A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.
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.
Li
Xinli
X
Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, Honolulu, Hawaii 96813, USA.
Su
Vivian
V
Kurata
Wendy E
WE
Jin
Chengshi
C
Lau
Alan F
AF
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D016601:RNA-Binding Proteins; D051381:Rats; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D018031:Connexin 43; C500216:GJA1 protein, human; D009687:Nuclear Proteins; C498506:PSMD4 protein, human; C490781:Psmd4 protein, mouse; D016601:RNA-Binding Proteins; C421907:UBQLN4 protein, human; D046988:Proteasome Endopeptidase Complex; C119011:26S proteasome non-ATPase regulatory subunit 13