The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCalpha

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKCalpha and PKCdelta mediated the apoptotic effect of PMA, whereas only PKCalpha was involved in the effect of the DAG-lactone. The PKCalpha selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKCalpha and PKCdelta. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKCalpha to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKCdelta predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.     

PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA)

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.     

Translocation of the classic protein kinase C isoforms in porcine oocytes: implications of protein kinase C involvement in the regulation of nuclear activity and cortical granule exocytosis

Protein kinase C (PKC) is a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The subcellular localization of classical PKCalpha, -betaI, and -gamma in the process of porcine oocyte maturation, fertilization, and parthenogenetic activation and their involvement in cortical granule (CG) exocytosis were investigated. The results of Western blot showed that PKCalpha, -betaI, and -gamma were expressed in the oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Confocal microscopy revealed that the three PKC isoforms were concentrated in the GV but evenly distributed in the cytoplasm of MII eggs. PKCalpha and -gamma were translocated to the plasma membrane soon after sperm penetration. cPKCs migrated into the pronucleus in fertilized eggs. Following treatment with a PKC activator, phorbol 12-myristate 13-acetate (PMA), CGs were released and PKCalpha and -gamma were translocated to the membrane. The CG exocytosis and PKC redistribution induced by PMA could be blocked by the PKC inhibitor staurosporine. Parthenogenetic stimulation with ionophore A23187 or electrical pulse also induced cPKC translocation and CG exocytosis. Eggs injected with PKCalpha isoform-specific antibody failed to undergo CG exocytosis after PMA treatment or fertilization. The results suggest that cPKCs, especially the alpha-isotype, regulate nuclear function and CG exocytosis in porcine eggs.     

Individual C1 domains of PKD3 in phorbol ester-induced plasma membrane translocation of PKD3 in intact cells

Protein kinase D3 is a novel member of the serine/threonine kinase family PKD. The regulatory region of PKD contains a tandem repeat of C1 domains designated C1a and C1b that bind diacylglycerol and phorbol esters, and are important membrane targeting modules. Here, we investigate the activities of individual C1 domains of PKD3 and their roles in phorbol ester-induced plasma membrane translocation of PKD3. Truncated C1a of PKD3 binds [(3)H]phorbol 12, 13-dibutyrate with high affinity, but no binding activity is detected for C1b. Meanwhile, mutations in C1a of truncated C1ab of PKD3 lead to the loss of binding affinity, while these mutations in C1b have little impact, indicating that C1a is responsible for most of the phorbol ester-binding activities of PKD3. C1a and C1b of the GFP-tagged full length PKD3 are then mutated to assess their roles in phorbol ester-induced plasma membrane translocation in intact cells. At low concentration of phorbol 12-myristate 13-acetate (PMA), the plasma membrane translocations of the C1a and C1ab mutants are significantly impaired, reflecting an important role of C1a in this process. However, at higher PMA concentrations, all C1 mutants exhibit increased rates of translocation as compared to that of wild-type PKD3, which parallel their enhanced activation by PMA, implying that PKD3 kinase activity affects membrane targeting. In line with this, a constitutive active PKD3-GFP translocates similarly as wild-type PKD3, while a kinase-inactive PKD3 shows little translocation up to 2 muM PMA. In addition, RO 31-8220, a potent PKC inhibitor that blocks PMA-induced PKD3 activation in vivo, significantly attenuates the plasma membrane translocation of wild-type PKD3 at different doses of PMA. Taken together, our results indicate that both C1a and the kinase activity of PKD3 are necessary for the phorbol ester-induced plasma membrane translocation of PKD3. PKC, by directly activating PKD3, regulates its plasma membrane localization in intact cells.     

Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver

Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors G?6850 (1 micromol/liter) and G?6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. G?6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.     

Specific subcellular targeting of PKCα and PKCε in normal and tumoral lactotroph cells by PMA-mitogenic stimulus

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCα and PKCε in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCα to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCε in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCα could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.     

The role of protein kinase C activation in signal transmission by interleukin 2

Role of protein kinase C (PKC) in interleukin (IL) 2-induced proliferation was investigated by utilizing two murine IL 2-dependent cell lines, CT6 and CTLL-2 cell lines. CT6 cells showed a marked proliferative response to phorbol 12-myristate 13-acetate (PMA), while CTLL-2 did not. PMA induced PKC translocation from cytosol to membrane only in a PMA-responsive cell line. IL 2 failed to stimulate PKC translocation in both cell lines. H-7, a potent and specific PKC inhibitor, however, inhibited the proliferation of both cell lines induced by IL 2. Taken collectively, IL 2 may induce PKC activation without its translocation.     

Protein kinase C translocation in intact vascular smooth muscle strips.

Using intact muscle strips from the bovine carotid artery, the time course of translocation of protein kinase C (PKC) from the cytosol to the membrane fraction was measured in response to various agonists that induce contractile responses. PKC activity was assessed by Ca2+/phospholipid-dependent phosphorylation of histone. Exposure of the muscle strips to phorbol ester (12-deoxyphorbol 13-isobutyrate) induced a rapid and sustained translocation of PKC from the cytosol to the membrane fraction, and a slowly developing but sustained contractile response. Histamine induced a comparable initial translocation of PKC to the membrane which then decreased somewhat to a stable plateau significantly above basal values. Histamine also led to a rapid and sustained increase in tension. Angiotensin I, which caused a rapid but transient contraction, induced a rapid initial translocation of PKC to the membrane. The membrane-associated PKC then declined to a stable plateau significantly lower than that seen after a histamine-induced response, and only slightly above the basal value. Endothelin, which induced a sustained contraction, caused a sustained translocation of PKC from the cytosol to the membrane. In contrast, although exposure to 35 mM-KCl induced a rapid and sustained contraction, it caused only a transient translocation of PKC; the membrane-associated PKC returned to its basal value within 20 min. These results demonstrate that PKC in intact smooth muscle can be rapidly translocated to the membrane and remains membrane-bound during sustained phorbol ester- or agonist-induced contractions, but that such a sustained translocation of PKC does not occur during prolonged stimulation with KCl.     

Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting

We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80-85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-epsilon. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.     

Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Cysteine-rich domains (Cys-domains) are ~50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes.     

Phorbol esters and calcium-mobilizing hormones increase membrane-associated protein kinase C activity in rat hepatocytes

Vasopressin, angiotensin II, epinephrine (alpha 1-adrenergic action) and phorbol 12-myristate 13-acetate (PMA) induce increases in membrane-associated protein kinase C activity concomitant with decreases in the cytosolic activity. The data indicate that the calcium-mobilizing hormones and the active phorbol ester induce translocation from the cytosol to the plasma membrane of this protein kinase. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked the translocation to the membrane of this protein kinase induced by PMA and vasopressin.     

Differential irreversible insertion of protein kinase C into phospholipid vesicles by phorbol esters and related activators.

Incubation of protein kinase C (PKC) alpha with phorbol 12,13-dibutyrate and phospholipid vesicles promoted a time-dependent irreversible insertion of the enzyme into the vesicles and the generation of a calcium-independent kinase activity. Calcium neither caused insertion nor influenced the insertion induced by the phorbol ester. The effect was strongly dependent on the phosphatidylserine concentration in the vesicle and could also be supported by other anionic phospholipids. An analysis of the structure-activity relations of PKC activators for the calcium-independent kinase activity revealed marked relative differences in potencies for binding and for insertion. Compounds such as phorbol 13-myristate 12-acetate and mezerein were very efficient at inducing insertion. In contrast, 12-deoxyphorbol esters and diacylglycerol were relatively inefficient at inducing insertion, requiring higher concentrations than expected from their binding affinities. The insertion of PKC alpha depended substantially on the length of the aliphatic esters in the 12- and 13-positions of the phorbol derivatives, and once again, potencies for insertion and binding were not directly proportional. Our findings suggest two different sites for ligand interaction on the molecule of PKC alpha with different structure-activity requirements. We speculate that the differential ability of compounds to promote insertion could contribute to the documented marked differences in the biological behavior of PKC activators.     

Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.     

Phenylarsine oxide and H2O2 plus vanadate induce reverse translocation of phorbol-ester-activated PKCbetaII

The intracellular localization of protein kinase C (PKC) is important for the regulation of its biological activity. Recently, it was reported that, whereas phorbol esters such as PMA induce prolonged translocation of PKC to the plasma membrane, with physiological stimuli, the translocation of PKC is transient and followed by rapid return to the cytoplasm. In addition, this membrane dissociation of PKC was shown to require both the kinase activity of PKC and the phosphorylation of its carboxyl terminus autophosphorylation sites. However, the detailed molecular mechanism of PKC reverse translocation remains obscure. We demonstrated that in porcine polymorphonuclear leucocytes (PMNs), phenylarsine oxide (PAO), a putative protein tyrosine phosphatase (PTPase) inhibitor, induced reverse translocation of PMA-stimulated PKCbetaII. Hydrogen peroxide (H(2)O(2)) in combination with vanadate, both of which are PTPase inhibitors, also induced reverse translocation of PKCbetaII. H(2)O(2) or vanadate alone had little effect on PMA-induced PKCbetaII translocation. Furthermore, genistein and ethanol, which are inhibitors of tyrosine kinase and phospholipase D, respectively, prevented the PKCbetaII reverse translocation induced by the PTPase inhibitors. These results indicate, for the first time, that the tyrosine phosphorylation/phospholipase D pathway may be involved in the process of membrane dissociation of PKC.     

Membrane targeting and cytoplasmic sequestration in the spatiotemporal localization of human protein kinase C alpha

In order to map the molecular determinants that dictate the subcellular localization of human protein kinase C alpha (hPKCalpha), full-length and deletion mutants of hPKCalpha were tagged with the green fluorescent protein (GFP) and transiently expressed in GH3B6 cells. We found that upon thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate stimulation, hPKCalpha-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKCalpha failed to translocate in single cells despite the presence of TRH receptors, as attested by the TRH-induced rise in intracellular calcium concentration in these cells. TRH-stimulated translocation of hPKCalpha-GFP from the cytoplasm to cell-cell contacts was a biphasic process: a fast (measured in seconds) and transient phase, followed by a slower (approximately 1 hour) and long lasting phase. The latter and the translocation induced by phorbol 12-myristate 13-acetate absolutely required the N-terminal V1 region. In contrast to the full-length hPKCalpha, the N-terminal regulatory domain alone or associated with the V3 hinge region was spontaneously and uniformly localized at the plasma membrane of single and apposed cells. However, treatment with the calcium chelator BAPTA/AM induced a differential cytoplasmic/nuclear redistribution of the regulatory domain, depending on its association with V3, which suggests the existence of a mechanism controlling the cytoplasmic sequestration of inactive hPKCalpha and involving the V3 region. By using other deletion mutants, we were able to map the sequence required for this sequestration to the C2+V3 regions. This work points to the existence of a complex interplay between membrane targeting and cytoplasmic sequestration in the control of the spatiotemporal localization of hPKCalpha.     

Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-kappaB and tumor necrosis factor alpha in LLC-PK1 cells

Fumonisin B1 (FB1), the most potent of the fumonisin mycotoxins, is a carcinogen and causes a wide range of species-specific toxicoses. FB1 modulates the activity of protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases that play important role in modulating a variety of biologic responses ranging from regulation of cell growth to cell death. Although it has been demonstrated that FB1 induces apoptosis in many cell lines, the precise mechanism of apoptosis is not fully understood. In this study, we investigated the membrane localization of various PKC isoforms, PKC enzyme activity, and its downstream targets, namely nuclear factor-kappa B (NF-kappaB), tumor necrosis factor alpha (TNFalpha), and caspase 3, in porcine renal epithelial (LLC-PK1) cells. FB1 repressed cytosol to membrane translocation of PKC-alpha, -delta, -epsilon, and -zeta isoforms over 24-72 h. The FB1-induced membrane PKC repression was corroborated by a concentration-dependent decrease in total PKC activity. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) for this duration also resulted in repressed PKC membrane localization and activity comparable to FB1. Exposure of cells to FB1 (10 microM) was associated with inhibition of cytosol to nuclear translocation of NF-kappaB and NF-kappaB-DNA binding at 72 h. The expression of TNFalpha was significantly inhibited at 24 and 48 h in response to 1 and 10 microM FB1. Increased caspase 3 activity was observed in LLC-PK1 cells exposed to > or =1 microM FB1 at 48 h. PMA also increased the caspase 3 activity at 24 and 48 h. Results suggest that FB1-induced apoptosis involves the activation of caspase 3, which is associated with the repression of PKC and possibly its down-stream effectors, NF-kappaB and TNFalpha.     

Differential localization of conventional protein kinase C isoforms during mouse oocyte development

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.     

The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon

The catalytic domain of overexpressed protein kinase C (PKC)-delta mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-delta/epsilonV5 and -epsilon/deltaV5, by swapping the V5 domains of PKC-delta and -epsilon. PKC-delta/epsilonV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-delta's functionality. The other chimera, PKC-epsilon/deltaV5, endowed inactive PKC-epsilon with nearly all PKC-delta's apoptotic ability, confirming the importance of PKC-delta in this function. Green fluorescent protein (GFP)-tagged PKC-deltaV5 and -epsilon/deltaV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-epsilon and -delta/epsilonV5 showed significantly less, indicating that the V5 region of PKC-delta contains determinants critical to its nuclear distribution. PKC-epsilon/deltaV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-epsilon, implicating the PKC-epsilonV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-delta and -epsilon.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.