Phosphorylation-dependent regulation of the Rac-specific GTPase activating protein ARHGAP25
Wisniewski Éva
János Szentágothai Neurosciences
Dr. Bereczki Dániel
SE Élettani Intézet könyvtára
2023-09-27 15:00:00
Cellular and Molecular Physiology
Dr. Hunyady László
Ligeti Erzsébet, Dr. Csépányi-Kömi Roland
Dr. Bőgel Gábor
Dr. Gombos Imre
Dr. Sőti Csaba János
Dr. Pomozi Viola
Dr. Tóth József
Small GTPases of the Rho family, such as Rac, Rho, and Cdc42, play an essential role in the actin-cytoskeleton organization, thereby regulating cell adhesion, migration, and immune cell responses. ARHGAP25, a Rac-specific GTPase activating protein, can accelerate Rac’s slow, endogenous GTP hydrolysis and control its cyclic operation and effector functions. We previously described ARHGAP25’s important role in phagocytosis, superoxide production, and transendothelial migration. Moreover, its complex role in tumor behavior has recently been recognized, rendering the post-translational modifications of ARHGAP25 all the more relevant.
During my Ph.D. studies, I examined ARHGAP25’s phosphorylation and the effect of phosphorylation on its enzymatic GAP activity. We identified eleven phosphorylated amino acids using mass spectrometry and in silico database screening. In collaboration, we reported that phosphorylation occurs on serine 363 and plays a role in the mobilization of hematopoietic stem cells and progenitor cells (HPSCs) from murine bone marrow. The limitations of currently available GAP assays led us to develop a new, in vitro bioluminescence-resonance energy transfer (BRET)-based method, which utilizes the specific molecular interaction between Rac and CRIB, the latter capable of binding only the active, GTP-bound form of Rac. We verified that this new method has comparable sensitivity to other established GAP assays, and we could reproduce the data we obtained with the previously used filter-binding assay using radiolabeling. Our new approach proved to be a safer and cheaper alternative, providing high temporal resolution and simultaneously enabling the measurement of an increased number of samples. In addition, the BRET-based GAP assay allowed us to demonstrate that the phosphorylation of ARHGAP25 with neutrophil cytosol kinases mitigates its enzymatic activity. Using phosphodeletion (serine-to-alanine) mutants, we revealed the selective role of serine residues S363 and S488, but not S379-380, in the phosphorylation-dependent regulation of ARHGAP25’s GAP activity.
