Potential role of the human Ha‐<i>ras</i> oncogene in the inhibition of gap junctional intercellular communication

El‐Fouly, Mohamed H.; Trosko, James E.; Chang, Chia‐Cheng; Warren, Stephen T.

doi:10.1002/mc.2940020305

Cited by 39 publications

(14 citation statements)

References 27 publications

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“…On the other hand, rus and rut while reducing either homologous or heterologous GJIC [19][20][21] (unpublished observations), appear to have affected the protein in a quite different manner. The connexin protein in the rus-transformed cells does not seem to be present in the nucleus and appears only occasionally in plasma membran+associated plaques.…”

Section: Resultsmentioning

confidence: 89%

“…Fischer 344 rat liver epithelial cells (WB-F344) [18] transfected or transduced with a variety of oncogenes associated with the neomycin drug-resistance marker (WB-neu, WB-src, WB-myc, WB-ras, and WB-myc/ ras) or with the neomycin drug-resistance marker alone (WB-neo) were used for this study [20][21][22]241. The mutant cell line WB-aB1 was derived from WB-F344 and isolated by Oh et al on the basis of its lack of GJIC [23].…”

Section: Cell Culturementioning

confidence: 99%

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Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

et al. 1996

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Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and t o what degree these transformations disrupted gap-junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed bysrc, neu, ras, and myclras all displayed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src-and neu-transformed cells, but not in the ras-and myclras-transformed cells. Nuclei isolated from WB-neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB-neo control cells, supporting the idea that the nuclear-localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC-deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane-localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein.Cells expressing rat previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showed a marked reduction in GJlC and plasma membrane-associated Cx43 immunostaining. These data suggest that altered localization of the gap-junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJlC in neoplastically transformed rat liver epithelial cells.

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Section: Resultsmentioning

confidence: 89%

Section: Cell Culturementioning

confidence: 99%

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

et al. 1996

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“…The protein product of the ras oncogene family is the pp21 vras with G protein-like activity (192). The Harvey ras oncogene (V-Ha-ras) pp21 v " Haras inhibits junctional communication (193) and metabolic cooperation (194). Interestingly, in our hands a temperature-sensitive v-kiras transformed cell line, also expressing a pp21 vk '" ras Glike protein, does not appear to affect communication in a detectable manner (our unpublished observation).…”

Section: May 1990mentioning

confidence: 78%

The Hormone-Induced Regulation of Contact-Dependent Cell-Cell Communication by Phosphorylation*

1990

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Although there is insufficient evidence to propose an elaborate paradigm for the regulation of connexon gating, a simple model emerges from results of studies done to date. Basically, this centers around the most consistent findings: namely, that activation of pkA has an enhancing effect on cell communication while activation of pkC decreases that process. This fits well the reported phenomena associated with gap junctions, particularly those involving growth control. For example growth factors, including tumor promoters which work via pkC, usually reduce cell-cell communication whereas agents that decrease growth often raise cellular cAMP levels, which can lead to increased communication. It can be argued that this model is too simple because it fails to take into account other intracellular agents that are thought to alter junctional gating: cytoplasmic acidification, cellular free Ca2+, tyrosine protein kinases, and tentatively, pkG. Proton and Ca2+ transporting systems are mainly activated by serine/threonine protein kinases such as pkA and pkC. Some ion channels are not regulated by phosphorylation but instead are modulated by other ions. However, at the moment there is no evidence as to which ion-specific channels mediate the changes in cellular pH or Ca2+ that cause a loss in communication. Neither is it known whether pH or Ca2+ levels are in vivo regulators of the junctions. This is especially so as fairly high levels of injected Ca2+ pass through the gap junctions of viable cells. The role of tyrosine protein kinases in connexon gating may involve interaction with the pkA and pkC regulatory cascades. For example, the pkA inhibitor protein (pkI) is 80-90% inactivated when tyrosine-phosphorylated by the EGF receptor or pp50v-src (D. Walsh, personal communication). In this situation, activity of the C subunit of pkA could be enhanced, or the lifetime of its catalytic activity extended. In some systems, pp60v-src is known to activate the pkC pathway. Thus, tyrosine protein kinases may invoke pkA and pkC pathways; however, the amplitude of enzyme activation and the temporal kinetics of this process are unknown. The fact that gap junctions are regulated at the transcription level and probably at the protein level by protein kinases is of major interest. This is especially so as the only known molecular mechanism that gap junctional communication mediates is the activation of cAMP-dependent protein kinases by hormone-induced signals passed from receptor-bearing cells to receptorless partners.

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“…Reduced GJC is a characteristic of cells transformed by several oncogenes (24), including SV-40 (33), polyomavirus middle T antigen (34), v-ras (35,36), v-mos (37), and v-fps (38), as well as v-src. The effects of Src on GJC have been studied extensively (17-20, 30, 32, 39 -41).…”

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confidence: 99%

Tyrosine Phosphorylation of Connexin 43 by v-Src Is Mediated by SH2 and SH3 Domain Interactions

Kanemitsu

Loo

Simon

et al. 1997

Journal of Biological Chemistry

185

160

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Reduction of gap junctional communication in v-The Rous sarcoma virus transforming protein, v-Src, and its cellular homologue, c-Src, are protein tyrosine kinases associated with the plasma membrane (1). Expression of v-Src leads to neoplastic cell transformation, accompanied by increased tyrosine phosphorylation of cellular proteins (2-4). Signaling through Src is due, in part, to protein-protein interactions mediated by SH2 1 and SH3 domains (5-7). Mutations in either the SH2 or SH3 domain of c-Src can lead to increased tyrosine kinase activity and oncogenic potential (5,8,9). SH2 domains bind Tyr(P) residues in target proteins. The specificity of binding is determined by residues immediately C-terminal to the Tyr(P) (6, 10, 11). SH3 domains bind short proline-rich peptide motifs (6,(12)(13)(14). Studies using phage display libraries to identify peptides that bind SH3 domains showed that a minimal PXXP consensus sequence is required for binding (15,16).In addition to playing a role in cell growth and transformation, the Src protein tyrosine kinase has been implicated in regulating GJC (17)(18)(19)(20). Gap junctions are membrane channels which mediate the intercellular passage of ions, second messengers, and small molecules (21). It has been proposed that growth regulatory molecules pass between cells through gap junctions (22)(23)(24)(25).Gap junctions are formed by specialized proteins termed connexins, arranged in the cell membrane so that each connexin has four membrane spanning regions, a cytoplasmic loop, two extracellular loops, and cytoplasmic N-and C-terminal ends. Among the 13 connexin family members identified to date, the C-terminal tail is the most divergent region. In some cases this region contains consensus protein kinase phosphorylation sites (26 -28). Connexins 32 and 43 are phosphoproteins (29 -32), however, Cx43 is phosphorylated on tyrosine by Src, but Cx32 is not (32). Comparison of the amino acid sequences of connexins 32 and 43 revealed that putative SH3-binding regions and tyrosine phosphorylation sites in Cx43 were absent in connexin 32 (26).Reduced GJC is a characteristic of cells transformed by several oncogenes (24), including SV-40 (33), polyomavirus middle T antigen (34), v-ras (35, 36), v-mos (37), and v-fps (38), as well as v-src. The effects of Src on GJC have been studied extensively (17-20, 30, 32, 39 -41). Several lines of evidence suggest that tyrosine phosphorylation of Cx43 is important in the regulation of GJC by v-Src. First, cells infected with temperaturesensitive Rous sarcoma virus show reduced levels of GJC (17,19), correlated with a rapid increase in tyrosine phosphorylation of Cx43 (40), at the permissive temperature. Second, expression of v-Src in communication-competent paired Xenopus oocytes expressing Cx43, but not in oocytes expressing Cx32, leads to reduced GJC, which depends on phosphorylation of Cx43 on tyrosine 265 (32). Finally, purified Src phosphorylates Cx43 in vitro at sites that are phosphorylated in vivo in v-src transformed Rat-1 fibroblasts (41)...

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Potential role of the human Ha‐ras oncogene in the inhibition of gap junctional intercellular communication

Cited by 39 publications

References 27 publications

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

The Hormone-Induced Regulation of Contact-Dependent Cell-Cell Communication by Phosphorylation*

Tyrosine Phosphorylation of Connexin 43 by v-Src Is Mediated by SH2 and SH3 Domain Interactions

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