ArhGAP15, a novel human RacGAP protein with GTPase binding property¹

Abstract

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed speci¢city towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and overexpression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress ¢bers and cell contraction. These morphological e¡ects can be attenuated by the co-expression of dominant negative Rac1 N17 . HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1. ß

Genetic and expression analysis of cattle identifies candidate genes in pathways responding to Trypanosoma congolense infection

“…ARHGAP15 is a RAC binding protein, and the mutation is at the proximal end of the RAC binding domain (PS50238) of the protein (10), suggesting a possible mechanism for any effect of the mutation on function. RAC1 is a GTPase that is active when binding GTP and inactive when binding GDP; the RhoGAP domain of ARHGAP15 accelerates the intrinsic rate of hydrolysis of GTP to GDP, thereby inactivating RAC1 (10). To determine whether the ARHGAP15 282H→P polymorphism might cause a difference in the rate of GTP hydrolysis by RAC1, the two alleles were expressed in vitro, and the rate of GTP hydrolysis was measured by following the change in absorbance at 360 nm caused by an increase in γP i abundance (Fig.…”

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase γ

“…Our results are in agreement with a role of PIP3 in controlling the duration rather than the triggering of Rac activity (7) and suggest an involvement of PI3K in mediating GAP activation in response to chemoattractants. PI3K signaling is a known activator of GAPs (38) and PIP3 might modulate those RacGAPs that, like ArhGAP15, are switched on by PH-domainmediated plasma membrane recruitment (24). However, RacGAP were found modulated even after cell lysis, thus suggesting that membrane translocation is accompanied by other events, possibly involving more stable modifications like, for example, RacGAP phosphorylation.…”

“…To find a potential PI3K␥-regulated RacGAP, bioinformatic analysis of coexpression data was performed (23). This approach led to the identification of ArhGAP15, a leukocyte-expressed PH-domain containing GTPase-activating protein (GAP) for Rac, triggered by plasma membrane translocation (24). The expression of a myctagged ArhGAP15 by lentiviral transduction in wild-type BMDMs (expression analysis not shown) revealed that this protein, although increasing cell rounding and blocking polarization, was in the cytoplasm in resting conditions but localized to plasma membrane after C5a stimulation (Fig.…”

ArhGAP15, a Rac-specific GTPase-activating Protein, Plays a Dual Role in Inhibiting Small GTPase Signaling

“…ArhGAP15, a protein known to possess Rac-GAP activity (25), also appeared among the proteins that became phosphorylated only in the presence of Pak2 and [␥-32 P]ATP (Fig. 1A).…”