Takehiko Ueyama

Involvement of Rac1 in Activation of Multicomponent Nox1- and Nox3-Based NADPH Oxidases

ABSTRACT Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47 phox and p67 phox or homologous regulators called Noxo1 and Noxa1, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxa1 depends on Noxo1, through tail-to-tail interactions between these proteins. Noxa1 can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxa1. However, membrane targeting of Noxa1 is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxa1 mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22 phox transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22 phox binding sites (SH3 domains) of the Nox organizers (p47 phox or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67 phox or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67 phox or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.

Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation

Dual oxidases (Duoxl and Duox2) are plasma membrane‐targeted hydrogen peroxide generators that support extracellular hemoperoxidases. Duox activator 2 (Duoxa2), initially described as an endoplasmic reticulum resident protein, functions as a maturation factor needed to deliver active Duox2 to the cell surface. However, less is known about the Duoxl/ Duoxal homologues. We identified four alternatively spliced Duoxal variants and explored their roles in Duox subcellular targeting and reconstitution. Duoxl and Duox2 are functionally rescued by Duoxa2 or the Duoxal variants that contain the third coding exon. All active maturation factors are cotransported to the cell surface when coexpressed with either Duoxl or Duox2, consistent with detection of endogenous Duoxal on apical plasma membranes of the airway epithelium. In contrast, the Duoxa proteins are retained in the endoplasmic reticulum when expressed without Duox. Duoxl/Duoxala and Duox2/Duoxa2 pairs produce the highest levels of hydrogen peroxide, as they undergo Golgi‐based carbohydrate modifications and form stable cell surface complexes. Cross‐functioning pairs that do not form stable complexes produce less hydrogen peroxide and leak superoxide. These findings suggest Duox activators not only promote Duox maturation, but they function as part of the hydrogen peroxide‐generating enzyme.—Morand, S., Ueyama, T., Tsujibe, S., Saito, N., Korzeniowska, A., Leto, T. L. Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation. FASEB J. 23, 1205–1218 (2009)

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca2+ signals through store-operated Ca2+ (SOC) channels, activated by the depletion of Ca2+ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca2+ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca2+ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca2+ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

Enzymological Analysis of Mutant Protein Kinase Cγ Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca2+ Homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cγ (PKCγ). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCγ kinase activity. To determine the effect of these mutations on PKCγ function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCγs. We found that wild-type PKCγ, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCγ. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCγ. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCγs was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ɛ in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ɛ, but not PKC-δ, concentrated around the beads. PKC-ɛ accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ɛ was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ɛ, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ɛ expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (ɛRD) and the first variable region (ɛV1) of PKC-ɛ inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ɛ and ɛRD, but decreased in cells expressing ɛV1, suggesting that the ɛRD and ɛV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ɛ in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.

Superoxide production at phagosomal cup/phagosome through βI protein kinase C during FcγR-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcγR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. βI PKC, εPKC, and diacylglycerol kinase (DGK) β dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47phox, an essential cytosolic component of NADPH oxidase and a substrate for βI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O2−) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O2− production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of βI PKC is involved in O2− production, and that O2− production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKβ plays a prominent role in regulation of O2− production during FcγR-mediated phagocytosis.

Subtype- and species-specific knockdown of PKC using short interfering RNA (siRNA)

RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human alphaPKC and human deltaPKC and applied them into mammalian cells to suppress the expression of endogenous alphaPKC and deltaPKC, respectively. Each siRNA for alpha or deltaPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of deltaPKC reached maximum 48-72h after the transfection of siRNA. In addition, the siRNA targeting rat deltaPKC suppressed endogenous and exogenous rat deltaPKCs but not human deltaPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species.

Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis

We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40phox interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47phox and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341–360. Furthermore, we demonstrated sequential interactions of p67phox with phagosomes involving adaptor proteins, p47phox and p40phox, during FcγR-mediated phagocytosis. Although p67phox is not targeted to phagosomes by itself, p47phox functions as an adaptor for the ternary complex (p47phox-p67phox-p40phox) in early stages of phagocytosis before phagosome closure, while p40phox functions in later stages after phagosomal closure. Interestingly, a mutated “open” form of p40phox linked p47phox to closed phagosomes and prolonged p47phox and p67phox retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.

Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes

Phagocytes spontaneously ruffle as they probe their environment and take up antigens. These cells are uniquely enriched in phosphatidic acid, which is necessary for ruffling and macropinocytosis.

Isoform-Specific Membrane Targeting Mechanism of Rac during FcγR-Mediated Phagocytosis: Positive Charge-Dependent and Independent Targeting Mechanism of Rac to the Phagosome

Rac1 and Rac2 are capable of stimulating superoxide production in vitro, but their targeting and functional mechanisms are still unknown. In the present study, we found that Rac1, 2, and 3 all accumulate at the phagosome during FcγR-mediated phagocytosis, and that the order of accumulation (Rac1 > Rac3 > Rac2) depends on the net positive charge in their polybasic (PB) regions (183–188 aa). Although all GFP-tagged prenylated PB regions of Rac isoforms (GFP-Rac(PB)) and GFP-tagged prenylated 6 Ala (GFP-6A) accumulated during phagocytosis, GFP-Rac2(PB) and GFP-6A showed weak accumulation at the phagosome through a linear structure connecting the phagosome and endomembranes. The PB region of Rac1 showed strong phospholipid interaction with PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and phosphatidic acid, however, that of Rac2 did not. Constitutively active Rac2, GFP-Rac2(Q61L), was predominantly localized at the endomembranes; these endomembranes fused to the phagosome through the linear structure during phagocytosis, and this accumulation mechanism did not depend on positive charge in the PB region. Our conclusion is that Rac1 directly targets to the phagosome using the positively charged PB region and this accumulation mechanism is likely enhanced by the phospholipids. In addition to this mechanism, Rac2 has a positive charge-independent mechanism in which Rac2 initially targets to endomembranes and then these endomembranes fuse to the phagosome.