The PC motif : a novel and evolutionarily conserved sequence involved in interaction between p40 phox and p67phox, SH3 domain‐containing cytosolic factors of the phagocyte NADPH oxidase

Nakamura, Rika; Sumimoto, Hideki; Mizuki, Kazuhito; Hata, Ken‐ichiro; Ago, Tetsuro; Kitajima, Shigetaka; Takeshige, Koichiro; Sakaki, Yoshiyuki; Ito, Takashi

doi:10.1046/j.1432-1327.1998.2510583.x

Cited by 75 publications

(92 citation statements)

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“…The region of p40 phox involved was reported as the PC motif (residues 287-302) (Nakamura et al, 1998;Ito et al, 2001), now referred as the OPR-PC-AID (OPCA) motif (Ponting et al, 2002). Later, Wilson et al (2003) showed that Thr337 and Pro339 in the region C-terminal region also participate in the heterodimerization.…”

Section: Discussionmentioning

confidence: 99%

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

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101

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In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

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Section: Discussionmentioning

confidence: 99%

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

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101

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“…Our data show that p40 phox and p67 phox form a very tight complex with a dissociation constant in the low nanomolar range. This interaction has been previously mapped to a region between the two SH3 domains in p67 phox , which contains the recently identified BP1 domain and its target sequence, the PC motif (phox and Cdc; also known as the OPR motif)), which resides in the C-terminal part of p40 phox (50,51). p40 phox has often been described as the primary binding partner for p47 phox in the resting state due to an interaction of its SH3 domain with the Pro-rich region in the C terminus of p47 phox .…”

Section: Discussionmentioning

confidence: 99%

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Lapouge¹,

Smith²,

Groemping³

et al. 2002

Journal of Biological Chemistry

135

142

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The phagocyte NADPH oxidase is a multiprotein enzyme whose subunits are partitioned between the cytosol and plasma membrane in resting cells. Upon exposure to appropriate stimuli multiple phosphorylation events in the cytosolic components take place, which induce rearrangements in a number of protein-protein interactions, ultimately leading to translocation of the cytoplasmic complex to the membrane. To understand the molecular mechanisms that underlie the assembly and activation process we have carried out a detailed study of the protein-protein interactions that occur in the p40-p47-p67 phox complex of the resting oxidase. Here we show that this complex contains one copy of each protein, which assembles to form a heterotrimeric complex. The apparent high molecular weight of this complex, as observed by gel filtration studies, is due to an extended, non-globular shape rather than to the presence of multiple copies of any of the proteins. Isothermal titration calorimetry measurements of the interactions between the individual components of this complex demonstrate that p67 phox is the primary binding partner of p47 phox in the resting state. These findings, in combination with earlier reports, allow us to propose a model for the architecture of the resting complex in which p67 phox acts as the bridging molecule that connects p40 phox and p47 phox .

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“…1), the binding assays for an aPKC-ZIP/p62 complex formation have been performed using a much longer construct of PKC -(1-107) (7,9,32,38,39). Interestingly, p40 phox PB1 required more C-terminally extended residues out of the alignment in order to interact with p67 phox PB1, whereas Cdc24p PB1 did not require such a residual region to bind with Bem1p PB1 (35). These results imply diversity and mutual specificity in the PB1-PB1 interaction.…”

Section: Preparation Of Pkc -(16 -99)-complementarymentioning

confidence: 99%

“…Type I contains a motif of 28 amino acid residues with highly conserved acidic and hydrophobic residues that is named a PC motif (35). This name is derived from the fact that the motif occurs in mammalian p40 phox and budding yeast Cdc24p.…”

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confidence: 99%

Solution Structure of Atypical Protein Kinase C PB1 Domain and Its Mode of Interaction with ZIP/p62 and MEK5

Hirano

Yoshinaga

Ogura

et al. 2004

Journal of Biological Chemistry

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Atypical protein kinase C (aPKC) has been implicated in several signaling pathways such as cell polarity, cell survival, and cell differentiation. In contrast to other PKCs, aPKC is unique in having the PB1 (Phox and Bem 1) domain in the N terminus. The aPKC PB1 domain binds with ZIP/p62, Par6, or MEK5 through a PB1-PB1 domain interaction that controls the localization of aPKC. Here, we determined the three-dimensional structure of the PB1 domain of PKC by NMR and found that the PB1 domain adopts a ubiquitin fold. The OPCA (OPR, PC, and AID) motif inserted into the ubiquitin fold was presented as a ␤␤␣ fold in which the side chains of conserved Asp residues were oriented to the same direction to form an acidic surface. This structural feature suggested that the acidic surface of the PKC PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKC -ZIP/p62 complex by mutational analysis. Interestingly, in the PKC PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motifpresenting surface, suggesting dual roles for the PKC PB1 domain in that it could interact with either the conserved lysine residue or the acidic residues on the OPCA motif of the target PB1 domains.As part of the ongoing study of atypical protein kinase C (aPKC), 1 isoform PKC was first cloned in 1988 (1), followed by the cloning of isoform PKC / (2, 3). Now, aPKC has been suggested as playing a crucial role in signal transduction pathways such as cell polarity, cell differentiation, and cell survival (4, 5). In contrast to conventional PKC, aPKC contains only a single C1 domain but is devoid of a C2 domain in the Nterminal regulatory region. A tandem repeat of the C1 domain is known to be a target of diacylglycerol, and the C2 domain is a binding site for Ca 2ϩ . However, because of the lack of a C2 domain and the incomplete C1 domain in aPKC, neither Ca 2ϩ nor diacylglycerol activates aPKC. Instead, aPKC contains a PB1 domain at the N terminus.In PKC signaling, the regulation of both the catalytic activity and the spatial localization of PKC is essential. PKC is known to translocate to a specific region in response to activation signals and then phosphorylate specific target proteins. Such spatial localization of PKC in cells is partially regulated by either PKC-binding scaffold proteins or anchoring proteins (6). In particular, aPKC binds with PB1 domain-containing proteins such as ZIP/p62 (7-9), Par6 (10 -13), or MEK5 (14) through the PB1-PB1 domain interaction. Thus, the PB1-mediated interaction between aPKC and the scaffolding or anchoring proteins plays a crucial role in exerting the biological functions of aPKC (4).ZIP/p62 homologues were isolated in different biological contexts as ZIP, p62, and EBIAP (7,15,16). Recently, it has been demonstrated that ZIP/p62 is involved in the NF B activation pathway in association with aPKC. ZIP/p62 is a scaffold protein of aPKC for the activation of NF B downstream of extracellular signals such as tumor necrosis fac...

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The PC motif : a novel and evolutionarily conserved sequence involved in interaction between p40 ^phox and p67^phox, SH3 domain‐containing cytosolic factors of the phagocyte NADPH oxidase

Cited by 75 publications

References 1 publication

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Solution Structure of Atypical Protein Kinase C PB1 Domain and Its Mode of Interaction with ZIP/p62 and MEK5

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The PC motif : a novel and evolutionarily conserved sequence involved in interaction between p40 phox and p67phox, SH3 domain‐containing cytosolic factors of the phagocyte NADPH oxidase

Cited by 75 publications

References 1 publication

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Solution Structure of Atypical Protein Kinase C PB1 Domain and Its Mode of Interaction with ZIP/p62 and MEK5

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Product

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The PC motif : a novel and evolutionarily conserved sequence involved in interaction between p40 ^phox and p67^phox, SH3 domain‐containing cytosolic factors of the phagocyte NADPH oxidase

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox