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A novel BRET-Based GAP assay reveals phosphorylation-dependent regulation of the RAC-specific GTPase activating protein ARHGAP25.
Wisniewski, Éva; Czárán, Domonkos; Kovács, Fanni; Bahurek, Eniko; Németh, Afrodité; Sasvári, Péter; Szanda, Gergo; Pettkó-Szandtner, Aladár; Klement, Eva; Ligeti, Erzsébet; Csépányi-Kömi, Roland.
Afiliación
  • Wisniewski É; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Czárán D; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Kovács F; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Bahurek E; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Németh A; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Sasvári P; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Szanda G; Department of Physiology, Semmelweis University, Budapest, Hungary.
  • Pettkó-Szandtner A; Laboratory of Proteomics Research, Biological Research Centre, Szeged, Hungary.
  • Klement E; Laboratory of Proteomics Research, Biological Research Centre, Szeged, Hungary.
  • Ligeti E; Single Cell Omics ACF, Hungarian Centre of Excellence for Molecular Medicine, Szeged, Hungary.
  • Csépányi-Kömi R; Department of Physiology, Semmelweis University, Budapest, Hungary.
FASEB J ; 36(11): e22584, 2022 11.
Article en En | MEDLINE | ID: mdl-36190314
ARHGAP25, a RAC-specific GTPase activating protein (GAP), is an essential regulator of phagocyte effector functions such as phagocytosis, superoxide production, and transendothelial migration. Furthermore, its complex role in tumor behavior has recently been recognized. We previously demonstrated that phosphorylation of serine 363 in ARHGAP25 regulates hematopoietic stem cells and progenitor cells in mouse bone marrow. However, the significance of other potential phosphorylation sites of ARHGAP25 remained unknown. Now, we developed a novel, real-time bioluminescence resonance energy transfer (BRET) assay to monitor the GAP activity of ARHGAP25 in vitro. Using this approach, we revealed that phosphorylation of S363 and S488, but not that of S379-380, controls ARHGAP25's RACGAP activity. On the other hand, we found in granulocyte-differentiated human PLB-985 cells that superoxide production and actin depolymerization are regulated by residues S363 and S379-380. The present data demonstrate the value of our BRET-GAP assay and show that different phosphorylation patterns regulate ARHGAP25's GAP activity and its effect on superoxide production and phagocytosis.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Superóxidos / Proteínas Activadoras de GTPasa Límite: Animals / Humans Idioma: En Revista: FASEB J Asunto de la revista: BIOLOGIA / FISIOLOGIA Año: 2022 Tipo del documento: Article País de afiliación: Hungria Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Superóxidos / Proteínas Activadoras de GTPasa Límite: Animals / Humans Idioma: En Revista: FASEB J Asunto de la revista: BIOLOGIA / FISIOLOGIA Año: 2022 Tipo del documento: Article País de afiliación: Hungria Pais de publicación: Estados Unidos