Dataset Information

Mapping of phosphorylation target sites of obscurin-kin 1 within the cytoplasmic domain of N-cadherin by LC-MS/MS

ABSTRACT: To test our hypothesis that obscurin-kin1 modulates, at least in part, the mechanochemical coupling of cardiomyocytes via phosphorylation of N-cadherin, which has been shown to play a pivotal role in these processes, we set forth to identify the potential phosphorylation target sites of obscurin-kin1 within the cytoplasmic domain of N-cadherin. To this end, we used the baculovirus system to produce catalytically active kin1 coupled to 6xHis-tag (His-Kin1-CA) and the bacterial system to produce an N-cadherin construct containing a portion of the cytoplasmic domain (aa 786-880, accession no. P15116) tagged to 6xHis (His-Ncad786-880), which is enriched in Ser residues predicted to be potent phosphorylation target sites by EXPASY/GPS2.1. Following affinity purification of the recombinant proteins, His-Ncad786-880 was subjected to an in vitro kinase assay in the presence of His-Kin1-CA or control baculovirus preparation infected with empty vector that had undergone the same purification process as His-Kin1-CA. The reaction mixtures were subsequently subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. An N-cadherin peptide encompassing amino acids 786-807 containing a single phosphorylation event at Ser-788 was repeatedly identified in the His-Ncad786-880 reaction mixture when treated with His-Kin1-CA, but not in the control preparation

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Li Wang

LAB HEAD: Aikaterini Kontrogianni-Konstantopoulos

PROVIDER: PXD031003 | Pride | 2024-05-23

REPOSITORIES: Pride

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Publications

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

Wang Li L Tsakiroglou Panagiotis P Gonzales Rex R Cho Suhan S Li Amy A Dos Remedios Cristobal C Wright Nathan N Kontrogianni-Konstantopoulos Aikaterini A

JCI insight 20240208 3

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 co ...[more]

PMID: 38127465

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Project description:Background: Negative-pressure wound therapy has become widely available in modern chronic wound cares. Accelerated keratinocyte (HaCaT cell) movements and decreased E-cadherin expressions induced by the applied negative pressure gradient of 125 mmHg (NP) have been reported in previous studies. However, the molecular mechanism for E-cadherin regulations under NP remains unexplored. We highlighted the signal transduction involved in NP-induced E-cadherin regulation for keratinocytes in the study. Results: Microarray results showed that catenin 1 (CTNND1) gene, the gene encoding the p120-catenin (p120ctn) in cell-cell junctions, was significantly (p = 0.0005) upregulated in HaCaT cells under NP for 12 hours in comparison with those at ambient pressure (AP). Cell fractionations and immunoblotting data showed that NP increased p120ctn phosphorylation, and resulted in p120ctn translocation from the plasma membrane to the cytoplasm. Fluorescent stains suggested that NP diminished the co-localization of p120ctn and E-cadherin on the plasma membrane. Similar phenomenon was also observed in the cells with overexpressed p120ctn at NP. Interestingly, overexpression of dN- p120ctn, a deletion mutant of p120ctn without the N-terminal phosphorylation sites, restored the adherens junctions (AJ) at NP. Knockdown of p120ctn with lentiviral small hairpin RNA not only diminished E-cadherin expressions but also accelerated cell movement at AP. Conclusions: Phosphorylation of p120ctn is endowed to respond rapidly to NP and contributes to the AJ disassembly. This NP-induced E-cadherin downregulation can possibly accelerate wound-healing process. Conclusions: Phosphorylation of p120ctn is endowed to respond rapidly to NP and contributes to the AJ disassembly. This NP-induced E-cadherin downregulation can possibly accelerate wound-healing process. HaCaT cells under negative pressure (NP) gradient of 125 mmHg for 12 hours in comparison with those at ambient pressure (AP) were tested for RNA extraction and hybridization on Affymetrix microarrays. The experiments have been biological replicated three times. The differential expression gene between NP and AP were selected and validated with qPCR method.

			Action	DRS
	1.fea	Other
	1.p	Other
	1.strail	Other
	2.fea	Other
	2.p	Other

Dataset Information

Mapping of phosphorylation target sites of obscurin-kin 1 within the cytoplasmic domain of N-cadherin by LC-MS/MS

Dataset's files

Publications

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

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