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M Cordenonsi

Publications and source records attributed to M Cordenonsi.

5 recordsLinked to original sources
PubMed2000

Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin.

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.

Animals↗
PubMed1999

Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin.

We characterized the sequence and protein interactions of cingulin, an M(r) 140-160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1-439) and tail (1,326-1,368) domains and a central alpha-helical rod domain (440-1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH(2)-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1 (K(d) approximately 5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377-1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH(2)-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

Amino Acid Sequence↗
PubMed1999

Xenopus laevis occludin. Identification of in vitro phosphorylation sites by 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 Xenopus laevis embryos. However, nothing is known about the identity of specific occludin kinase(s) and occludin phosphorylation site(s). Furthermore, nothing is known about the interaction of occludin with cingulin, a cytoplasmic plaque component of tight junctions. Here we report the isolation and sequencing of a complete X. laevis occludin cDNA, and experiments aimed at mapping X. laevis occludin in vitro phosphorylation site(s) and characterizing occludin interaction with cingulin. The sequence of Xenopus occludin is homologous to that of occludins from other species, with identities ranging from 41% to 58%. Bacterially expressed domain E of Xenopus occludin (amino acids 247-493) was a good substrate for protein kinase CK2 (stoichiometry 10.8%, Km 8.4 microM) but not for CK1 kinase, protein kinase A, cdc2 kinase, MAP kinase or syk kinase. Residues Thr375 and Ser379 were identified as potential CK2 phosphorylation sites in this region based on sequence analysis. Mutation of Ser379 to aspartic acid or alanine reduced phosphorylation by CK2 by approximately 50%, and double mutation of Ser379 into aspartic acid and Thr375 into aspartic acid essentially abolished phosphorylation. Glutathione S-transferase (GST) pull-down experiments using extracts of Xenopus A6 epithelial cells showed that constructs of GST fused to wild-type and mutant forms of the C-terminal region of X. laevis occludin associate with several polypeptides, and immunoblot analysis showed that one of these polypeptides is cingulin. GST pull-down experiments using in vitro translated, full-length Xenopus cingulin indicated that cingulin interacts directly with the C-terminal region of occludin.

Amino Acid Sequence↗
PubMed1997

Occludin dephosphorylation in early development of Xenopus laevis.

Using immunoblot and immunofluorescence analysis with a cross-reacting antiserum, we identified Xenopus laevis occludin as a 57-61 kDa antigen colocalized with cingulin in epithelial junctions of embryos. Occludin was completely extracted from unfertilized eggs and embryos with a solution containing 0.1% Triton X-100 and 1% NP40. Maternal occludin in unfertilized eggs migrated by SDS-PAGE as a 61 kDa protein. In fertilized eggs and in early cleavages up to blastula stage 8 it migrated as a series of polypeptides with 57-60 kDa. In gastrulae, neurulae and tailbud stage embryos, it migrated as a 57 kDa polypeptide. The electrophoretic mobility downshift was specifically reproduced by treatment of extracts with acid phosphatase, indicating that it is due to dephosphorylation. The correlation of occludin dephosphorylation with the de novo assembly of tight junction in native epithelia of Xenopus embryos suggests a possible role of occludin dephosphorylation in the events leading to tight junction assembly. To identify kinases which can phosphorylate occludin, recombinant chicken occludin (cytoplasmic domain) was subjected to in vitro phosphorylation. Occludin was phosphorylated on serine and threonine residues by protein kinase CK2 and p34cdc2/cyclin B complex, but was not significantly phosphorylated by mitogen-activated protein kinase, protein kinase CK1 and p38Syk tyrosine kinase. We noted that occludin sequences contain a motif matching the activation loop of the cytoplasmic domain of insulin receptor kinase.

Animals↗