Thr203 of claudin-1, a putative phosphorylation site for MAP kinase, is required to promote the barrier function of tight junctions.
Mitogen-activated protein kinase (MAPK) modulates the barrier function of tight junctions. We identified a putative phosphorylation site for MAPK at around Thr203 (PKPTP) in claudin-1, and determined the biological significance of this site. To this end, using the rat lung endothelial cell line RLE, we generated cells expressing doxycycline (Dox)-inducible wild-type claudin-1 and its mutant with substitution of Thr203 to Ala, and named them RLE:rtTA:CL1 and RLE:rtTA:CL1T203A, respectively. We herein show, by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin, that functional tight junctions were reconstituted in both cells by Dox-induced expression of claudin-1. Interestingly, the barrier functions of tight junctions were less developed in RLE:rtTA:CL1T203A cells compared with RLE:rtTA:CL1 cells. Consistently, levels of both detergent-insoluble claudin-1 protein and its threonine-phosphorylation after Dox treatment were low in RLE:rtTA:CL1T203A cells compared to RLE:rtTA:CL1 cells. Furthermore, pretreatment with the MAPK inhibitor PD98059 markedly suppressed the barrier function and amount of detergent-insoluble claudin-1 in Dox-exposed RLE:rtTA:CL1 cells, whereas it marginally influenced those in RLE:rtTA:CL1T203A cells. These findings indicate that Thr203 of claudin-1 is required to enhance the barrier function of claudin-1-based tight junctions, probably via its phosphorylation and subsequent integration into tight junctions.
Fujibe
Masato
M
Department of Pathology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Chiba
Hideki
H
Kojima
Takashi
T
Soma
Tamotsu
T
Wada
Takuro
T
Yamashita
Toshihiko
T
Sawada
Norimasa
N
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D062445:Claudin-1; D017931:DNA Primers; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D019108:Tight Junctions / Q000502:physiology*; D014162:Transfection
D062445:Claudin-1; C568113:Cldn1 protein, mouse; C568114:Cldn1 protein, rat; D017931:DNA Primers; D008565:Membrane Proteins; D011994:Recombinant Proteins; D010769:Phosphothreonine; D013912:Threonine; D020928:Mitogen-Activated Protein Kinases