Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda; Johnson, Keith R.; Mehta, Parmender P.

doi:10.1091/mbc.e12-07-0537

Cited by 69 publications

(93 citation statements)

References 98 publications

(88 reference statements)

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“…However, these S373D cells contain larger gap junctions and have excellent intercellular communication, therefore it seems unlikely that S373 phosphorylation necessarily and directly leads to gap junction turnover. Our preferred model would involve subsequent Cx43 phosphorylation at S279, S282 and/or S368, consistent with their reported roles in gap junction disassembly (Johnson et al, 2013;Lampe, 1994).…”

Section: Discussionsupporting

confidence: 71%

Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

Dunn

Lampe

2013

Journal of Cell Science

124

177

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The proteins that form vertebrate gap junctions, the connexins, are highly regulated and have short (,2 hour) half-lives. Phosphorylation of connexin43 (Cx43) affects gap junction assembly, channel gating and turnover. After finding dramatic effects on gap junctions with Akt inhibitors, we created an antibody specific for Cx43 phosphorylated on S373, a potential Akt substrate. We found S373 phosphorylation in cells and skin or heart almost exclusively in larger gap-junctional structures that increased dramatically after wounding or hypoxia. We were able to mechanistically show that Akt-dependent phosphorylation of S373 increases gap junction size and communication by completely eliminating the interaction between Cx43 and ZO-1. Thus, phosphorylation on S373 acts as a molecular 'switch' to rapidly increase gap-junctional communication, potentially leading to initiation of activation and migration of keratinocytes or ischemic injury response in the skin and the heart, respectively.

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Section: Discussionsupporting

confidence: 71%

Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

Dunn

Lampe

2013

Journal of Cell Science

124

177

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“…Another recent study in HeLa cells identified two tyrosine-based AP-2-binding motifs in the Cx43 C-terminus (Y 265 AYF and Y 286 KLV), one of which is a Src-dependent phosphorylation site, that cooperate to mediate gap junction endocytosis (Fong et al, 2013). Mutation of the Y 286 KLV motif alone in a yeast-two-hybrid screen (no post-translational modification) using the Cx43CT as bait against the AP-2 m2 subunit inhibited this interaction (Johnson et al, 2013). Taken together, these studies suggest that the phosphorylation of Y265 enables the interaction with AP-2 to mediate Cx43 endocytosis.…”

Section: Discussionmentioning

confidence: 99%

“…The control cells were treated in the same way without 1% Triton X-100. The coverslips were fixed and immunostained as described previously (Johnson et al, 2013).…”

Section: Scrape-loading Assaymentioning

confidence: 99%

Tyrosine phosphatase TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

Spagnol

Naslavsky

et al. 2014

Journal of Cell Science

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Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperaturesensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phosphospecific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCa and PKCd, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health.

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“…Stock cultures were maintained weekly by seeding 5 ϫ 10 5 cells per 10-cm dish in 10 ml of complete culture medium with a medium change at day 3 or 4 as described (18,20). New stocks were initiated after 10 passages.…”

Section: Methodsmentioning

confidence: 99%

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Katoch

Mitra

Ray

et al. 2015

Journal of Biological Chemistry

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Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

Abstract: In tumor cells that coexpress Cx43 and Cx26, the assembly of Cx43 is selectively impaired due to endocytosis. Assembly can be restored upon expressing a Cx43 sorting-motif mutant and mutants that cannot be phosphorylated on Ser-279 or Ser-282.

Cited by 69 publications

References 98 publications

Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

Tyrosine phosphatase TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

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