Studying the ShcD and ERK interaction under acute oxidative stress conditions in melanoma cells.

AHMED, Samrein, AMER, Sara, EMAD, Mira, RAHMANI, Mohamed and PRIGENT, Sally A (2019). Studying the ShcD and ERK interaction under acute oxidative stress conditions in melanoma cells. The international journal of biochemistry & cell biology, 112, 123-133.

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Abstract

The newly identified melanoma-associated adaptor ShcD was found to translocate to the nucleus upon hydrogen peroxide treatment. Therefore, the aim of this study was to identify the ShcD network in melanoma cells under oxidative stress. LC-MS/MS and GFP-trap were performed to study the ShcD phosphorylation status during acute severe oxidative stress. ShcD was found to be phosphorylated at threonine-159 (Thr159) in response to 5 mM H₂O₂ treatment. The GPS 2.1 phosphorylation prediction program predicted that the Thr159Pro motif, housed in the N-terminus of the ShcD-CH2 domain, is a potential phosphorylation site for MAPKs (ERK, JNK or p38). Co-immunoprecipitation experiments revealed that ShcD mainly interacts with ERK in B16 and MM138 melanoma cells under both hydrogen peroxide-untreated and -treated conditions. Moreover, ShcD interacts with both phosphorylated and un-phosphorylated ERK, although the interaction between ShcD and phospho-ERK was primarily observed after H₂O₂ treatment. A MEK inhibitor (U0126) enhanced the interaction between ShcD and unphosphorylated ERK under oxidative stress conditions. Furthermore, Thr159 was mutated to either alanine (A) or glutamic acid (E) to study whether the threonine phosphorylation state influences the ShcD/ERK interaction. Introducing the T159E mutation obliterated the ShcD/ERK interaction. To identify the functional impact of the ShcD/ERK interaction on cell survival signalling under oxidative stress conditions, caspase 3/7 assays and 7AAD cell death assays were used. The ShcD/ERK interaction promoted anti-survival signalling upon exposure to hydrogen peroxide, while U0126 treatment reduced death signalling. Our data also showed that the death signalling initiated by the ShcD/ERK interaction was accompanied by p21 phosphorylation. In summary, these data identified ShcD, via its interaction with ERK, as a proapoptotic protein under oxidative stress conditions.

Item Type:	Article
Uncontrolled Keywords:	Cell Line, Tumor; Animals; Humans; Mice; Melanoma, Experimental; Extracellular Signal-Regulated MAP Kinases; MAP Kinase Signaling System; Amino Acid Motifs; Oxidative Stress; Cyclin-Dependent Kinase Inhibitor p21; Shc Signaling Adaptor Proteins; HEK293 Cells; Protein Domains; ERK; Hydrogen peroxide; Oxidative stress; ShcD; Amino Acid Motifs; Animals; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; MAP Kinase Signaling System; Melanoma, Experimental; Mice; Oxidative Stress; Protein Domains; Shc Signaling Adaptor Proteins; 0601 Biochemistry and Cell Biology; 1101 Medical Biochemistry and Metabolomics; 1116 Medical Physiology; Biochemistry & Molecular Biology; 3101 Biochemistry and cell biology; 3205 Medical biochemistry and metabolomics
Identification Number:	https://doi.org/10.1016/j.biocel.2019.05.009
Page Range:	123-133
SWORD Depositor:	Symplectic Elements
Depositing User:	Symplectic Elements
Date Deposited:	25 Apr 2024 15:15
Last Modified:	25 Apr 2024 15:15
URI:	https://shura.shu.ac.uk/id/eprint/33587

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