Cordenonsi M et al. (1997), Occludin dephosphorylation in early development...

XB-ART-15430

J Cell Sci 1997 Dec 01;110 ( Pt 24):3131-9. doi: 10.1242/jcs.110.24.3131.

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Occludin dephosphorylation in early development of Xenopus laevis.

Cordenonsi M , Mazzon E , De Rigo L , Baraldo S , Meggio F , Citi S .

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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.

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Species referenced: Xenopus laevis
Genes referenced: camp ccnb1.2 cdk1 cgn csnk1a1 csnk2b ins insr krt8.1 mapk1 ocln

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	Fig. 1. Immunofluorescence localization of occludin (ROC-896 antigen) in Xenopus laevis embryos. Embryos at stages 6 (32-cell) (A,B), 11 (midgastrula) (C,D), 30 (tailbud) (E,F) were labelled with rabbit immune serum (A,C,E) or preimmune serum (B,D,F), followed by FITC-labelled secondary antibody. Note the junctional localization of the antigen at all stages, and the lack of junctional reactivity using preimmune serum. Pigment granules are visible as small black dots in A,B,C. The diffuse cytoplasmic fluorescence in D is probably due to autofluorescence of yolk particles. Bar, 50 mm.
	Fig. 2. Double immunofluorescence localization of cingulin (A) and occludin (B) in Xenopus embryos. Stage 30 embryo ectoderm was stained with anti-Xenopus cingulin mouse immune serum M902 (A) and with ROC-896 rabbit immune serum (B), followed by TRITC-labelled anti-mouse (A) or FITC-labelled anti-rabbit (B) secondary antibodies. The diffuse cytoplasmic labeling seen in B was hardly detected when a different secondary antibody was used. Bar, 30 mm.
	Fig. 3. Immunoblot analysis of Xenopus embryo (gastrula) extract with preimmune rabbit (ROC- 896) serum (P) and immune serum (I). Note that a polypeptide of approximate molecular size of 57 kDa is specifically recognized by the immune serum. White triangles on the right indicate cross-reacting polypeptides that are weakly stained by the antiserum (approximate molecular mass 150, 100, 45, 38 kDa).
	Fig. 4. Solubility of Xenopus laevis occludin as determined by immunoblotting analysis of fractions with ROC-896 antiserum. (A) Supernatant fractions from unfertilized eggs and gastrulae embryos after biochemical fractionation and centrifugation (see Materials and Methods): F, floating fraction; C, cytoplasmic soluble protein supernatant fraction; M, membrane protein supernatant fraction; T, total extract. Note that occludin is detected in the total, membrane, and (weakly) in the floating fractions of both eggs and embryos, and weakly in the cytoplasmic soluble fraction of unfertilized eggs only. The polypeptides of approximately 45 kDa and 38 kDa are probably degradation products, since their presence and amount was variable from experiment to experiment. Note that the apparent molecular mass of the antigen in unfertilized eggs was higher than in gastrulae (see also Figs 5 and 6). (B) Supernatant (C/S, M/S, S/S) and pellet (C/P, M/P, S/P) fractions from neurulae after extraction with lysis buffer ‘C’ (no detergents, see Materials and Methods) (lanes C), or with lysis buffer ‘M’ (with Triton X-100 and NP-40, lanes M), or with lysis buffer ‘S’ (with Triton X-100, NP-40 and SDS, lanes S). Note that apparently complete extraction of occludin is obtained with non-ionic detergents. Numbers on the left indicate apparent molecular sizes based on the migration of prestained molecular mass markers.
	Fig. 5. Electrophoretic shift of Xenopus occludin during early development. Immunoblot analysis with ROC-896 of extracts from unfertilized eggs (V), 16-cell embryo (16), 32-cell embryo (32), early blastula stage 7 (B7), midblastula stage 8 (B8), gastrula stage 11 (G), neurula stage 17 (N), and tailbud stage 28 (TB). Numbers on the left indicate approximate molecular mass, determined on the basis of the migration of prestained standards. Arrows on the right indicate the migration of the slower and faster mobility forms.
	Fig. 6. Time course of occludin dephosphorylation by acid phosphatase. Immunoblot analysis of occludin from extracts of unfertilized Xenopus eggs with (+) or without (-) incubation with acid phosphatase for 0, 15, 30, 45 and 60 minutes. G, untreated gastrulae extract; V, untreated unfertilized eggs extract. Numbers on the left indicate migration of molecular size standards. Black arrow on the right indicates the slow migrating form of occludin. White triangle on the right indicates the migration range of the faster migrating forms of occludin.
	Fig. 7. Specificity of dephosphorylation of occludin by acid phosphatase. Immunoblot analysis of samples with (+) or without (-) acid phosphatase (P’ase), phosphatase inhibitor phenylarsine oxide 20 mM (inhibit), unfertilized Xenopus eggs extract (Extract) after 0 and 60 minutes incubation at 37°C. (0-), extract prior to addition of 10´ dephosphorylation buffer. Stage 8, untreated extract from stage 8 blastula Xenopus embryos. Numbers on the left indicate approximate molecular mass, determined on the basis of the migration of prestained standards. Arrows on the right indicate the migration of the slower and faster mobility forms of occludin. Note that acid phosphatase migrates with an apparent size of 45 kDa, and comigrates with the putative degradation product of occludin.
	Fig. 8. SDS-PAGE (A) and corresponding [32P] autoradiogram (B) of samples containing GST alone (lanes 1, 3, 5, 7 in A and corresponding lanes 1¢, 3¢, 5¢, 7¢ in B) and GST-(chicken occludin Cterminal cytoplasmic tail)(lanes 2, 4, 6, 8 in A and corresponding lanes 2¢, 4¢, 6¢, 8¢ in B) after incubation with p34cdc2/cyclin B (cdc2)(lanes 1, 2 and 1¢, 2¢), mitogen-activated protein kinase (MAPK)(lanes 3, 4, and 3¢, 4¢), protein kinase CK2 (CK2)(lanes 5, 6, and 5¢, 6¢) and cAMP-dependent protein kinase (pKA)(lanes 7, 8, and 7¢, 8¢) for 15 minutes at 37°C. Note that GST-ChOc is phosphorylated by cdc2 and CK2, and is not significantly phosphorylated by MAPK and pKA (see also Table 1). Also note that pKA (slower migrating polypeptide) is autophosphorylated. The apparent molecular mass of GST and GST-ChOc was 29 kDa and 48 kDa, respectively.