RESUMEN
ßPix activates Nox1, an O2--generating NADPH oxidase, through Rac activation. In this study, we found that S525E mutation of ßPix eliminated its Nox1-activating ability in transfected Caco-2 cells. Unexpectedly, affinity for Rac was not diminished but rather enhanced by S525E mutation, and guanine nucleotide exchange factor (GEF) activity was not altered. The N-terminal fragment (amino acids 1-400) showed similar Rac-binding and GEF activity to wild-type ßPix. In contrast, the C-terminal fragment (amino acids 408-646) had higher Rac-binding activity, particularly for Rac-GTP, than wild-type ßPix, and showed no GEF activity. These data suggest that a second Rac-binding site within the C-terminal region is opened by phosphorylation of Ser-525. The site may bind not only Rac-GDP but also Rac-GTP released from the N-terminal catalytic region, which interrupts Rac-GTP translocation to the membrane where Nox1 resides. If one considers that S340E mutation enhances Nox1 activation (Kaito et al., 2014), the present study suggests that ßPix can also play an inhibitory role in O2- production, depending on the sites of phosphorylation.
Asunto(s)
Mutación Missense , NADPH Oxidasa 1/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Superóxidos/metabolismo , Sustitución de Aminoácidos , Células CACO-2 , Activación Enzimática/genética , Humanos , NADPH Oxidasa 1/genética , Fosforilación/genética , Dominios Proteicos , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Rac is an activating factor for Nox1, an O2(-)-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by ßPix has been demonstrated in cell lines. We examined the effects of ßPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. ßPix expression enhanced O2(-) production in resting cells and cells stimulated with EGF or phorbol ester. ßPix(S340E) further enhanced O2(-) production, while ßPix(S340A) eliminated the ßPix effect. ßPix(S340E), but not ßPix(S340A), had higher affinity and GEF activity for Rac than wild-type ßPix. These results suggest that ßPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.
