Connexins are transmembrane proteins that create intercellular aqueous channels connecting the cytoplasms of adjacent cells. Gap junctions permit the passive transfer of small molecules directly from cell to cell and play a critical role in the function of normal organs, such as the depolarizing current in the heart, electrotonic conduction in nerves, metabolic homeostasis in the lens and ear, and embryonic development. Disruption of gap junction function by mutations in connexin genes causes several human diseases, including peripheral neuropathy (CMTX), deafness, cataract formation, skin diseases, and may contribute to the development and maintenance of cancer. Understanding the mechanisms that regulate the function of these unique intercellular channels is an important goal. This proposal will continue our investigation of the regulation of connexin43 (Cx43) channels by phosphorylation induced by the Src tyrosine kinase oncoprotein and begin the exploration of another regulatory mechanism involving the interaction of novel proteins with Cx43.
Specific Aim 1 will investigate the involvement of the P-Y247 site and other domains of Cx43 in the Src-induced disruption of Cx43 gap junction channels.
Specific Aim 2 will investigate the interaction between Cx43 and CIP75 and the role CIP75 may play in the proteasomal degradation of Cx43 in mammalian cells.
Specific Aim 3 will investigate the interaction between Cx43 and CIP85 and the possible role of CIP85 in the vesicular trafficking and degradation of Cx43. The novel CIP75 and CIP85 proteins contain multiple interaction domains that may contribute to the modulation of Cx43 levels and function.
These aims will be accomplished by: in vitro and cellular protein-protein interaction studies, subcellular localization of proteins in fixed and live cells by laser confocal microscopy, analysis of site-specific or deletion mutants, inhibition of protein function by siRNA, dominant negative inhibitors, and the analysis of exogenous wild-type and mutant proteins introduced into cells by DMA transfection, retroviral infection, or direct protein transduction.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA052098-17
Application #
7404563
Study Section
Special Emphasis Panel (ZRG1-SSS-U (04))
Program Officer
Ault, Grace S
Project Start
1990-12-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
17
Fiscal Year
2008
Total Cost
$274,628
Indirect Cost
Name
University of Hawaii
Department
Type
Organized Research Units
DUNS #
965088057
City
Honolulu
State
HI
Country
United States
Zip Code
96822
Kopanic, Jennifer L; Schlingmann, Barbara; Koval, Michael et al. (2015) Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75). Biochem J 466:571-85
Su, Vivian; Lau, Alan F (2014) Connexins: mechanisms regulating protein levels and intercellular communication. FEBS Lett 588:1212-20
Su, Vivian; Hoang, Christina; Geerts, Dirk et al. (2014) CIP75 (connexin43-interacting protein of 75 kDa) mediates the endoplasmic reticulum dislocation of connexin43. Biochem J 458:57-67
Cochrane, Kimberly; Su, Vivian; Lau, Alan F (2013) The connexin43-interacting protein, CIP85, mediates the internalization of connexin43 from the plasma membrane. Cell Commun Adhes 20:53-66
Su, Vivian; Lau, Alan F (2012) Ubiquitination, intracellular trafficking, and degradation of connexins. Arch Biochem Biophys 524:16-22
Dunn, Clarence A; Su, Vivian; Lau, Alan F et al. (2012) Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability. J Biol Chem 287:2600-7
Su, Vivian; Cochrane, Kimberly; Lau, Alan F (2012) Degradation of connexins through the proteasomal, endolysosomal and phagolysosomal pathways. J Membr Biol 245:389-400
Su, Vivian; Nakagawa, Reid; Koval, Michael et al. (2010) Ubiquitin-independent proteasomal degradation of endoplasmic reticulum-localized connexin43 mediated by CIP75. J Biol Chem 285:40979-90
Kieken, Fabien; Spagnol, Gaƫlle; Su, Vivian et al. (2010) NMR structure note: UBA domain of CIP75. J Biomol NMR 46:245-50
Cochrane, Kimberly; Berestecky, John M; Kitamura, Carolynn et al. (2009) Monoclonal antibodies against the connexin43-interacting protein CIP85. Hybridoma (Larchmt) 28:355-61

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