The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog

Heather S. Duffy, Ionela Iacobas, Kylie Hotchkiss, Bethany J. Hirst-Jensen, Alejandra Bosco, Nadine Dandachi, Rolf Dermietzel, Paul L. Sorgen, David C. Spray

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.

Original languageEnglish (US)
Pages (from-to)9789-9796
Number of pages8
JournalJournal of Biological Chemistry
Volume282
Issue number13
DOIs
StatePublished - Mar 30 2007

Fingerprint

Connexins
Hooks
Liver
Membranes
Tumor Suppressor Proteins
Hepatocytes
Membrane Microdomains
src Homology Domains
Confocal microscopy
Confocal Microscopy
Yeast
Cell Cycle
Membrane Proteins
Proteins
Yeasts
Western Blotting
Cells
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog. / Duffy, Heather S.; Iacobas, Ionela; Hotchkiss, Kylie; Hirst-Jensen, Bethany J.; Bosco, Alejandra; Dandachi, Nadine; Dermietzel, Rolf; Sorgen, Paul L.; Spray, David C.

In: Journal of Biological Chemistry, Vol. 282, No. 13, 30.03.2007, p. 9789-9796.

Research output: Contribution to journalArticle

Duffy, HS, Iacobas, I, Hotchkiss, K, Hirst-Jensen, BJ, Bosco, A, Dandachi, N, Dermietzel, R, Sorgen, PL & Spray, DC 2007, 'The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog', Journal of Biological Chemistry, vol. 282, no. 13, pp. 9789-9796. https://doi.org/10.1074/jbc.M605261200
Duffy, Heather S. ; Iacobas, Ionela ; Hotchkiss, Kylie ; Hirst-Jensen, Bethany J. ; Bosco, Alejandra ; Dandachi, Nadine ; Dermietzel, Rolf ; Sorgen, Paul L. ; Spray, David C. / The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 13. pp. 9789-9796.
@article{af5ec9443ed04f90b8bb2e993dcbf240,
title = "The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog",
abstract = "Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.",
author = "Duffy, {Heather S.} and Ionela Iacobas and Kylie Hotchkiss and Hirst-Jensen, {Bethany J.} and Alejandra Bosco and Nadine Dandachi and Rolf Dermietzel and Sorgen, {Paul L.} and Spray, {David C.}",
year = "2007",
month = "3",
day = "30",
doi = "10.1074/jbc.M605261200",
language = "English (US)",
volume = "282",
pages = "9789--9796",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "13",

}

TY - JOUR

T1 - The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog

AU - Duffy, Heather S.

AU - Iacobas, Ionela

AU - Hotchkiss, Kylie

AU - Hirst-Jensen, Bethany J.

AU - Bosco, Alejandra

AU - Dandachi, Nadine

AU - Dermietzel, Rolf

AU - Sorgen, Paul L.

AU - Spray, David C.

PY - 2007/3/30

Y1 - 2007/3/30

N2 - Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.

AB - Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.

UR - http://www.scopus.com/inward/record.url?scp=34248224743&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34248224743&partnerID=8YFLogxK

U2 - 10.1074/jbc.M605261200

DO - 10.1074/jbc.M605261200

M3 - Article

VL - 282

SP - 9789

EP - 9796

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 13

ER -