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.
Cordenonsi
M
M
Dipartimento di Biologia, Universita' degli Studi di Padova, Padova, Italy.
Mazzon
E
E
De Rigo
L
L
Baraldo
S
S
Meggio
F
F
Citi
S
S
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D014982:Xenopus laevis / Q000196:embryology
D008565:Membrane Proteins; D062793:Occludin; D010744:Phosphoric Monoester Hydrolases