A single point mutation in the V3 region affects protein kinase Calpha targeting and accumulation at cell-cell contacts

Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Calpha (PKCalpha) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKCalpha (hPKCalpha) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCalpha, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca(2+)](i) increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKCalpha wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and beta-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKCalpha translocates but did not accumulate at the plasma membrane and beta-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKCalpha at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCalpha and the actin microfilament network.     

Cytoskeletal reorganizations in human umbilical vein endothelial cells as a result of cytokine exposure.

Treatment of HUVECs in culture with several cytokines and phorbol esters caused reorganizations of the actin and microtubule networks, as well as a redistribution of focal contract proteins. However, expression of the cytoskeletal proteins which link cells, via integrins, to the substrate, was not significantly affected. Indirect immunofluorescence microscopy of endothelial cells after treatment with interleukin-1 alpha and beta, gamma-interferon, tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, and phorbol 12,13-dibutyrate allowed us to observe reductions in the areas of cell-cell contact, redistribution of the stress fiber network, and concomitant changes in focal contacts. Microtubule arrays in TNF-treated cells became bundled. Phorbol esters induced formation of microtubule organizing centers not seen in resting or TNF-treated HUVECs. Talin was distributed along stress fibers and not exclusively in focal contacts. Vitronectin receptor was observed in focal contacts, occasionally at cell-cell contacts, and in vesicular structures close to the lumenal surface, after both types of treatment. Although these morphological changes were easily observed by indirect immunofluorescence, no quantitative differences in specific cytoskeletal proteins were detected by immunoblots and [35S]cysteine metabolic labeling experiments.     

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.     

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.     

Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4).

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4-GFP (T4-GFP) was clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259-470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259-470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259-470) recruited full-length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259-470). Thus, it seems that individual T4(259-470) mutant molecules are sufficient to direct the respective TRAF4-T4(259-470) heteromeric complexes into the nucleus. In cells forming cell-cell contacts, TRAF4 was recruited to the sites of contact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-kappaB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-kappaB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidly up-regulated. In Jurkat T-cells deficient for I-kappaB kinase gamma (IKKgamma, also known as NEMO), an essential component of the NF-kappaB-inducing-IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-kappaB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-kappaB pathway is involved in up-regulation of TRAF4 in T-cells.     

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.     

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.     

Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

Cooperation between PKC-alpha and PKC-epsilon in the regulation of JNK activation in human lung cancer cells

Phorbol esters can induce activation of two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. Unlike ERK activation, JNK activation by phorbol esters is somehow cell-specific. However, the mechanism(s) that contribute to the cell-specific JNK activation remain elusive. In this study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. In NSCLC cells, PMA induced JNK activation in a time- and dose-dependent manner. JNK activation was attenuated by protein kinase C (PKC) down-regulation through prolonged pre-treatment with PMA and significantly inhibited by PKC inhibitors G?6976 and GF109203X. Subcellular localization studies demonstrated that PMA induced translocation of PKC-alpha, -betaII, and -epsilon isoforms, but not PKC-delta, from the cytosol to the membrane. Analysis of various PKC isoforms revealed that PKC-epsilon was exclusively absent in the SCLC cell lines tested. Ectopic expression of PKC-epsilon in SCLC cells restored PMA activation of JNK signaling only in the presence of PKC-alpha, suggesting that PKC-alpha and PKC-epsilon act cooperatively in regulating JNK activation in response to PMA. Furthermore, using dominant negative mutants and pharmacological inhibitors, we define that a putative Rac1/Cdc42/PKC-alpha pathway is convergent with the PKC-epsilon/MEK1/2 pathway in terms of the activation of JNK by PMA.     

Phorbol esters, but not epidermal growth factor or insulin, rapidly decrease soluble protein kinase C activity in rat hepatocytes

Exposure of freshly isolated rat hepatocytes to tumor-promoting phorbol esters like phorbol 12-myristate 13-acetate resulted in a time- and concentration-dependent translocation of protein kinase C from the soluble to the particulate fraction of the cells. No such disappearance of soluble protein kinase C activity was observed with either epidermal growth factor or insulin, indicating that activation of protein kinase C is not necessarily involved in the short-term metabolic action of physiological growth factors on rat hepatocytes.     

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.     

Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate

Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 microM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethanolamine. Exposure of cells to phorbol 12-myristate 13-acetate induces an increase of the activity of the enzymes CTP: ethanolaminephosphate cytidylyltransferase and ethanolaminephosphotransferase. These effects are accompanied by a decrease of the pool size of ethanolaminephosphate and CDPethanolamine and an increase of the level of diacylglycerols after 30 min of incubation in the presence of phorbol 12-myristate 13-acetate. Upon prolonged incubation, the CDPethanolamine and diacylglycerol pools are restored to the level found in untreated cells. These results indicate that stimulation of phosphatidylethanolamine synthesis by phorbol 12-myristate 13-acetate is probably exerted at the level of CTP : ethanolaminephosphate cytidylytransferase, although there may be an additional effect on the subsequent step of phosphatidylethanolamine synthesis, the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerols.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylated form of MacMARCKS is essential to LFA-1-dependent cell-cell adhesion of U937 monocytic cells.

MacMARCKS (MRP, F52), a protein kinase C (PKC) substrate, is involved in the activation of beta2-integrin. To determine the role of the PKC-mediated phosphorylation of MacMARCKS in this process, human U937 monocytic cells were transfected with cDNAs encoding wild type or mutant MacMARCKS. We observed that the expression of the exogenous wild type MacMARCKS greatly enhanced LFA-1-mediated cell-cell adhesion in U937 cells treated with phorbol 12-myristate 13-acetate (PMA). This MacMARCKS-stimulated adhesion depended on the phosphorylation status of MacMARCKS: whereas phosphorylated MacMARCKS enhanced adhesion, unphosphorylated MacMARCKS inhibited it. However, phosphorylated MacMARCKS alone could not induce LFA-1-mediated cell-cell adhesion unless phorbol esters were added, suggesting that the phosphorylation of other proteins might also be involved. Okadaic acid, a phosphatase inhibitor, induced LFA-1-mediated cell-cell adhesion only in the cells expressing wild type or phosphorylated MacMARCKS and not in the cells expressing unphosphorylated MacMARCKS. Therefore, we conclude that the phosphorylated form of MacMARCKS is essential to LFA-1-mediated cell-cell adhesion.     

p23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function

There is emerging evidence that C1 domains, motifs originally identified in PKC isozymes and responsible for binding of phorbol esters and diacylglycerol, interact with the Golgi/endoplasmic reticulum protein p23 (Tmp21). In this study, we investigated whether PKCδ, a kinase widely implicated in apoptosis and inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. Using a yeast two-hybrid approach, we found that the PKCδ C1b domain associates with p23 and identified two key residues (Asp(245) and Met(266)) implicated in this interaction. Interestingly, silencing p23 from LNCaP prostate cancer cells using RNAi markedly enhanced PKCδ-dependent apoptosis and activation of PKCδ downstream effectors ROCK and JNK by phorbol 12-myristate 13-acetate. Moreover, translocation of PKCδ to the plasma membrane by phorbol 12-myristate 13-acetate was enhanced in p23-depleted LNCaP cells. Notably, a PKCδ mutant that failed to interact with p23 triggered a strong apoptotic response when expressed in LNCaP cells. In summary, our data compellingly support the concept that C1 domains have dual roles both in lipid and protein associations and provide strong evidence that p23 acts as an anchoring protein that retains PKCδ at the perinuclear region, thus limiting the availability of this kinase for activation in response to stimuli.     

Phorbol Esters Increase Dopamine Transporter Phosphorylation and Decrease Transport Vmax

Abstract: Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport V _max in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK₁ cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport V _max by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter V _max suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.