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.
