Phosphorylation of Cx43 residue Y313 by Src contributes to blocking the interaction with Drebrin and disassembling gap junctions.
Connexin43
Drebrin
Gap junctions
Intercellular communication
Phosphorylation
Src
Journal
Journal of molecular and cellular cardiology
ISSN: 1095-8584
Titre abrégé: J Mol Cell Cardiol
Pays: England
ID NLM: 0262322
Informations de publication
Date de publication:
01 2019
01 2019
Historique:
received:
26
02
2018
revised:
26
10
2018
accepted:
13
11
2018
pubmed:
19
11
2018
medline:
10
1
2020
entrez:
19
11
2018
Statut:
ppublish
Résumé
Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Y → F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.
Identifiants
pubmed: 30448479
pii: S0022-2828(18)31168-4
doi: 10.1016/j.yjmcc.2018.11.008
pmc: PMC8961861
mid: NIHMS1787602
pii:
doi:
Substances chimiques
Antibodies
0
Connexin 43
0
Neuropeptides
0
drebrins
0
Phosphotyrosine
21820-51-9
src-Family Kinases
EC 2.7.10.2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
36-49Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM103427
Pays : United States
Organisme : NIGMS NIH HHS
ID : P30 GM106397
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM072631
Pays : United States
Organisme : NCRR NIH HHS
ID : S10 RR027301
Pays : United States
Informations de copyright
Copyright © 2018 Elsevier Ltd. All rights reserved.
Références
Methods. 2000 Feb;20(2):196-204
pubmed: 10671313
FEBS Lett. 2014 Apr 17;588(8):1423-9
pubmed: 24508467
Mol Membr Biol. 2002 Apr-Jun;19(2):121-36
pubmed: 12126230
J Cell Biol. 1990 Nov;111(5 Pt 1):2077-88
pubmed: 2172261
Int J Biochem Cell Biol. 2004 Jul;36(7):1171-86
pubmed: 15109565
Cell Commun Adhes. 2001;8(4-6):265-9
pubmed: 12064600
J Cell Sci. 2014 Aug 1;127(Pt 15):3269-79
pubmed: 24849651
Am J Forensic Med Pathol. 2012 Sep;33(3):215-21
pubmed: 22182986
Biochim Biophys Acta. 2013 Jan;1828(1):51-68
pubmed: 22342665
Circ Res. 2017 Oct 13;121(9):1069-1080
pubmed: 28923791
Biophys J. 1996 Mar;70(3):1294-302
pubmed: 8785285
Am J Physiol Cell Physiol. 2003 Feb;284(2):C511-20
pubmed: 12388103
Cell Rep. 2013 Nov 14;5(3):611-8
pubmed: 24210816
J Cell Biol. 2007 Dec 17;179(6):1301-9
pubmed: 18086922
J Membr Biol. 2012 Aug;245(8):495-506
pubmed: 22965738
J Biol Chem. 2001 Mar 16;276(11):8544-9
pubmed: 11124251
Cell Commun Adhes. 2007 Sep-Oct;14(5):211-26
pubmed: 18163231
Methods Mol Biol. 2004;278:313-52
pubmed: 15318002
Circ Res. 1998 Jul 13;83(1):27-32
pubmed: 9670915
Cell Regul. 1990 Dec;1(13):989-1002
pubmed: 1966893
Nucleic Acids Res. 2012 Jan;40(Database issue):D261-70
pubmed: 22135298
Mol Cell Biol. 1990 Apr;10(4):1754-63
pubmed: 1690850
Cell Commun Adhes. 2008 May;15(1):75-84
pubmed: 18649180
Circ Res. 1999 Oct 15;85(8):672-81
pubmed: 10521240
Circ Res. 2002 Jul 26;91(2):104-11
pubmed: 12142342
Biochemistry. 2012 May 29;51(21):4331-42
pubmed: 22558917
Cardiovasc Res. 2008 Oct 1;80(1):9-19
pubmed: 18519446
J Cell Biol. 2001 Aug 20;154(4):815-27
pubmed: 11514593
Exp Cell Res. 2007 Dec 10;313(20):4083-90
pubmed: 17956757
J Clin Invest. 2000 Jan;105(2):161-71
pubmed: 10642594
J Biomol NMR. 1995 Nov;6(3):277-93
pubmed: 8520220
Front Pharmacol. 2013 Oct 22;4:130
pubmed: 24155720
J Biol Chem. 2002 Nov 22;277(47):44962-8
pubmed: 12270943
J Cell Biol. 1999 Mar 8;144(5):1033-45
pubmed: 10085299
J Biol Chem. 2016 Apr 1;291(14):7637-50
pubmed: 26841867
J Bioenerg Biomembr. 1996 Aug;28(4):359-68
pubmed: 8844333
J Biol Chem. 2012 Aug 10;287(33):27771-88
pubmed: 22718765
J Cell Sci. 2006 Sep 1;119(Pt 17):3634-42
pubmed: 16931598
J Biol Chem. 1995 May 26;270(21):12751-61
pubmed: 7539006
J Biol Chem. 2001 Jan 19;276(3):1780-8
pubmed: 11035005
Exp Cell Res. 2009 Dec 10;315(20):3587-97
pubmed: 19835873
Int J Mol Sci. 2018 May 24;19(6):
pubmed: 29882937
J Am Coll Cardiol. 2014 Mar 11;63(9):928-34
pubmed: 24361364
Curr Biol. 2004 Apr 20;14(8):650-8
pubmed: 15084279
J Biol Chem. 2016 Jul 22;291(30):15867-80
pubmed: 27235399
J Cell Sci. 2014 Jan 15;127(Pt 2):455-64
pubmed: 24213533
Circ Res. 2011 Jun 10;108(12):1459-66
pubmed: 21527737
J Biol Chem. 1997 Sep 5;272(36):22824-31
pubmed: 9278444
Annu Rev Biochem. 1996;65:475-502
pubmed: 8811187
PLoS One. 2016 Jun 09;11(6):e0157073
pubmed: 27280719
Biochim Biophys Acta. 2005 Jun 10;1711(2):154-63
pubmed: 15955300
Mol Biol Cell. 2011 May;22(9):1516-28
pubmed: 21411628
J Biol Chem. 2013 Aug 23;288(34):24857-70
pubmed: 23828237
J Am Coll Cardiol. 2011 Nov 22;58(22):2332-9
pubmed: 22093512
J Membr Biol. 2007 Jun;217(1-3):35-41
pubmed: 17629739
Circ Res. 2009 May 8;104(9):1103-12
pubmed: 19342602