Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions

Ito, Takashi; Matsui, Yasuji; Ago, Tetsuro; Ota, Kazuhisa; Sumimoto, Hideki

doi:10.1093/emboj/20.15.3938

Cited by 165 publications

(212 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

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

101

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“…is expected from the sequence alignment of the PB1 domain (Fig. 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).…”

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

confidence: 99%

“…DNA fragments encoding PKC and ZIP/p62 were prepared as described previously (9). Human PKC -(16 -99) was expressed in Escherichia coli strain BL21 (DE3) cells as a glutathione S-transferase (GST) fusion protein.…”

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

confidence: 99%

“…The PB1 domain is evolutionarily conserved from yeasts to humans, especially in the context of cell polarity-related proteins (9,33) and is classified into two types, type I and type II (34). Type I contains a motif of 28 amino acid residues with highly conserved acidic and hydrophobic residues that is named a PC motif (35).…”

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confidence: 99%

“…The PC motif is also known as OPR (octicosapeptide repeat) (36) or AID (atypical PKC interaction domain) (4) and is generically named the OPCA (OPR, PC, and AID) motif (33). Type I utilizes acidic residues on the OPCA motif as an interaction site, whereas type II utilizes a conserved lysine residue on the side opposite to the OPCA motif-presenting surface as its interaction site, suggesting that the PB1-PB1 interaction is mainly electrostatic (9, 37) but also highly specific and exclusive (9). Moreover, some PB1 domains such as aPKC and ZIP/ p62 contain the OPCA motif and the conserved lysine residue and are classified as type I and type II ( Fig.…”

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confidence: 99%

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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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Characterization of a novel TFG variant causing autosomal recessive pure hereditary spastic paraplegia

Hsiao,

Tsai,

Shen

et al. 2024

Ann Clin Transl Neurol

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ObjectiveTFG mutations have previously been implicated in autosomal recessive hereditary spastic paraplegia (HSP), also known as SPG57. This study aimed to investigate the clinical and molecular features of TFG mutations in a Taiwanese HSP cohort.MethodsGenetic analysis of TFG was conducted in 242 unrelated Taiwanese HSP patients using a targeted resequencing panel covering the entire coding regions of TFG. Functional assays were performed using an in vitro cell model to assess the impact of TFG variants on protein function. Additionally, other representative TFG mutant proteins were examined to understand the broader implications of TFG mutations in HSP.ResultsThe study identified a novel homozygous TFG c.177A>C (p.(Lys59Asn)) variant in a family with adolescent‐onset, pure form HSP. Functional analysis revealed that the Lys59Asn TFG variant, similar to other HSP‐associated TFG mutants, exhibited a low affinity between TFG monomers and abnormal assembly of TFG homo‐oligomers. These structural alterations led to aberrant intracellular distribution, compromising TFG's protein secretion function and resulting in decreased cellular viability.InterpretationThese findings confirm that the homozygous TFG c.177A>C (p.(Lys59Asn)) variant is a novel cause of SPG57. The study expands our understanding of the clinical and mutational spectrum of TFG‐associated diseases, highlighting the functional defects associated with this specific TFG variant. Overall, this research contributes to the broader comprehension of the genetic and molecular mechanisms underlying HSP.

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Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions

Cited by 165 publications

References 44 publications

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

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

Characterization of a novel TFG variant causing autosomal recessive pure hereditary spastic paraplegia

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Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions

Cited by 165 publications

References 44 publications

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

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

Characterization of a novel TFG variant causing autosomal recessive pure hereditary spastic paraplegia

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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