Protein kinase C mu is negatively regulated by 14-3-3 signal transduction proteins

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3tau isoform has been shown to associate with protein kinase C theta and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3tau interacts with protein kinase C mu (PKCmu), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCmu and 14-3-3tau can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKCmu-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKCmu deletion mutants, the 14-3-3tau binding region is mapped within the regulatory C1 domain. Binding of 14-3-3tau to PKCmu is significantly enhanced upon phorbol ester stimulation of PKCmu kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3tau is not a substrate of PKCmu. In contrast 14-3-3tau strongly down-regulates PKCmu kinase activity in vitro. Moreover, overexpression of 14-3-3tau significantly reduced phorbol ester induced activation of PKCmu kinase activity in intact cells. We therefore conclude that 14-3-3tau is a negative regulator of PKCmu in T cells.     

The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28

We investigated the role of protein kinase C theta (PKCtheta) in the activation of the NF-kappaB cascade in primary human CD4(+) lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca(2+)-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4(+) T cells. The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells. Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.     

Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.     

Selective association of protein kinase C with 14-3-3 zeta in neuronally differentiated PC12 Cells. Stimulatory and inhibitory effect of 14-3-3 zeta in vivo

The 14-3-3 protein is a family of highly conserved acidic proteins found in a wide range of eukaryotes from yeast to mammals. 14-3-3 acts as an adapter protein and interacts with signaling molecules including protein kinase C (PKC). Although 14-3-3 zeta was originally characterized as an endogenous PKC inhibitor, it was reported to activate PKC in vitro, but the in vivo regulation of PKC by 14-3-3 is still not well understood. To examine the regulation of PKC by 14-3-3 in the cell, we have generated a sub-cell line, PC12-B3, that stably expresses FLAG epitope-tagged 14-3-3 zeta isoform in PC12 cells. Here we show that PKC-alpha and PKC-epsilon become associated with 14-3-3 zeta when the cells are neuronally differentiated by nerve growth factor. We found that the immunoprecipitate by anti-FLAG antibody contains constitutive and autonomous Ca(2+)-independent non-classical PKC activity. In contrast, the FLAG immunoprecipitate has no Ca(2+)-dependent classical PKC activity despite the fact that PKC-alpha is present in the FLAG immunoprecipitate from differentiated PC12-B3 cells. Our results show that the association with 14-3-3 zeta has distinct effects on classical PKC and non-classical PKC activity.     

Resistance to experimental autoimmune encephalomyelitis and impaired IL-17 production in protein kinase C theta-deficient mice

The protein kinase C theta (PKC theta) serine/threonine kinase has been implicated in signaling of T cell activation, proliferation, and cytokine production. However, the in vivo consequences of ablation of PKC theta on T cell function in inflammatory autoimmune disease have not been thoroughly examined. In this study we used PKC theta-deficient mice to investigate the potential involvement of PKC theta in the development of experimental autoimmune encephalomyelitis, a prototypic T cell-mediated autoimmune disease model of the CNS. We found that PKC theta-/- mice immunized with the myelin oligodendrocyte glycoprotein (MOG) peptide MOG(35-55) were completely resistant to the development of clinical experimental autoimmune encephalomyelitis compared with wild-type control mice. Flow cytometric and histopathological analysis of the CNS revealed profound reduction of both T cell and macrophage infiltration and demyelination. Ex vivo MOG(35-55) stimulation of splenic T lymphocytes from immunized PKC theta-/- mice revealed significantly reduced production of the Th1 cytokine IFN-gamma as well as the T cell effector cytokine IL-17 despite comparable levels of IL-2 and IL-4 and similar cell proliferative responses. Furthermore, IL-17 expression was dramatically reduced in the CNS of PKC theta-/- mice compared with wild-type mice during the disease course. In addition, PKC theta-/- T cells failed to up-regulate LFA-1 expression in response to TCR activation, and LFA-1 expression was also significantly reduced in the spleens of MOG(35-55)-immunized PKC theta-/- mice as well as in in vitro-stimulated CD4+ T cells compared with wild-type mice. These results underscore the importance of PKC theta in the regulation of multiple T cell functions necessary for the development of autoimmune disease.     

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.     

Critical role of novel Thr-219 autophosphorylation for the cellular function of PKCtheta in T lymphocytes

Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.     

Lipid raft targeting of hematopoietic protein tyrosine phosphatase by protein kinase C theta-mediated phosphorylation

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.     

The binding of PRAS40 to 14-3-3 proteins is not required for activation of mTORC1 signalling by phorbol esters/ERK

PRAS40 binds to the mTORC1 (mammalian target of rapamycin complex 1) and is released in response to insulin. It has been suggested that this effect is due to 14-3-3 binding and leads to activation of mTORC1 signalling. In a similar manner to insulin, phorbol esters also activate mTORC1 signalling, in this case via PKC (protein kinase C) and ERK (extracellular-signal-regulated kinase). However, phorbol esters do not induce phosphorylation of PRAS40 at Thr(246), binding of 14-3-3 proteins to PRAS40 or its release from mTORC1. Mutation of Thr(246) to a serine residue permits phorbol esters to induce phosphorylation and binding to 14-3-3 proteins. Such phosphorylation is apparently mediated by RSKs (ribosomal S6 kinases), which lie downstream of ERK. However, although the PRAS40(T246S) mutant binds to 14-3-3 better than wild-type PRAS40, each inhibits mTORC1 signalling to a similar extent. Our results show that activation of mTORC1 signalling by phorbol esters does not require PRAS40 to be phosphorylated at Thr(246), bind to 14-3-3 or be released from mTORC1. It is conceivable that phorbol esters activate mTORC1 by a distinct mechanism not involving PRAS40. Indeed, our results suggest that PRAS40 may not actually be involved in controlling mTORC1, but rather be a downstream target of mTORC1 that is regulated in response only to specific stimuli, such as insulin.     

Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with G?6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and G?6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with G?6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.     

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and phospholipase C gamma 1 are required for NF-kappa B activation and lipid raft recruitment of protein kinase C theta induced by T cell costimulation

The NF-kappaB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)theta. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)gamma1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-kappaB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCgamma1 failed to activate NF-kappaB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCgamma1 expression restored CD3/CD28-induced IkappaB kinase (IKK) activity as well as NF-kappaB DNA binding and transactivation. PKCtheta activated NF-kappaB in SLP-76- and PLCgamma1-deficient cells, showing that PKCtheta is acting further downstream. In contrast, Vav1-induced NF-kappaB activation was normal in SLP-76(-) cells, but absent in PLCgamma1(-) cells. CD3/CD28-stimulated recruitment of PKCtheta and IKKgamma to lipid rafts was lost in SLP-76- or PLCgamma1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCtheta to the immunological synapse strictly relied on the presence of SLP-76 and PLCgamma1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCgamma1 for NF-kappaB activation and raft translocation of PKCtheta and IKKgamma.     

Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells

In this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCalpha, a conventional Ca(2+)-dependent PKC, and to PKCtheta, a novel Ca(2+)-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCalpha activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.