The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42

PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. Here we describe an unexpected link between these two systems. We have identified a family of mammalian Par6 proteins that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homologue of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3.     

Solution structure of atypical protein kinase C PB1 domain and its mode of interaction with ZIP/p62 and MEK5

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 PKCiota 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 betabetaalpha 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 PKCiota PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKCiota-ZIP/p62 complex by mutational analysis. Interestingly, in the PKCiota PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motif-presenting surface, suggesting dual roles for the PKCiota 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.     

A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935), we previously showed that p47(phox), a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22(phox), a subunit of the NADPH oxidase component flavocytochrome b(558), also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22(phox). We identified two protein kinase families capable of phosphorylating p22(phox): 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47(phox) and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47(phox). In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22(phox). These findings suggest that phosphorylation of p22(phox) by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.     

Structure of a cell polarity regulator, a complex between atypical PKC and Par6 PB1 domains

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCiota and Par6alpha PB1 domains to a resolution of 1.5 A. Both PB1 domains adopt a ubiquitin fold. PKCiota PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6alpha PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCiota PB1 and the conserved lysine residue on the Par6alpha PB1. Structural comparison of the PKCiota and Par6alpha PB1 complex with the p40phox and p67phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.     

Association of the atypical protein kinase C-interacting protein p62/ZIP with nerve growth factor receptor TrkA regulates receptor trafficking and Erk5 signaling

Previous work demonstrated an essential role for the atypical protein kinase C interacting protein, p62, in neurotrophin survival and differentiation signaling. Here we show that p62 interacts not only with TrkA but also with TrkB and TrkC, which are the primary receptors for brain-derived neurotrophic factor and neurotrophin-3. The interaction of p62 with TrkA requires the kinase activity of TrkA. Mapping analysis indicates that p62 does not compete with Shc for binding to TrkA, and p62 association was confined to the juxtamembrane region of TrkA, amino acids 472-493. By immunofluorescence the colocalization of p62 and TrkA was observed 30 min post-nerve growth factor treatment within overlapping vesicular structures. Upon subcellular fractionation, activated TrkA colocalized to an endosomal compartment and p62 was coassociated with the receptor post-nerve growth factor stimulation. Moreover, an absence of p62 blocked internalization of TrkA without an effect on phosphorylation of either TrkA or MAPK; however, Erk5 signaling was selectively abrogated. We propose that p62 plays a novel role in connecting receptor signals with the endosomal signaling network required for mediating TrkA-induced differentiation.     

The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway

The atypical protein kinase C (aPKC)-interacting protein, p62, has previously been shown to interact with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). Here we demonstrate that the inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this we show that p62 selectively interacts with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, we demonstrate that the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6.     

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase

The plant hormone abscisic acid (ABA) triggers production of reactive oxygen species (ROS) in guard cells via the AtrbohD and AtrbohF NADPH oxidases, leading to stomatal closure. The ABA-activated SnRK2 protein kinase open stomata 1 (OST1) (SRK2E/SnRK2.6) acts upstream of ROS in guard cell ABA signaling. Here, we report that OST1 phosphorylates Ser13 and Ser174 on AtrbohF. In addition, substitution of Ser174 to Ala results in a ∼40% reduction in the phosphorylation of AtrbohF by OST1. We also show that OST1 physically interacts with AtrbohF. These results provide biochemical evidence suggesting that OST1 regulates AtrbohF activity.

Structured summary

MINT-7260179, MINT-7260147, MINT-7260165: OST1 (uniprotkb:Q940H6) phosphorylates (MI:0217) ATRBOHF (uniprotkb:O48538) by protein kinase assay (MI:0424)MINT-7260208: OST1 (uniprotkb:Q940H6) and ATRBOHF (uniprotkb:O48538) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)     

Lectin-mediated retention of p62 facilitates p62-E1 heterodimerization in endoplasmic reticulum of Semliki Forest virus-infected cells

The Semliki Forest virus (SFV) spike subunits p62 and E1 are made from a common coding unit in the order p62-E1. The proteins are separated by the host signal peptidase upon translocation into the endoplasmic reticulum (ER). Shortly thereafter, p62 and E1 form heterodimers. Heterodimerization preferentially occurs between subunits derived from the same translation product, so-called cis heterodimerization. As the p62 protein has the capacity to leave the ER in the absence of E1, it has been postulated that there exists a retention mechanism for the p62 protein, putatively at or near the translocon, in the ER in order to promote cis heterodimerization (B. U. Barth and H. Garoff, J. Virol. 71:7857-7865, 1997). Here we show that there exists such a mechanism, that it is at least in part mediated by the ER chaperones calnexin and calreticulin, and that the retention is important for efficient cis heterodimerization.     

Positioning atypical protein kinase C isoforms in the UV-induced apoptotic signaling cascade.

Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzyme-like protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade.     

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A-protein kinase C cross talk

Dopamine cellular signaling via the D(1) receptor (D(1)R) involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D(1)R-mediated inhibition of NADPH oxidase activity involves cross talk between PKA and a specific PKC isoform(s). In HEK-293 cells heterologously expressing human D(1)R (HEK-hD(1)), fenoldopam, a D(1)R agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D(1)R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two PKA inhibitors, H89 (10μmol/L; 88±8.1%) and Rp-cAMP (10μmol/L; 97.7±6.7%), and two PKC inhibitors, bisindolylmaleimide I (1μmol/L; 94±6%) and staurosporine (10nmol/L; 93±8%), which by themselves had no effect (n=4-8/group). The inhibitory effect of PMA (1μmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%; n=5 or 6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθ(S676), an effect that was also blocked by H89. Fenoldopam (1μmol/L) also increased PKCθ(S676) in HEK-hD(1) and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross talk between PKA and PKCθ plays an important role in the D(1)R-mediated negative regulation of NADPH oxidase activity in human kidney cells.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages.

Activation of the superoxide-generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium-free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester- and zymosan-induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na+/H+ antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide-generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.     

Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.     

HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex

Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.     

Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.     

The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex

The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-kappaB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCzeta. Ajuba recruits TRAF6 to p62 and in vitro activates PKCzeta activity and is a substrate of PKCzeta. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-kappaB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-kappaB activity following IL-1 stimulation. We propose that Ajuba is a new cytosolic component of the IL-1 signaling pathway modulating IL-1-induced NF-kappaB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex.     

RGSZ1 interacts with protein kinase C interacting protein PKCI-1 and modulates mu opioid receptor signaling

Protein kinase C interacting protein (PKCI-1) was identified among the potential interactors from a yeast two hybrid screen of human brain library using N terminal of RGSZ1 as a bait. The cysteine string region, unique to the RZ subfamily, contributes to the observed interaction because PKCI-1 interacted with N-terminus of RGS17 and GAIP, but not with that of RGS2 or RGS7 where cysteine string motif is absent. The interaction between RGSZ1 and PKCI-1 was confirmed by coimmunoprecipitation and immunofluorescence. PKCI-1 and RGSZ1 could be detected by coimmunoprecipitation using 14-3-3 antibody in cells transfected with PKCI-1 or RGSZ1 respectively, but when transfected with PKCI-1 and RGSZ1 together, only RGSZ1 could be detected. Phosphorylation of Galphaz by protein kinase C (PKC) reduces the ability of the RGS to effectively function as GTPase accelerating protein for Galphaz, and interferes with ability of Galphaz to interact with betagamma complex. We investigated the roles of 14-3-3 and PKCI-1 in phosphorylation of Galphaz. Phosphorylation of Galphaz by PKC was inhibited by 14-3-3 and the presence of PKCI-1 did not provide any further inhibition. PKCI-1 interacts with mu opioid receptor and suppresses receptor desensitization and PKC related mu opioid receptor phosphorylation [W. Guang, H. Wang, T. Su, I.B. Weinstein, J.B. Wang, Mol. Pharmacol. 66 (2004) 1285.]. Previous studies have also shown that mu opioid receptor co-precipitates with RGSZ1 and influence mu receptor signaling by acting as effector antagonists [J. Garzon, M. Rodriguez-Munoz, P. Sanchez-Blazquez, Neuropharmacology 48 (2005) 853., J. Garzon, M. Rodriguez-Munoz, A. Lopez-Fando, P. Sanchez-Blazquez Neuropsychopharmacology 30 (2005) 1632.]. Inhibition of cAMP by mu opioid receptor was significantly reduced by RGSZ1 and this effect was enhanced in combination with PKCI-1. Our studies thus provide a link between the previous observations mentioned above and indicate that the major function of PKCI-1 is to modulate mu opioid receptor signaling pathway along with RGSZ1, rather than directly mediating the Galphaz RGSZ1 interaction.     

JFC1, a novel tandem C2 domain-containing protein associated with the leukocyte NADPH oxidase

We have employed a yeast two-hybrid system to screen a B lymphoblast-derived cDNA library, searching for regulatory components of the NADPH oxidase. Using as bait the C-terminal half of p67(phox), which contains both Src homology 3 domains, we have cloned JFC1, a novel human 62-kDa protein. JFC1 possesses two C2 domains in tandem. The C2A domain shows homology with the C2B domain of synaptotagmins. JFC1 mRNA was abundantly expressed in bone marrow and leukocytes. The expression of JFC1 in neutrophils was restricted to the plasma membrane/secretory vesicle fraction. We confirmed JFC1-p67(phox) association by affinity chromatography. JFC1-containing beads pulled down both p67(phox) and p47(phox) subunits from neutrophil cytosol, but when the recombinant proteins were used, only p67(phox) bound to JFC1, indicating that JFC1 binds to the cytosolic complex via p67(phox) without affecting the interaction between p67(phox) and p47(phox). In contrast to synaptotagmins, JFC1 was unable to bind to inositol 1,3,4,5-tetrakisphosphate but did bind to phosphatidylinositol 3,4,5-trisphosphate and to a lesser extent to phosphatidylinositol 3,4-diphosphate. From the data presented here, it is proposed that JFC1 is acting as an adaptor protein between phosphatidylinositol 3-kinase products and the oxidase cytosolic complex.     

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.