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Occludin dephosphorylation in early development of Xenopus laevis
M Cordenonsi1, E Mazzon, L De Rigo
1Dipartimento di Biologia, Universita' degli Studi di Padova, Padova, Italy.
Insights
Xenopus laevis occludin, a key protein in epithelial junctions, undergoes dephosphorylation during tight junction assembly. This dephosphorylation is linked to changes in occludin
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Tight junctions are crucial for epithelial barrier function and are dynamic structures.
- Occludin is a major component of tight junctions, but its role during assembly is not fully understood.
- Xenopus laevis embryos provide a model system for studying early embryonic development and tissue formation.
Purpose of the Study:
- To investigate the changes in occludin during Xenopus embryonic development and tight junction assembly.
- To identify the molecular mechanisms, specifically phosphorylation, regulating occludin function.
- To determine which kinases are involved in occludin phosphorylation.
Main Methods:
- Immunoblot and immunofluorescence analysis were used to identify and localize occludin in Xenopus embryos.
- Differential extraction with detergents (Triton X-100, NP40) was employed to characterize occludin solubility.
- SDS-PAGE and acid phosphatase treatment were used to analyze changes in occludin molecular weight and phosphorylation status.
- In vitro phosphorylation assays with recombinant chicken occludin (cytoplasmic domain) were performed to identify specific kinases.
Main Results:
- Xenopus laevis occludin was identified as a 57-61 kDa antigen localized to epithelial junctions.
- Occludin exhibited a downshift in electrophoretic mobility from 61 kDa in unfertilized eggs to 57 kDa in later embryonic stages, correlating with dephosphorylation.
- Protein kinase CK2 and the p34cdc2/cyclin B complex were identified as kinases capable of phosphorylating occludin in vitro.
- Mitogen-activated protein kinase, protein kinase CK1, and p38Syk tyrosine kinase did not significantly phosphorylate occludin.
Conclusions:
- Occludin dephosphorylation is associated with the de novo assembly of tight junctions in Xenopus embryos, suggesting a regulatory role.
- Specific kinases, including CK2 and p34cdc2/cyclin B, phosphorylate occludin, potentially modulating its function during junction formation.
- Further research into the phosphorylation sites and regulatory networks of occludin is warranted to fully elucidate its role in epithelial development.
Abstract:
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.