Xenopus laevis occludin - Identification of in vitro phosphorylation sitesby protein kinase CK2 and association with cingulin

Occludin is a protein component of the membrane domain of tight junctions, and has been shown to be phosphorylated in vivo in cultured cells and Xenop us laevis embryos. However, nothing is known about the identity of specific occludin kinase(s) and occludin phosphorylation site(s). Furthermore, noth ing is known about the interaction of occludin with cingulin, a cytoplasmic plaque component of tight junctions. Here we report the isolation and sequ encing of a complete X. laevis occludin cDNA, and experiments aimed at mapp ing X. leavis occludin in vitro phosphorylation site(s) and characterizing occludin interaction with cingulin. The sequence of Xenopus occludin is hom ologous to that of occludins from other species, with identities ranging fr om 41% to 58%. Bacterially expressed domain E of Xenopus occludin (amino ac ids 247-493) was a good substrate for protein kinase CK2 (stoichiometry 10. 8%, K-m 8.4 mu m) but not for CK1 kinase, protein kinase A, cdc2 kinase, MA P kinase or syk kinase. Residues Thr375 and Ser379 were identified as poten tial CK2 phosphorylation sites in this region based on sequence analysis. M utation of Ser379 to aspartic acid or alanine reduced phosphorylation by CK 2 by approximate to 50%, and double mutation of Ser379 into aspartic acid a nd Thr375 into aspartic acid essentially abolished phosphorylation. Glutath ione S-transferase (GST) pull-down experiments using extracts of Xenopus A6 epithelial cells showed that constructs of GST fused to wild-type and muta nt forms of the C-terminal region of X. laevis occludin associate with seve ral polypeptides, and immunoblot analysis showed that one of these polypept ides is cingulin. GST pull-down experiments using in vitro translated, full -length Xenopus cin,cingulin indicated that cingulin interacts directly wit h the C-terminal region of occludin.