Thr203 of claudin-1, a putative phosphorylation site for MAP kinase, is required to promote the barrier function of tight junctions
| Autor: | Takuro Wada, Hideki Chiba, Norimasa Sawada, Tamotsu Soma, Takashi Kojima, Masato Fujibe, Toshihiko Yamashita |
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| Rok vydání: | 2003 |
| Předmět: |
MAPK/ERK pathway
Threonine Biology Transfection Polymerase Chain Reaction Tight Junctions Mice Claudin-1 Animals Protein kinase A Claudin Barrier function DNA Primers Binding Sites Tight junction Base Sequence Membrane Proteins Cell Biology Molecular biology Recombinant Proteins Cell biology Endothelial stem cell Kinetics Phosphothreonine Paracellular transport Phosphorylation Mitogen-Activated Protein Kinases |
| Zdroj: | Experimental cell research. 295(1) |
| ISSN: | 0014-4827 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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