Abstract
rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.
Highlights
Post-translational Processing of rac p21s Is Important Both for Their Interaction with the GDP/GTP ExchangeProteins and for Their Activation of NADPH Oxidase*
We examined whether post-translational processing of racl and rac2 p21s was necessary for their interaction with smg GDS and rho GDI and for their p21s (3 pmol each) were assayed in the presence of smg GDS or rho GDI by the filtration method usinga nitrocellulose filter as described [11,16, 18, 22].The reactionsfor smg GDS andrho GDI were carried out for 20 min a t 25 and 30 "C, respectively
We have previously shown that the NADPHoxidase activity in our system is dependent on the simultaneous presence of the GTPyS-bound form of the purified SOCI, and recombinant p47-phox and p67-phox, and thesolubilized membrane fraction of the differentiated HL-60 cells, and that deletion of one of these components decreases the NADPH oxidase activity [11].The GTPyS-boundform of processed racl- and rac2 p21s substituted for the purified SOCI and stimulated the NADPH oxidase activity in the presence of recombinant p47-phox and p67-phox and the solubilized membrane fraction, butunprocessed racl andrac2 p21s were far lesseffective (Fig. 3)
Summary
Was sonicated and centrifuged at 100,000 x g for 1 h. These fractions were collected fraction asdescribed previously [23] It was confirmed in two different ways that ther a c l and rac p21swhich were purified from the soluble and membrane fractions were post-translationally unprocessed and processed, respectively. The membrane fraction contained post-translationally processed the r a c l or rac p21 cDNA were labeled with [3H]mevalonoracl p21 and unidentified endogenous small G proteins. A major peak of ["S]GTPyS-binding activity appeared in Fraction8s4-90 with about 40% yield These fractions were collected and used as post-translationally processed racl p21. Aminorpeak of [35S]GTPyS-binding activity, which might be derived from unidentified endogenous small tone, endogenous small G proteins in the membrane fraction appearedto be labeled to a smallextent, since thefaint radioactive band with a M , value slightly higher than those of rac p21swas observed
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