Regulation of protein kinase C inactivation by Fas-associated protein with death domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

Involvement of protein kinase C in phorbol ester-induced sensitization of HeLa cells to cis-diamminedichloroplatinum(II)

We have investigated the effect of tumor promoting phorbol esters on the antiproliferative actions of several antitumor agents. Pretreatment of HeLa cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a significant (9-fold) increase in cellular sensitivity to cis-diamminedichloroplatinum(II) (CP). TPA also sensitized HeLa cells to melphalan (2.5-fold) but had no effect on the antiproliferative activity of bleomycin, doxorubicin, vincristine, or mitomycin C. The sensitization of HeLa cells by TPA was concentration-dependent up to 1 nM and paralleled the activation of protein kinase C by TPA measured in vitro. The maximum stimulation of protein kinase C (6-fold) was observed with 10 nM TPA. 4 alpha-Phorbol 12,13-didecanoate neither activated protein kinase C nor sensitized HeLa cells to CP. 4-O-Methyl-TPA, which does not affect cell cycle distribution of HeLa cells, also sensitized these cells to CP by 6-fold and activated protein kinase C by 3-fold. Inhibitors of protein kinase C, such as palmitoylcarnitine and sphingosine, antagonized PDBu-induced sensitization of HeLa cells to CP. The maximum sensitization of HeLa cells to CP required prolonged pretreatment (greater than or equal to 24 h) with phorbol esters but could not be explained by down-regulation of protein kinase C. For example, 4-O-methyl-TPA caused no down-regulation of protein kinase C. Moreover, TPA caused substantial down-regulation of protein kinase C (1% of control) in A-253 cells but failed to sensitize A-253 cells to CP. TPA (100 nM), however, activated protein kinase C in A-253 cells by 5.5-fold. Therefore, activation of protein kinase C by TPA appears to be necessary but not sufficient for cellular sensitization to CP. The sensitization of HeLa cells by TPA was associated with a concentration- and time-dependent increase in cellular platinum content. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) blocked sensitization of HeLa cells to CP as well as the increase in platinum content caused by a 24-h pretreatment with PDBu.     

Mechanisms mediating caspase activation in cell death

The initial activation of a caspase in a caspase cascade is a crucial event that determines whether a cell will ultimately undergo cell death. Although each cell contains a number of different caspases, only a small subset may be required for apoptosis in response to a specific stimulus. It now seems that each caspase cascade has two types of caspases involved, the upstream or class I caspases, and the downstream or class II caspases. Class I caspases are characterised by long amino-terminal prodomains that carry specific protein - protein interaction domains which mediate oligomerisation of caspases, often assisted by specific adaptor molecules. Oligomerisation appears to be sufficient for autocatalytic activation of class I caspases. Once the first caspase in the pathway has been activated, it processes downstream caspases initiating a cascade of amplifying events that lead to the apoptotic death of a cell. This article reviews our current understanding of mechanisms that mediate the activation of caspases.     

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.     

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-α, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-α, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gsα, but not by protein kinase C-α. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gsα- or forskolin-mediated stimulation. J. Cell. Biochem. 64:492–498. © 1997 Wiley-Liss, Inc.     

Regulation of caspase 9 through phosphorylation by protein kinase C zeta in response to hyperosmotic stress

Caspase 9 is a critical component of the mitochondrial or intrinsic apoptotic pathway and is activated by Apaf-1 following release of cytochrome c from mitochondria in response to a variety of stimuli. Caspase 9 cleaves and activates effector caspases, mainly caspase 3, leading to the demise of the cell. Survival signaling pathways can impinge on this pathway to restrain apoptosis. Here, we have identified Ser144 of human caspase 9as an inhibitory site that is phosphorylated in a cell-free system and in cells in response to the protein phosphatase inhibitor okadaic acid. Inhibitor sensitivity and interactions with caspase 9 indicate that the predominant kinase that targets Ser144 is the atypical protein kinase C isoform zeta (PKCzeta). Prevention of Ser144 phosphorylation by inhibition of PKCzeta or mutation of caspase 9 promotes caspase 3 activation. Phosphorylation of serine 144 in cells is also induced by hyperosmotic stress, which activates PKCzeta and regulates its interaction with caspase 9, but not by growth factors, phorbol ester, or other cellular stresses. These results indicate that phosphorylation and inhibition of caspase 9 by PKCzeta restrain the intrinsic apoptotic pathway during hyperosmotic stress. This work provides further evidence that caspase 9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis.     

Poly(I:C)-induced tumour cell death leads to DC maturation and Th1 activation

Dendritic cells (DCs) have the ability to generate peptide epitopes for MHC class I molecules derived from apoptotic tumour cells for direct recognition by cytotoxic T cells. This function has lead to DCs being used in vaccine strategies. In this study, we investigate the effect of inducing apoptosis in tumour cell lines using IFN-γ and poly(I:C), the subsequent maturation of the endocytosing DC and its ability to direct the resulting T cell response. We show that uptake of poly(I:C)-induced apoptotic tumour cells leads to DC maturation and activation with a Th1 cell polarising capacity. In contrast, these effects are not seen by DCs loaded with γ-irradiated apoptotic tumour cells. We propose that the manner in which tumour cells are induced to die can have a profound effect on the endocytosing DC and the resulting T cell response.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

Background: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop.Results: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC βII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC α and βII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme.Conclusions: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

Regulation of interleukin receptor-associated kinase (IRAK) phosphorylation and signaling by iota protein kinase C

We have previously shown that the activity of the interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for nerve growth factor (NGF)-induced activation of NF-kappaB and cell survival ((2002) J. Biol. Chem. 277, 28010-28018). Herein we demonstrate that NGF induces co-association of IRAK with atypical protein kinase C iota (PKC) and that the iota PKC.IRAK complex is recruited to the p75 neurotrophin receptor. Recruitment of IRAK to the receptor was dependent upon the activity of the iota PKC. Moreover, transfection of kinase-dead iota PKC blocked both NGF- and IL-1-induced IRAK activation and the activity of NF-kappaB. Hence, iota PKC lies upstream of IRAK in the kappaB pathway. Examining the primary structure of IRAK, we identified three putative PKC phosphorylation sites; iota PKC selectively phosphorylated peptide 1 (RTAS) within the death domain domain at Thr66, which is highly conserved among all IRAK family members. Mutation of Thr66 to Ala impaired the autokinase activity of IRAK and reduced its association with iota PKC but not TRAF6, resulting in impaired NGF- as well as IL-1-induced NF-kappaB activation. These findings provide insight into the underlying mechanism whereby IRAK regulates the kappaB pathway and reveal that IRAK is a substrate of iota PKC.     

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.     

Receptor for activated C kinase (RACK) and protein kinase C (PKC) in egg activation

In somatic cells, translocation of PKCs is facilitated by receptor for activated C kinase (RACK); however its involvement in egg activation is still elusive. We have followed the translocation pattern of conventional and novel PKCs (cPKCs and nPKCs, respectively) upon egg activation. Confocal microscopy indicated the expression and localization of RACK1, a specific receptor protein for cPKCs. Activation of MII eggs, led to translocation to the egg cortex of PKCα, βII and δ and the co-translocation of RACK1, with both PKCα and PKCβII. The association of PKC and actin, both known to be involved in cortical granules exocytosis (CGE) with RACK1, was demonstrated by co-immunoprecipitation. Egg activation resulted in an increased RACK1 level along with a decreased level of PKCβII. Based on these results, we suggest that upon egg activation, RACK1 shuttles activated cPKCs to the egg cortex, thus facilitating CGE.     

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.