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Dev Dyn
2022 Dec 01;25112:1952-1967. doi: 10.1002/dvdy.508.
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Phosphorylation of serine residues S252, S268/S269, and S879 in p120 catenin activates migration of presomitic mesoderm in gastrulating zebrafish embryos.
Kupai A
,
Nakahara H
,
Voss KM
,
Hirano MS
,
Rodriguez A
,
Lackey DL
,
Murayama JF
,
Mathieson CJ
,
Shan B
,
Horton EC
,
Curtis GH
,
Huang J
,
Hille MB
.
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BACKGROUND: Cadherin-associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis.
RESULTS: We knocked down endogenous p120 catenin-δ1 with an antisense RNA-splice-site morpholino (Sp-MO) causing defects in axis elongation. These defects were rescued by co-injections of mRNAs for wildtype mouse p120 catenin-δ1-3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not.
CONCLUSIONS: We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.
Department of Biology, University of Washington, Mary Gates Research Scholarships for Research Funds, The Fan Shu Properties, The Phyllis C. Burr Trust
FIGURE 1
Roles of S268, S269, and S288 in p120 catenin activity. (A) Schema of the amino acid residues studied in this article. (B) Embryo examples used for scoring the rescue of the Sp-MO defect at 24 hours post fertilization (hpf) after co-injection of p120 catenin mRNA at the one-cell stage. (C-E) Percentage of injected embryos in each scoring category. Numbers above the bars indicate the total number of embryos observed. Numbers within the bars indicate the total number of female fish from which eggs were injected. Numbers after the labels in the horizontal axis indicate separate experiments performed with the experiments numbered in order of performance. Similar numbers indicate tests done simultaneously. The data in (C) were from a single day; those in (D) were from three separate experiments and numbered in the order they were performed; those in (E) were from three separate experiments. Statistical significance is shown with the longer bar indicating the injection to which the other injections are compared using Kruskal-Wallis multiple comparison test, ***P < .001, **P < .01, *P < .05, ns = not significant (P > .05). (F-H) Western blots of protein extracts from embryos injected with the indicated mRNAs. All p120 catenin proteins migrated at 110-120 kDa
FIGURE 2
Role of the double-mutants S268/S269 and S268/S288 in p120 catenin activity. (A,B) Percentage of embryos showing the noted phenotypes after injection of mutant p120 catenin mRNAs. Some embryos were injected with only mutant mRNA. Numbers above the bars indicate the total number of embryos observed. Numbers within the bars indicate the total number of female fish from which eggs were injected. Numbers after the labels in the horizontal axis indicate separate experiments performed with the experiments numbered in order of performance. Similar numbers indicate tests done simultaneously. The data in (A) were from five separate experiments numbered in the order they were performed and those in (B) from two injection experiments. Statistical significance is shown, with the longer bar indicating the injection to which the other injections are compared using Kruskal-Wallis multiple comparison test, ***P < .001, ns = not significant (P > .05). (C,D) Western blots of protein extracts from embryos injected with the mRNAs indicated. All p120 catenin proteins migrated at 110-120 kDa. Slashes indicate blots from the same gel that was moved to be proximal.
FIGURE 3
Roles of S252 and T310 in axis extension. (A,B) Percentage of embryos showing the noted phenotypes after injection of mutant p120 catenin mRNA. Some embryos were injected with only mutant mRNA. Scoring is as indicated in Figure 1B color key. Numbers above the bars indicate the total number of embryos observed. Numbers within the bars indicate the total number of female fish from which eggs were injected. Numbers after the labels in the horizontal axis indicate separate experiments performed, with the experiments numbered in order of performance. Similar numbers indicate tests done simultaneously. The data in (A) are from eight separate experiments and those in (B) from six separate experiments. Statistical significance is shown, with the longer bar indicating the sample to which the other samples were compared using Kruskal-Wallis multiple comparison test, ***P < .001, ns = not significant between all injections indicated (P > .05). (C,D) Western blots of protein extracts from embryos injected with the mRNAs indicated. All p120 catenin proteins migrated at 110-120 kDa. Slashes indicate blots from the same gel that was moved to be proximal.
FIGURE 4
Role of the C-terminal region of p120 catenin in axis extension, S879, T916, S905, and T906. (A,B,E) Percentage of embryos showing the noted phenotypes after injection mutant p120 catenin mRNAs. Some embryos were injected with only mutant mRNA. Scoring is as indicated in Figure 1B color key. Numbers above the bars indicate the total number of embryos observed. Numbers within the bars indicate the total number of female fish from which eggs were injected. Numbers after the labels in the horizontal axis indicate the specific experiments used in each bar. The data in (A) are from three separate experiments numbered in the order they were performed, those in (B) are from seven separate experiments; those in (E) are from three separate experiments. (C,D) Western blots of protein extracts from embryos injected with the mRNAs indicated. Statistical significance was determined using Kruskal-Wallis multiple comparison test, ***P < .001**P < .01, ns = not significant between all injections indicated (P > .05). (F-H) Western blots of protein extracts from embryos injected with the indicated mRNAs. All p120 catenin proteins migrated at 110-120 kDa. Slashes indicate blots from the same gel that was moved to be proximal.
FIGURE 5
Relative concentrations of mRNAs for p120 catenin isoforms formed during the development of zebrafish. To show the changes in mRNA concentrations during development at 28°C, the relative amounts of each isoform were measured by semi-quantitative PCR of cDNA isolated from Oblong, Bud, and 2-Somite stages. Specific primers were used for each isoform. Translational elongation factor 1a (Ef1a) was used as a loading control, for comparison of cDNAs at each stage. The values were transposed from stained DNA gels and the bar graph in the study by Hsu et al5 (Supplement Figure S2D). CTNND2b = p120 catenin-δ2b (orange), CTNND1 = p120 catenin-δ1 (blue), ARVCF (grey), CTNND2a = p120 catenin-δ2a (yellow)
FIGURE 6
A proposed model for the signaling pathway of p120 catenin in zebrafish embryos as regulated by phosphorylation on serine residues. Left side, adhesive state: Unphosphorylated serine residues are adjacent to cadherin and the serine threonine kinases, which are represented by protein kinase C (PKC). Although cadherin is depicted as a monomer, it was recently shown to dimerize when p120 catenin bids at its juxta-membrane position.59 Right side, motile state: There is phosphorylation on S252, S268, and/or S879, p120 catenin binds VAV2 and subsequently to Cdc42 or Rac1 GTPases leading to filopodial-lamellipodial extension that results in the progression of cells toward the dorsal axis and embryo elongation. Phosphorylation of T310, S905, and/or T916 would augment binding to VAV2 but it is not required. VAV2, which diffuses laterally (arrows) while intermittently attaching to phospholipids in the plasma membrane,21, 24 can bind to p120 catenin attached to E-cadherin or to p120 catenin in the region of actin polymerization. Dorsal migration is stimulated by Wnt diffusing from the dorsal prechordal area.
