OCCLUDIN DEPHOSPHORYLATION IN EARLY DEVELOPMENT OF XENOPUS-LAEVIS

Using immunobot and immunofluorescence analysis with a cross-reacting antiserum, we identified Xenopus laevis occludin as a 57-61 kDa antige n colocalized with cingulin in epithelial junctions of embryos, Occlud in was completely extracted from unfertilized eggs and embryos with a solution containing 0.1% Triton X-100 and 1% NP40. Maternal occludin i n unfertilized eggs migrated by SDS-PAGE as a 61 kDa protein, In ferti lized eggs and in early cleavages up to blastula stage 8 it migrated a s a series of polypeptides with 57-60 kDa. In gastrulae, neurulae and tailbud stage embryos, it migrated as a 57 kDa polypeptide. The electr ophoretic mobility downshift was specifically reproduced by treatment of extracts with acid phosphatase, indicating that it is due to dephos phorylation. The correlation of occludin dephosphorylation with the de novo assembly of tight junction in native epithelia of Xenopus embryo s suggests a possible role of occludin dephosphorylation in the events leading to tight junction assembly, To identify kinases which can pho sphorylate occludin, recombinant chicken occludin (cytoplasmic domain) was subjected to in vitro phosphorylation. Occludin was phosphorylate d on serine and threonine residues by protein kinase CK2 and p34(cdc2) /cyclin B complex, but was not significantly phosphorylated by mitogen -activated protein kinase, protein kinase CK1 and p38(Syk) tyrosine ki nase, We noted that occludin sequences contain a motif matching the ac tivation loop of the cytoplasmic domain of insulin receptor kinase.