Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Differential requirement for p56lck in HIV-tat versus TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal kinase, and apoptosis

HIV-tat protein, like TNF, activates a wide variety of cellular responses, including NF-kappa B, AP-1, c-Jun N-terminal kinase (JNK), and apoptosis. Whether HIV-tat transduces these signals through the same mechanism as TNF is not known. In the present study we investigated the role of the T cell-specific tyrosine kinase p56lck in HIV-tat and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, an isogeneic lck-deficient T cell line. Treatment with HIV-tat protein activated NF-kappa B, degraded I kappa B alpha, and induced NF-kappa B-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56lck kinase. These effects were specific to HIV-tat, as activation of NF-kappa B by PMA, LPS, H2O2, and TNF was minimally affected. p56lck was also found to be required for HIV-tat-induced but not TNF-induced AP-1 activation. Similarly, HIV-tat activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. HIV-tat also induced cytotoxicity, activated caspases, and reactive oxygen intermediates in Jurkat cells, but not in JCaM1 cells. HIV-tat activated p56lck activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56lck tyrosine kinase reversed the HIV-tat-induced NF-kappa B activation and cytotoxicity. Overall, our results demonstrate that p56lck plays a critical role in the activation of NF-kappa B, AP-1, JNK, and apoptosis by HIV-tat protein but has minimal or no role in activation of these responses by TNF.     

Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta.

Protein kinase C zeta (zeta PKC) is critically involved in the control of a number of cell functions, including proliferation and nuclear factor kappa B (NF-kappa B) activation. Previous studies indicate that zeta PKC is an important step downstream of Ras in the mitogenic cascade. The stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase (MAPK), which is required for cell growth. MAPK is activated by phosphorylation by another kinase named MAPK kinase (MEK), which is the substrate of a number of Ras-activated serine/threonine kinases such as c-Raf-1 and B-Raf. We show here that MAPK and MEK are activated in vivo by an active mutant of zeta PKC, and that a kinase-defective dominant negative mutant of zeta PKC dramatically impairs the activation of both MEK and MAPK by serum and tumour necrosis factor (TNF alpha). The stimulation of other kinases, such as stress-activated protein kinase (SAPK) or p70S6K, is shown here to be independent of zeta PKC. The importance of MEK/MAPK in the signalling mechanisms activated by zeta PKC was addressed by using the activation of a kappa B-dependent promoter as a biological read-out of zeta PKC.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

Mechanism of inhibition of Raf-1 by protein kinase A.

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.     

Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.     

Tumor necrosis factor signal transduction. Cell-type-specific activation and translocation of protein kinase C

We have investigated the changes in protein kinase C (PKC) activity after treatment of several cell lines with TNF. Binding studies with [3H]phorbol dibutyrate (PBt2) on whole cells revealed rapid and transient activation of PKC in Jurkat, K562, and U937 cells with a maximum of phorbol ester binding at 6 min after TNF treatment. As shown by Scatchard analysis, the TNF-induced increase of [3H]PBt2 binding reflected increments of phorbol ester binding site numbers rather than greater binding affinities. Upon subfractionation of TNF-treated U937 cells a transient increase of PBt2 binding in the membrane fraction was accompanied by a long term loss of PBt2-binding in the cytosol, indicating a TNF-induced translocation of PKC from the cytosol to the cell membrane. With histone III-S as a substrate, the determination of specific PKC activity revealed similar kinetics of PKC translocation in U937 cells. TNF also induced PKC translocation in K562 and Jurkat cells. However, although TNF caused long term down-regulation of cytosolic PKC activity in U937 cells, the cytosolic PKC activity only transiently decreased in both Jurkat and K562 cells and then recovered to near basal levels. In the human nonmalignant fibroblast cell line CCD18, PKC was not activated by TNF. Our data suggest that PKC activation may play a major role in TNF signal transduction in some, but not all target cells.     

Murine T-cell differentiation antigen CD8 is a direct substrate of protein kinase C

Murine T cell differentiation antigen CD8 alpha (Lyt-2) is phosphorylated in vivo after phorbol 12-myristate 13-acetate (PMA) treatment of cells. Concanavalin A,dibutyryl cAMP and calcium ionophore are unable to stimulate phosphate incorporation into CD8 alpha. Depletion of cellular protein kinase C (PKC) by prolonged PMA treatment abolished this phosphorylation, suggesting that PKC is required for this effect. Using the amino acid sequence derived from cloning CD8 alpha, peptides encompassing both possible intracellular phosphorylation sites were made and used to test the ability of various kinases to phosphorylate CD8 alpha sequences. Only the proximal serine peptide was a kinase substrate, and of PKC, cAMP-dependent kinase and the multifunctional calcium/calmodulin-dependent kinase, only PKC was able to phosphorylate this peptide. These studies provide the first definitive evidence that CD8 alpha is a direct substrate of PKC.     

Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells.

HL60 and EL4 cells incubated with tumor necrosis factor-alpha (TNF-alpha) plus staurosporin, a potent inhibitor of protein kinases, showed at least 2-fold increased levels of nuclear factor-kappa B (NF-kappa B) activity compared with TNF-alpha alone both during rapid NF-kappa B activation from the cytosolic pool and protein synthesis-dependent NF-kappa B activation. NF-kappa B activation by phorbol 12-myristate 13-acetate (PMA) and interleukin-1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-alpha-induced increase in mRNA for the p51 subunit of NF-kappa B but interfered with any phorbol ester (PMA)-induced increase in p51 mRNA. Thus, induction of NF-kappa B and p51 mRNA by TNF-alpha was not mediated by a staurosporin-sensitive factor, but NF-kappa B activation by TNF-alpha was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-R alpha (TNF receptor type alpha) after staurosporin-treatment correlated with increased induction of NF-kappa B by TNF-alpha. Staurosporin-treatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-kappa B activation by TNF-alpha, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-R alpha by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-kappa B activation by TNF-alpha. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-R alpha phosphorylation.     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

Possible role of Ca2(+)-independent protein kinase C isozyme, nPKC epsilon, in thyrotropin-releasing hormone-stimulated signal transduction: differential down-regulation of nPKC epsilon in GH4C1 cells

Protein kinase C (PKC) molecular species of GH4C1 cells were analyzed after separation by hydroxyapatite column chromatography. A novel Ca2(+)-independent PKC, nPKC epsilon, was identified together with two conventional Ca2(+)-dependent PKCs, PKC alpha and beta II by analysis of kinase and phorbol ester-binding activities, immunoblotting using isozyme-specific antibodies, and Northern blotting. These PKCs are down-regulated differently when cells are stimulated by outer stimuli; phorbol esters deplete PKC beta II and nPKC epsilon from the cells more rapidly than PKC alpha, whereas thyrotropin-releasing hormone (TRH) at 200 nM depletes nPKC epsilon exclusively with a time course similar to that induced by phorbol esters. However, translocation of PKC alpha and beta II to the membranes is elicited by both TRH and phorbol esters. These results suggest that TRH and phorbol ester activate PKC alpha and beta II differently but that nPKC epsilon is stimulated similarly by both stimuli. Thus, in GH4C1 cells, Ca2(+)-independent nPKC epsilon may play a crucial role distinct from that mediated by Ca2(+)-dependent PKC alpha and beta II in a cellular response elicited by both TRH and phorbol esters.     

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.     

IL-4 promotes anti-Ig-mediated protein kinase C translocation and reverses phorbol ester-mediated protein kinase C down-regulation in murine B cells

Co-stimulation of B lymphocytes with IL-4 plus nonmitogenic concentrations of anti-Ig antibodies, or protein kinase C (PKC) activators, drives resting B cells into DNA synthesis. Although cross-linking of the sIg receptors provokes the generation of the intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, the molecular mechanism utilized by IL-4R in murine B cells has not, as yet, been defined. In human B cells IL-4 has been shown to induce a transient rise in IP3 followed by a sustained elevation of cAMP. However, in murine B cells, IL-4 does not induce the release of IP3, Ca2+ mobilization, PKC translocation, or indeed modify signaling via the phosphoinositide pathway induced by ligation of sIg receptors. We now present evidence that, in murine B cells, IL-4 synergizes with nonmitogenic concentrations of anti-Ig to provoke translocation of PKC from the cytosol to membranes. In addition, the lymphokine up-regulates PKC levels and activity and prevents phorbol ester-induced PKC down-regulation in B cells. We therefore propose that (unknown) signals generated via IL-4R potentiate and/or prolong sIg-induced PKC activation. These observations may therefore provide a biochemical basis for explaining how IL-4 and anti-Ig synergize to induce B cell activation.     

Activation of cGMP-dependent protein kinase by protein kinase C

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.