Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation
Li Wang, Panagiotis Tsakiroglou, Rex Gonzales, Suhan Cho, Amy Li, Cristobal dos Remedios, Nathan Wright, Aikaterini Kontrogianni-Konstantopoulos
Li Wang, Panagiotis Tsakiroglou, Rex Gonzales, Suhan Cho, Amy Li, Cristobal dos Remedios, Nathan Wright, Aikaterini Kontrogianni-Konstantopoulos
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Research Article Cell biology

Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation

Abstract

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min–1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

Authors

Li Wang, Panagiotis Tsakiroglou, Rex Gonzales, Suhan Cho, Amy Li, Cristobal dos Remedios, Nathan Wright, Aikaterini Kontrogianni-Konstantopoulos

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Figure 5

Phosphorylation of N-cadherin at Ser-788 results in reduced binding to P120-catenin.

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Phosphorylation of N-cadherin at Ser-788 results in reduced binding to P...
(A) Equivalent amounts of recombinant GST-tagged WT (GST-Ncad746–906-WT), phosphomimic (GST-Ncad746–906-SE), and phosphoablated (GST-Ncad746–906-SA) N-cadherin proteins containing the cytoplasmic domain along with control GST protein, as shown by Commassie Blue staining, were tested in pull-down assays for their ability to precipitate and retain endogenous p120-catenin from adult mouse cardiac lysates. Phosphomimic GST-Ncad746–906-SE exhibited significantly reduced binding to p120-catenin compared with GST-Ncad746–906-WT and phosphoablated GST-Ncad746–906-SA proteins (n = 4); the binding of p120-catenin to GST-Ncad746–906-WT was set to 1, while binding of p120-catenin to either GST-Ncad746–906-SE or GST-Ncad746–906-SA was normalized to GST-Ncad746–906-WT. Statistical evaluation was performed with repeated measures 1-way ANOVA followed by Tukey’s multiple-comparison test, *P = 0.0203 for comparison of GST-Ncad746–906-SE vs. Ncad746–906-WT and *P = 0.0496 for comparison of GST-Ncad746–906-SE vs. GST-Ncad746–906-SA. This effect was specific to p120-catenin, as binding of β-catenin to GST-Ncad746–906-WT, GST-Ncad746–906-SE, or GST-Ncad746–906-SA was unaltered (n = 3). Statistical significance was determined with repeated measures 1-way ANOVA followed by Tukey’s multiple-comparison test. Noncontinuous lanes are separated with white space (please see supplemental material). (B) Coomassie Blue staining showing recombinant GST, GST-Ncad746–906-WT, GST-N-cad746–906-SE, and MBP-p120-catenin311–747 that were used in ITC experiments; noncontinuous lanes are separated with white space (please see supplemental material). An average binding affinity, KD, of 1.25 ± 0.03 μM was determined for GST-Ncad746–906-WT and MBP-p120-catenin311–747, whereas a KD of 196.85 ± 29.53 μM was calculated for GST-Ncad746–906-SE and MBP-p120-catenin311–747, consistent with the decreased binding observed in the pull-down assays; n = 3 independent ITC experiments. Data are expressed as mean ± SEM.