Dataset Information

Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes.

ABSTRACT: Cultured cardiomyocytes were used to study the turnover and post-translational modification of connexin43 (Cx43), a major gap junction protein in neonatal cardiac myocytes. Immunoprecipitation of [35S]Met-labelled lysates with anti-Cx43 antibodies followed by analysis using SDS/PAGE and fluorography revealed two bands, one at 40 kDa and the other at 42 kDa. Alkaline phosphatase treatment of [35S]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [32P]P1 incorporation into the 42 kDa band, but not into the band at 40 kDa. In addition, another alkaline phosphatase-sensitive phosphorylated form of Cx43 was identified at 44 kDa. In pulse-chase experiments, the half-life of Cx43 in cardiomyocytes was determined to be 1-2 h. Furthermore, the turnover rate of phosphate groups on Cx43 was found to be experimentally defined by the half-life of the protein. The observation that phosphate groups can remain with the protein throughout its life is consistent with the finding that in isolated adult rat heart gap junction plaques, Cx43 is primarily phosphorylated. We postulate that the rapid turnover of Cx43 and its multiple sites of phosphorylation play important roles in the regulation of cell-cell communication via gap junctions.

SUBMITTER: Laird DW

PROVIDER: S-EPMC1149880 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin-Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

Dataset Information

Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets