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 1

The codistribution of obscurin-B containing kin1 and N-cadherin at cell junctions is coordinately regulated by Ca2+.

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The codistribution of obscurin-B containing kin1 and N-cadherin at cell ...
(A–F) Ca2+ switch experiments combined with confocal imaging demonstrated that obscurin-B colocalizes with N-cadherin at cell junctions in primary cultures of ERVMs (A) and HEK293 cells (D) in the presence of Ca2+. Ca2+ removal for 4 hours resulted in loss of both obscurin-B and N-cadherin from cell junctions, with residual proteins exhibiting a punctate or diffuse cytoplasmic distribution in ERVMs (B) and HEK293 cells (E), respectively. Addition of Ca2+ in the medium for 1 hour restored the codistribution of obscurin-B and N-cadherin at cell junctions in both ERVMs (C) and HEK293 cells (F); n = 4 experiments for ERVM (4 optical fields per condition, per experiment) and n = 2 for HEK293 cells (5–6 optical fields per condition, per experiment). (G) Confocal evaluation of ERVM cultures transfected with control GFP and GFP-Kin1-CA indicated that exogenous GFP-Kin1-CA, but not GFP protein, colocalizes with endogenous N-cadherin at cell contact sites (arrowheads); n = 3 experiments. (H) Cell fractionation of HEK293 cells transfected with control GFP or GFP-Kin1-CA followed by immunoblotting analysis showed that approximately 36% of exogenous GFP-Kin1-CA is found in the membrane fraction, similarly to endogenous N-cadherin; the identity of the membrane and cytoplasmic fractions was confirmed by probing for Na/K ATPase and HSP90, respectively, which also served as loading controls within each fraction; n = 3 experiments. Scale bars: 20 μm (A and D–F) and 40 μm (G).