Dephosphorylation of Y228 and Y217 and phosphorylation of Y335 in p120 catenin activate convergent extension during zebrafish gastrulation

Abstract

Background The cadherin‐associated protein p120 catenin regulates convergent extension through interactions with cadherin proteins, Cdc42, and Rac1, as we previously showed in zebrafish (Danio rerio). Phosphorylation of p120 catenin changes the nature of its activity in vitro but is virtually unexplored in embryos. We used our previously developed antisense RNA splice‐site morpholino targeted to endogenous p120 catenin‐δ1 to cause defects in axis elongation probing the functions of three p120 catenin tyrosine‐phosphorylation sites in gastrulating zebrafish embryos. Results The morpholino‐induced defects were rescued by co‐injections with mouse p120 catenin‐δ1‐3A mRNAs mutated at residues Y228 and Y217 to a non‐phosphorylatable phenylalanine (F) or mutated at residue Y335 to a phosphomimetic glutamic acid (E). Co‐injection of the complementary mutations Y228E, Y217E, or Y335F mRNAs partially rescued embryos whereas dual mutation to Y228E‐Y217E blocked rescue. Immunopurification showed Y228F mutant proteins preferentially interacted with Rac1, potentially promoting cell migration. In contrast, the phosphomimetic Y228E preferentially interacted with E‐cadherin increasing adhesion. Both Y228F and Y335F strongly bind VAV2. Conclusions p120 catenin serves dual roles during gastrulation of zebrafish. Phosphorylation and dephosphorylation of tyrosine residues Y217, Y228, and Y335 precisely balance cell adhesion and cell migration to facilitate somite compaction and axis elongation.

Phosphorylation of serine residuesS252,S268/S269, andS879in p120 catenin activates migration of presomitic mesoderm in gastrulating zebrafish embryos

“…Dephosphorylation of tyrosine residues 112 or 217 of p120 catenin augments cell migration during convergent extension in Xenopus 15 . In the preceding paper, we show that dephosphorylation of tyrosine residues 217 and 228 of p120 catenin‐δ1‐3A (CTNND1‐3A) accomplishes the same in zebrafish embryos 16 …”

“…Moreover, a novel finding is that phosphorylation of either S268 or S269 was required for cell migration, consistent with the presence of only one of the residues in all but one of the isoforms of zebrafish and amphibians. Our companion paper shows that phosphorylation of Y217 and Y228 increases cell‐cell adhesion and disrupts cell migration, whereas phosphorylation of Y335 ameliorates cell migration 16 . The complexity of the regulation by phosphorylation at multiple sites by multiple kinases is remarkable.…”

“…(a) Many cell extracts were prepared as described in the preceding paper. 16 (b) Embryos were also dechorionated, poked to release the yolk, and pipetted into iced 500 μL hexane, and 500 μL ice cold methane was added immediately. 61 They were fixed with gentle shaking for 5 minutes followed by centrifugation at 90-360g for 0.5 minutes and rinsed twice in 500 μL of cold methanol with the same centrifugation.…”

“…Microinjection and analysis were performed as described in the preceding paper. 16 The statistical significance of rescue from Sp-MO was determined by the SPSS Laerd Statistics program for nonparametric pairwise comparisons of independent samples in a Kruskal-Wallis test. Significance was adjusted by the Bonferroni correction for multiple samples.…”

“…15 In the preceding paper, we show that dephosphorylation of tyrosine residues 217 and 228 of p120 catenin-δ1-3A (CTNND1-3A) accomplishes the same in zebrafish embryos. 16 In cell cultures, the phosphorylation of p120 catenin is known to occur on six serine (S) and two threonine (T) residues in the N-and C-terminal regions, which include S122, S252, S268, S288, T310, S312, S879, and T916. 17,18 Dephosphorylation of serine and threonine residues achieved by adding kinase inhibitors or by conversion of serine residues to alanine at S268, S288, or T310 increases adhesion, implying that phosphorylation at serine and threonine residues increases cell motility.…”