Cryptic Rac-binding and p21 -activated Kinase Phosphorylation Sites of NADPH Oxidase Component p67

Abstract

Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 Cdc42Hs/Rac -activated kinase (PAK) phosphorylates p67 phox amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67 phox in control of the NADPH oxidase.Members of the Rho family, Rac1, Cdc42Hs, and RhoA, play essential roles in growth factor-mediated changes in cell morphology associated with the formation of actin microfilaments and "focal complexes" (1-4). They also act downstream of Ras in distinct parts of the transformation process (5-8) and activate Jun N-terminal protein kinase (9, 10) and entry into the G 1 phase of the cell cycle (11). Rho family interacting proteins that may play a role as effectors in these signaling pathways include kinases (12-15), GTPase-activating proteins (e.g. phox are phosphorylated during oxidase activation (27-30), and the isolation and identification of the kinases involved are essential to gain a better understanding of the mechanism by which complex formation and oxidase activity are controlled in vivo. PAK has been shown to be activated by fMet-Leu-Phe in neutrophils and to phosphorylate p47 phox in vitro (31). Hitherto, kinases that can phosphorylate p67 phox have not been identified.We have been using the NADPH oxidase as a model protein complex to investigate the mechanism by which Rho family GTPases activate cellular pathways. In this study, the Rac1-p67 phox interaction has been investigated in more detail. Using binding assays, we show that the Rac1-binding site of p67 phox is cryptic, located between amino acids 170 and 199, and that the N-terminal fragment (amino acids 1-192) can be used as an inhibitor of the Rac signaling pathway. The binding sites in p67phox for Rac1 and p40 phox are distinct. Recombinant PAK purified from Escherichia coli can phosphorylate p67 phox ; the phosphorylation site(s) are cryptic and located adjacent to the Rac1-binding site. Deletion of either the polyproline-rich sequence (aa 1 226 -236) or the C-terminal SH3 domain (aa 460 -526) led to increases in Rac1 binding and PAK phosphorylation, suggesting that there is an intramolecular interaction between these two domains of p67 phox that gives rise to the crypticity. Taken together, these data suggest that unfolding of p67 phox , via disruption of a potential intramolecular "SH3 domain-polyproline" interaction, may play a role in control of the NADPH oxidase. MATERIALS AND METHODSCell Culture and Microinjection-Swiss 3T3 fibroblast cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotic/antimycotic (Life Technologies, Inc.) at 37°C and 5% CO 2 . Swiss 3T3 cells were serum-starved for 24 -48 h before being microinjected with p67 phox protein (0.1-1 mg/ml) and obser...

The Sorting Nexin, DSH3PX1, Connects the Axonal Guidance Receptor, Dscam, to the Actin Cytoskeleton

“…p47 phox contains two SH3 domains and an SH3 domain target sequence in addition to a PX domain. In both cases, a proposed conversion from intramolecular interactions to intermolecular SH3-polyproline interactions occurs in response to extracellular signals to up-regulate NADPH oxidase activity (47). The SH3 domain of DSH3PX1 is also capable of interactions involving sequences that span its PX domain.…”

Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

“…In addition, p67 phox fragment 1-192 inhibits Rac-mediated membrane ruffling but does not inhibit the Cdc42-mediated development of filopodia and RhoA-mediated stress fiber formation. 56 The Cdc42 binding to p67 phox observed initially 62 has been proved to be an artifact caused by the presence in the assay of a Cdc42-binding PAK-like protein. 56 Purified recombinant Cdc42N17 did not inhibit the cell-free NADPH oxidase system, indicating that the inhibition of NADPH oxidase activity when Cdc42N17 is expressed did not result from a direct competition between Cdc42N17 and Rac for the formation of an active complex.…”

“…32,33 Rac binds to p67 phox , to the flavocytochrome b 558 , and to the membrane through distinct regions. [55][56][57][58][59][60] The recent crystal structure of a fragment encompassing amino acids 1 to 203 of p67 phox in complex with Rac-GTP 60 reveals significant differences in the way p67 phox interacts with Rac, in comparison with other structures of Rho family effector complexes. Although Cdc42 does not normally activate NADPH oxidase, 31 it becomes active by replacing amino acids 27 and 30 within the effector loop by the corresponding residues of Rac.…”

Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

“…Recent studies have implicated Pak in the activation of NF-B in macrophages (22), tumor growth (23) and the pathogenesis of HIV (24,25). Paks can also catalyze the phosphorylation in vitro of both the 47-and 67-kDa subunits of the superoxide (O 2 Ϫ )-generating system of phagocytic leukocytes (NADPH-oxidase) (5,26). However, it is not known whether Paks participate in the phosphorylation of these oxidase subunits in vivo.…”