Abstract
Compromise in adherens junctions (AJs) is associated with several chronic inflammatory diseases. We reported previously that Janus kinase 3, a non-receptor tyrosine kinase, plays a crucial role in AJ formation through its interaction with β-catenin. In this report, we characterize the structural determinants responsible for Jak3 interactions with β-catenin and determine the functional implications of previously unknown tyrosine residues on β-catenin phosphorylated by Jak3. We demonstrate that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of β-catenin, where Jak3 directly phosphorylated three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. However, prior phosphorylation of β-catenin at Tyr654 was essential for further phosphorylation of β-catenin by Jak3. Interaction studies indicated that phosphorylated Jak3 bound to phosphorylated β-catenin with a dissociation constant of 0.28 μm, and although both the kinase and FERM (Band 4.1, ezrin, radixin, and moesin) domains of Jak3 interacted with β-catenin, the NTD domain of β-catenin facilitated its interactions with Jak3. Physiologically, Jak3-mediated phosphorylation of β-catenin suppressed EGF-mediated epithelial–mesenchymal transition and facilitated epithelial barrier functions by AJ localization of phosphorylated β-catenin through its interactions with α-catenin. Moreover, loss of Jak3-mediated phosphorylation sites in β-catenin abrogated its AJ localization and compromised epithelial barrier functions. Thus, we not only characterize Jak3 interaction with β-catenin but also demonstrate the mechanism of molecular interplay between AJ dynamics and EMT by Jak3-mediated NTD phosphorylation of β-catenin.
Highlights
Compromise in adherens junctions (AJs) is associated with several chronic inflammatory diseases
Because the N-terminal domain (NTD) of -catenin has four tyrosine residues, and we reported previously that, in human intestinal epithelial cells, Jak3 does not associate with Tyr142-phosphorylated -catenin [7], using an in vitro kinase assay, we determined whether mutation of Y142F of -catenin has an effect on Jak3-mediated tyrosine phosphorylation of the -catenin NTD
Because we reported previously that, in human intestinal epithelial cell (IEC), Jak3-associated -catenin had phosphorylation at Tyr654 of the C-terminal domain (CTD) [7], and Fig. 1 showed that the non-phosphorylated CTD of -catenin inhibited NTD phosphorylation by Jak3, using a phosphomimetic (Y654E) approach, we determined whether prior phosphorylation of -catenin at Tyr654 was necessary for the enhanced Jak3 interactions and further phosphorylation of -catenin
Summary
We reported that Jak facilitated intestinal mucosal barrier functions through its interactions with the adherens junction protein -catenin [7, 18, 22]. To further confirm that Jak3-mediated changes in ␣-catenin localization were due to its interactions with -catenin, using confocal microscopy, we showed that EGF activation of IECs led to loss in junctional localization of ␣-catenin (Fig. 4G, second row), which was significantly reversed by co-stimulation of these cells with IL-2 (Fig. 4G, third row), as indicated by strong yellow staining in the merged panel. To confirm that these effects were due to Jak activation, these experiments were performed in the presence of Jak inhibitor. The Y30E,Y64E,Y86E mutation that mimicked the phosphorylation state of these residues preserved the geometric flexibility of the NTD required for AJ localization of -catenin
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