Phosphorylation of Tyr-398 and Tyr-402 in occludin prevents its interaction with ZO-1 and destabilizes its assembly at the tight junctions.
Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.
Elias
Bertha C
BC
Department of Physiology, Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Suzuki
Takuya
T
Seth
Ankur
A
Giorgianni
Francesco
F
Kale
Gautam
G
Shen
Le
L
Turner
Jerrold R
JR
Naren
Anjaparavanda
A
Desiderio
Dominic M
DM
Rao
Radhakrishna
R
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D018938:Caco-2 Cells; D002645:Chickens; D004285:Dogs; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D062793:Occludin; D016877:Oxidants / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D019061:src-Family Kinases
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568987:Ocln protein, rat; D016877:Oxidants; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human