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Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes.

ABSTRACT: Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such as phosphatase and tensin homolog (PTEN). Our results showed that the antiproliferative effect of Cx43 is reduced when Csk and PTEN are silenced in glioma cells, suggesting the involvement of both enzymes. Confocal microscopy and immunoprecipitation assays confirmed that Cx43, in addition to c-Src, binds to PTEN and Csk in glioma cells transfected with Cx43 and in astrocytes. Pull-down assays showed that region 266-283 in Cx43 is sufficient to recruit c-Src, PTEN and Csk and to inhibit the oncogenic activity of c-Src. As a result of c-Src inhibition, PTEN was increased with subsequent inactivation of Akt and reduction of proliferation of human glioblastoma stem cells. We conclude that the recruitment of Csk and PTEN to the region between residues 266 and 283 within the C-terminus of Cx43 leads to c-Src inhibition.

SUBMITTER: Gonzalez-Sanchez A

PROVIDER: S-EPMC5226550 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes.

González-Sánchez Ana A Jaraíz-Rodríguez Myriam M Domínguez-Prieto Marta M Herrero-González Sandra S Medina José M JM Tabernero Arantxa A

Oncotarget 20160801 31

Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such ...[more]

PMID: 27391443

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Project description:In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

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Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes.

Publications

Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes.

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