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Author Spotlight: Developing Tools to Tune the Activity of Tyrosine Phosphatases
Published on: September 6, 2024
Role of Protein Tyrosine Phosphatase Epsilon (PTPε) in Leukotriene D4-Induced CXCL8 Expression
Fanny Lapointe1, Sylvie Turcotte1, Steeve Véronneau1
1Division of Immunology and Allergy, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Abstract:
Phosphorylation on tyrosine residues is recognized as an important mechanism for connecting extracellular stimuli to cellular events and defines a variety of physiologic responses downstream of G protein-coupled receptor (GPCR) activation. To date, few protein tyrosine phosphatases (PTPs) have been shown to associate with GPCRs, and little is known about their role in GPCR signaling. To discover potential cysteinyl-leukotriene receptor (CysLT1R)-interacting proteins, we identified protein tyrosine phosphatase ε (PTPε) in a yeast two-hybrid assay. Since both proteins are closely linked to asthma, we further investigated their association. Using a human embryonic kidney cell line 293 (HEK-293) cell line stably transfected with the receptor (HEK-LT1), as well as human primary monocytes, we found that PTPε colocalized with CysLT1R in both resting and leukotriene D4 (LTD4)-stimulated cells. Cotransfection of HEK-LT1 with PTPε had no effect on CysLT1R expression or LTD4-induced internalization, but it inhibited LTD4-induced CXC chemokine 8 (CXCL8) promoter transactivation, protein expression, and secretion. Moreover, reduced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), but not of p38 or c-Jun-N-terminal kinase 1 or 2 mitogen-activated protein kinases (MAPKs), was observed upon LTD4 stimulation of HEK-LT1 coexpressing cytosolic (cyt-) PTPε, but not receptor (R) PTPε The increased interaction of cyt-PTPε and ERK1/2 after LTD4 stimulation was shown by coimmunoprecipitation. In addition, enhanced ERK1/2 phosphorylation and CXCL8 secretion were found in LTD4-stimulated human monocytes transfected with PTPε-specific siRNAs, adding support to a regulatory/inhibitory role of PTPε in CysLT1R signaling. Given that the prevalence of severe asthma is increasing, the identification of PTPε as a new potential therapeutic target may be of interest.
Insights
Protein tyrosine phosphatase epsilon (PTPε) interacts with cysteinyl-leukotriene receptor 1 (CysLT1R) and inhibits its signaling pathway, suggesting PTPε as a potential therapeutic target for asthma.
Area of Science:
- Cellular signaling
- Molecular biology
- Immunology
Background:
- Tyrosine phosphorylation is crucial for cellular responses to external stimuli, particularly downstream of G protein-coupled receptor (GPCR) activation.
- The role of protein tyrosine phosphatases (PTPs) in GPCR signaling remains largely unexplored.
- Cysteinyl-leukotriene receptor 1 (CysLT1R) and protein tyrosine phosphatase epsilon (PTPε) are implicated in asthma pathogenesis.
Purpose of the Study:
- To identify proteins interacting with CysLT1R.
- To investigate the functional role of PTPε in CysLT1R signaling.
- To explore the potential of PTPε as a therapeutic target for asthma.
Main Methods:
- Yeast two-hybrid assay to identify interacting proteins.
- Cell-based assays using HEK-293 cells stably expressing CysLT1R (HEK-LT1) and human primary monocytes.
- Immunofluorescence microscopy for colocalization studies.
- Western blotting and ELISA for protein expression and secretion analysis.
- Coimmunoprecipitation to assess protein interactions.
- RNA interference (siRNA) to knockdown PTPε expression.
Main Results:
- PTPε was identified as a CysLT1R-interacting protein.
- PTPε colocalized with CysLT1R in both resting and stimulated cells.
- PTPε inhibited leukotriene D4 (LTD4)-induced CXCL8 promoter transactivation, protein expression, and secretion.
- Cytosolic PTPε, but not receptor PTPε, reduced LTD4-induced extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation.
- PTPε physically interacted with ERK1/2 upon LTD4 stimulation.
- Knockdown of PTPε in human monocytes enhanced LTD4-induced ERK1/2 phosphorylation and CXCL8 secretion.
Conclusions:
- PTPε negatively regulates CysLT1R signaling, specifically the LTD4-induced activation of the ERK1/2 pathway and subsequent CXCL8 production.
- The interaction between PTPε and CysLT1R, and its inhibitory effect on downstream signaling, highlights PTPε as a potential therapeutic target for asthma.
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