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A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.

ABSTRACT: The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.

SUBMITTER: Li X

PROVIDER: S-EPMC2877505 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.

Li Xinli X Su Vivian V Kurata Wendy E WE Jin Chengshi C Lau Alan F AF

The Journal of biological chemistry 20071213 9

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indi ...[more]

PMID: 18079109

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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

A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.

Publications

A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.

Similar Datasets

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