latter has been associated with acute, rather than chronic, gating of the channels immediately after v-src expression, a process that is mediated through a "ball-and-chain" mechanism. In this study we show that, while ERK1/2 is necessary for acute closure of gap junction channels, it is not sufficient. Rather, multiple pathways converge to regulate Cx43 coupling in response to expression of v-src, including parallel signaling through PKC and MEK1/2, with additional positive and negative regulatory effects mediated by PI3 kinase, distinguished by the involvement of Akt.
    Journal of Membrane Biology 09/2012; 245(8):495-506. DOI:10.1007/s00232-012-9500-0 · 2.46 Impact Factor
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