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Tyrosine phosphorylation site-specific interactome of adaptor protein Shc1 explored by synthetic protein domains

ABSTRACT: As a critical adaptor protein, Shc1 contains SH2 and PTB domains for scaffolding tyrosine phosphorylation-dependent protein complexes, therefore regulating growth factor receptor signaling networks. The three tyrosine phosphorylation sites (pY) on the CH1 linker region of SHC1 provide additional docking sites for recruiting additional dimension of signaling complexes. The affinity purification-mass spectrometry (AP-MS) method with synthetic phosphopeptides provides an unbiased discovery approach but is often limited by the length of synthetic peptides. Here, we chemically synthesized the CH1 region (Shc1CH1) covering 107 amino acids with the 239, 240, and 313 pY sites by multiple chemical peptide ligations. Combing with AP-MS and accurate label-free quantification, we explored the site-specific interactome of these distinct phospho-forms of Shc1 and identified the enhanced complex assembly around the double-phosphorylated Y239/204 sites. We also found that Plcg1 and Plcg2 interacted with pY313 strongly and specifically. Besides, we demonstrated that the AP-MS with the mouse Shc1CH1 probes could evaluate the subcellular EGFR/HER2 signaling in three human cell lines. Overall, long synthetic peptides assembled via peptide ligation widened the scope of peptide-based AP-MS approach for the analysis of site-specific interactomes of proteins with long phosphorylated docking sites.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Xiong Chen

LAB HEAD: Ruijun Tian

PROVIDER: PXD049113 | Pride | 2024-08-15

REPOSITORIES: Pride

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			Action	DRS
	16_01_4T1_Shc1_Noprobe.raw	Raw
	16_02_4T1_Shc1_YYYS1.raw	Raw
	16_03_4T1_Shc1_YYYS2.raw	Raw
	16_04_4T1_Shc1_YYYS3.raw	Raw
	16_05_4T1_Shc1_pYYYS1.raw	Raw

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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.

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Tyrosine phosphorylation site-specific interactome of adaptor protein Shc1 explored by synthetic protein domains

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