Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

The protein kinase TAK1 (transforming growth factor-beta-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38alpha MAPK down-regulates TAK1 and showed that p38alpha MAPK phosphorylates TAB1 at Ser(423) and Thr(431). In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser(372) and Ser(524)) and three on TAB3 (Ser(60), Thr(404) and Ser(506)) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser(372) and Ser(524) of TAB2 are not phosphorylated by pathways dependent on p38alpha/beta MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser(60) and Thr(404) of TAB3 appear to be phosphorylated directly by p38alpha MAPK, whereas Ser(506) is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38alpha MAPK. Studies using TAB1(-/-) MEFs indicate important roles for TAB1 in recruiting p38alpha MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser(60) and Thr(404) and in inhibiting the dephosphorylation of TAB3 at Ser(506). TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFalpha was able to stimulate detectable TAK1 activity in TAB1(-/-) MEFs. Surprisingly, the IL-1 and TNFalpha-stimulated activation of MAPK cascades and IkappaB (inhibitor of nuclear factor kappaB) kinases were similar in TAB1(-/-), MEKK3(-/-) [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFalpha-induced activation of these signalling pathways in TAB1(-/-) and MEKK3(-/-) MEFs.     

Tpl2 (tumor progression locus 2) phosphorylation at Thr290 is induced by lipopolysaccharide via an Ikappa-B Kinase-beta-dependent pathway and is required for Tpl2 activation by external signals

The serine-threonine protein kinase encoded by the tumor progression locus 2 (Tpl2) proto-oncogene transduces Toll-like receptor and death receptor signals in a variety of cell types. Here we show that Tpl2 undergoes phosphorylation at Thr(290) both in cells overexpressing Tpl2 and in cells stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha and that phosphorylation on this site parallels Tpl2 activation. Reconstitution of Tpl2(-/-) macrophages with wild type Tpl2 or Tpl2 T290D restored ERK activation by LPS, whereas reconstitution of the same cells with Tpl2 T290A did not, suggesting that phosphorylation at Thr(290) is required for the physiological activation of Tpl2 by external signals. Both the wild type Tpl2 and the kinase-inactive mutant Tpl2 K167M undergo Thr(290) phosphorylation, suggesting that Thr(290) may be a site of trans-phosphorylation rather than auto-phosphorylation. Pretreatment of 293 cells and primary macrophages with the Ikappa-B kinase-beta (IKKbeta) inhibitor PS-1145 blocked Tpl2 phosphorylation at Thr(290), suggesting that phosphorylation depends on IKKbeta, an obligatory positive regulator of Tpl2. We conclude that Tpl2 phosphorylation at Thr(290) is induced by LPS, depends on IKKbeta, and is required for the physiological activation of Tpl2 by external signals.     

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.     

A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa.

Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants and encodes a Ser/Thr protein kinase. We have mapped the cot-1 mutation to a single base change resulting in a His to Arg substitution at amino acid 351, which resides within the catalytic domain. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW COT1 isoform (67-kDa) present in the wild-type was not detected in cot-1 grown at the restrictive temperature. Similarly, this isoform was not detected in cot-3 or cot-5 strains, when grown at restrictive temperatures. Reduced levels of Ser/Thr kinase activity and an increase in type 1 and type 2B phosphatase (calcineurin) activities were measured in a cot-1 background. Apparent changes in the phosphorylation state of the p150(Glued) subunit of the dynactin cytoskeletal motor component (encoded by ro-3, a suppressor of cot-1) and evidence of in vitro physical interactions between COT1 and calcineurin indicate a functional linkage among COT1 kinase, type 2B phosphatase, and dynactin.     

Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.     

Cell elongation and branching are regulated by differential phosphorylation states of the nuclear Dbf2-related kinase COT1 in Neurospora crassa

Dysfunction of the Neurospora crassa nuclear Dbf2-related kinase COT1 leads to cessation of tip extension and massive induction of new sites of growth. To determine the role phosphorylation plays in COT1 function, we mutated COT1 residues corresponding to positions of highly conserved nuclear Dbf2-related phosphorylation sites. Analyses of the point-mutation cot-1 strains (mimicking non- and constitutively phosphorylated states) indicate the involvement of COT1 phosphorylation in the regulation of hyphal elongation and branching as well as asexual development by altering cell wall integrity and actin organization. Phosphorylation of COT1's activation segment (at Ser417) is required for proper in vitro kinase activity, but has only a limited effect on hyphal growth. In marked contrast, even though phosphorylation of the C-terminal hydrophobic motif (at Thr589) is crucial for all COT1 functions in vivo , the lack of Thr589 phosphorylation did not significantly affect in vitro COT1 kinase activity. Nevertheless, its regulatory role has been made evident by the significant increase observed in COT1 kinase activity when this residue was substituted in a manner mimicking constitutive phosphorylation. We conclude that COT1 regulates elongation and branching in an independent manner, which is determined by its phosphorylation state.     

Common inhibitory serine sites phosphorylated by IRS-1 kinases, triggered by insulin and inducers of insulin resistance

The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the IkappaB kinase beta (IKKbeta) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKKbeta shares a repertoire of seven potential target sites on IRS-1 with protein kinase C zeta (PKCzeta), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKKbeta and PKCzeta when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKCzeta overlap with IRS kinases triggered by inducers of insulin resistance, such as IKKbeta, to phosphorylate IRS-1 on common Ser sites.     

Mechanistic studies of the mitotic activation of Mos

The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2beta. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix alphaC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix alphaC.     

A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun

We have used phospho-specific antibodies to re-examine the multisite phosphorylation of c-Jun in murine RAW macrophages and embryonic fibroblasts. Our results indicate that JNK isoforms are required and sufficient for the phosphorylation of Thr91 and Thr93, as well as the phosphorylation of Ser63 and Ser73, in response to LPS or anisomycin in macrophages and TNFalpha or anisomycin in fibroblasts. However, the phorbol ester (TPA) and EGF-induced phosphorylation of Ser63 and Ser73 is mediated by ERK1/ERK2, as well as JNK1/JNK2, in fibroblasts from wild-type mice and by ERK1/ERK2 alone in fibroblasts from JNK-deficient mice. The phosphorylation of Thr239 is catalysed by GSK3 and the phosphorylation of Ser243 by an as yet unidentified protein kinase. The inhibition of GSK3 is not required for the dephosphorylation of Thr239 in response to LPS, and nor is the phosphorylation of Thr91 and Thr93 required for the TPA- or EGF-induced dephosphorylation of Thr239 in fibroblasts. The agonist-induced dephosphorylation of Thr239 may involve a conformational change that exposes Thr239 to dephosphorylation and/or the activation of a Thr239 phosphatase.     

Importance of autophosphorylation at Ser186 in the A-loop of salt inducible kinase 1 for its sustained kinase activity

Autophosphorylation is an important mechanism by which protein kinases regulate their own biological activities. Salt inducible kinase 1 (SIK1) is a regulator in the feedback cascades of cAMP-mediated gene expression, while its kinase domain also features autophosphorylation activity. We provide evidence that Ser186 in the activation loop is the site of autophosphorylation and essential for the kinase activity. Ser186 is located at the +4 position of the critical Thr residue Thr182, which is phosphorylated by upstream kinases such as LKB1. The relationship between phosphorylation at Ser186 and at Thr182 in COS-7 cells indicates that the former is a prerequisite for the latter. Glycogen synthase kinase-3beta (GSK-3beta) phosphorylates Ser/Thr residues located at the fourth position ahead of the pre-phosphorylated Ser/Thr residues, and inhibitors of GSK-3beta reduce the phosphorylation at Thr182. The results of an in vitro reconstitution assay also indicate that GSK-3beta could be the SIK1 kinase. However, overexpression and knockdown of GSK-3beta in LKB1-defective HeLa cells suggests that GSK-3beta alone may not be able to phosphorylate or activate SIK1, indicating that LKB1 may play a crucial role by phosphorylating SIK1 at Thr182, possibly as an initiator of the autophosphorylation cascade, and GSK-3beta may phosphorylate SIK1 at Thr182 by recognizing the priming-autophosphorylation at Ser186 in cultured cells. This may also be the case for the other isoform SIK2, but not for SIK3.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.     

Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha

TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38alpha at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre-incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the first residue phosphorylated by SAPK2a/p38alpha that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS-stimulated macrophages, and by proinflammatory cytokines or osmotic shock in epithelial KB cells or embryonic fibroblasts. The activation of TAK1 by TNF-alpha, IL-1 or osmotic shock was also enhanced in embryonic fibroblasts from SAPK2a/p38alpha-deficient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38alpha-mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38alpha but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).     

Resistance exercise decreases eIF2Bepsilon phosphorylation and potentiates the feeding-induced stimulation of p70S6K1 and rpS6 in young men

We investigated the effect of resistance exercise and feeding on the activation of signaling proteins involved in translation initiation. Nine young men (23.7+/-0.41 yr; BMI=25.5+/-1.0 kg/m2; means+/-SE) were tested twice after they performed a strenuous bout of unilateral resistance exercise, such that their contralateral leg acted as a nonexercised comparator, in either the fasted and fed [1,000 kJ, each 90 min (3 doses): 10 g protein, 41 g carbohydrate, 4 g fat] states. Muscle biopsies were obtained 6 h postexercise from both legs, resulting in four experimental conditions: rest-fasted, rest-fed, exercise-fasted, and exercise-fed. Feeding increased PKB/Akt (Ser473) phosphorylation (P<0.05), while exercise increased the phosphorylation of Akt and the downstream 70 kDa S6 protein kinase (p70S6K1, Thr389) and ribosomal protein S6 (rpS6, Ser235/236, Ser240/244; all P<0.05). The combination of resistance exercise and feeding increased the phosphorylation of p70S6K1 (Thr389) and rpS6 (Ser240/244) above exercise alone (P<0.05). Exercise also reduced phosphorylation of the catalytic epsilon subunit of eukaryotic initiation factor 2B (eIF2Bepsilon, Ser540; P<0.05). Mammalian target of rapamycin (mTOR, Ser2448), glycogen synthase kinase-3beta (GSK-3beta, Ser9), and focal adhesion kinase (FAK, Tyr576/577) phosphorylation were unaffected by either feeding or resistance exercise (all P>0.14). In summary, feeding resulted in phosphorylation of Akt, while resistance exercise stimulated phosphorylation of Akt, p70S6K1, rpS6, and dephosphorylation eIF2Bepsilon with a synergistic effect of feeding and exercise on p70(S6K1) and its downstream target rpS6. We conclude that resistance exercise potentiates the effect of feeding on the phosphorylation and presumably activation of critical proteins involved in the regulation of muscle protein synthesis in young men.     

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1): implication of protein kinase CK2.

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis

Bacterially expressed heterotrimeric (alpha1, beta1, and gamma1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the alpha-subunit, viz. Thr258 and Ser485 (Ser491 in the alpha2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the beta1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the alpha-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.     

Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O(2)(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O(2)(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O(2)(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I(A) PI3K, but not translocation of the p110gamma subunit of class I(B) PI3K, to the neutrophil membrane. Inhibition of protein kinase Czeta (PKCzeta) inhibited MBP-stimulated O(2)(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta (Thr(505)) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I(A) PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I(A) PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O(2)(-) production in response to MBP stimulation.