Tumor-promoting Phorbol Esters Research Articles

Protein kinase C pharmacology: refining the toolbox.

PKC (protein kinase C) has been in the limelight since the discovery three decades ago that it acts as a major receptor for the tumour-promoting phorbol esters. Phorbol esters, with their potent ability to activate two of the three classes of PKC isoenzymes, have remained the best pharmacological tool for directly modulating PKC activity. However, with the discovery of other phorbol ester-responsive proteins, the advent of various small-molecule and peptide modulators, and the need to distinguish isoenzyme-specific activity, the pharmacology of PKC has become increasingly complex. Not surprisingly, many of the compounds originally touted as direct modulators of PKC have subsequently been shown to hit many other cellular targets and, in some cases, not even directly modulate PKC. The complexities and reversals in PKC pharmacology have led to widespread confusion about the current status of the pharmacological tools available to control PKC activity. In the present review, we aim to clarify the cacophony in the literature regarding the current state of bona fide and discredited cellular PKC modulators, including activators, small-molecule inhibitors and peptides, and also address the use of genetically encoded reporters and of PKC mutants to measure the effects of these drugs on the spatiotemporal dynamics of signalling by specific isoenzymes.

Upregulation of PKCη by PKCε and PDK1 involves two distinct mechanisms and promotes breast cancer cell survival

BackgroundProtein kinase C (PKC) serves as the receptor for tumor-promoting phorbol esters, which are potent activators of conventional (c) and novel (n) PKCs. We recently showed that these activators induced selective upregulation of PKCη in breast cancer cells. The objective of this study is to understand unique regulation of PKCη and its importance in breast cancer. MethodsThe levels of PKC isozymes were monitored in breast cancer cells following treatment with inhibitors of kinases, proteasome and proteases by Western blotting. PKCε was introduced by adenoviral delivery. PKCη and PDK1 were depleted by siRNA silencing. Cell growth was determined by the MTT or clonal assay. ResultsThe general PKC inhibitors Gö 6983 and bisindolylmaleimide but not cPKC inhibitor Gö 6976 led to substantial PKCη downregulation, which was partly rescued by the introduction of nPKCε. Inhibition of phosphoinositide-dependent kinase-1 (PDK1) by Ly294002 or knockdown of PDK1 also led to downregulation of basal PKCη but had no effect on PKC activator-induced upregulation of PKCη. Proteasome inhibitors blocked PKCη downregulation triggered by PDK1 inhibition/depletion but not by Gö 6983. PKCη level increased in malignant but not in non-tumorigenic or pre-malignant cells in the progressive MCF-10A series associated with activated PDK1, and knockdown of PKCη inhibited breast cancer cell growth and clonogenic survival. ConclusionUpregulation of PKCη contributes to breast cancer cell growth and targeting either PKCε or PDK1 triggers PKCη downregulation but involves two distinct mechanisms. General significanceThe status of PKCη may serve as a potential biomarker for breast cancer malignancy.

Novel regulation of protein kinase C-η

Protein kinase C (PKC) is the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs, but persistent treatment with phorbol esters leads to downregulation of these PKCs. However, PKCη, a novel PKC isozyme, resists downregulation by tumor-promoting phorbol esters, but little is known about how PKCη level is regulated. Phosphorylation and dephosphorylation play an important role in regulating activity and stability of PKCs. In the present study, we have investigated the molecular mechanism of PKCη regulation. Several PKC activators, including phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate and indolactam V caused upregulation of PKCη, whereas the general PKC inhibitor Gö 6983, but not the conventional PKC inhibitor Gö 6976 led to the downregulation of PKCη. Upregulation of PKCη was associated with an increase in phosphorylation of PKCη. Silencing of phosphoinositide-dependent kinase-1, which phosphorylates PKCη at the activation loop, failed to prevent PKC activator-induced upregulation of PKCη. Knockdown of PKCε but not PKCα inhibited PKC activator-induced upregulation of PKCη. Thus, our results suggest that the regulation of PKCη is unique and PKCε is required for the PKC activator-induced upregulation of PKCη.

Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.

옥수수 뿌리에서 굴중성 반응과 에틸렌 생성에 미치는 Phorbol 12-myristate 13-acetate 조절 작용

암을 유발하는 phorbol ester로 알려진 Phorbol 12-myristate 13-acetate (PMA)는 동물세포에서 신호전달 효소의 하나인 protein kinase C (PKC)를 활성화시킨다. 본 연구에서는 옥수수 일차뿌리에서 PMA가 에틸렌 생성을 통하여 굴중성 반응을 조절하는 효과를 연구하였다. PMA는 8시간 동안 <TEX>$10^{-6}$</TEX> M과 <TEX>$10^{-4}$</TEX> M에서 농도 의존적으로 뿌리 생장과 굴중성 반응을 촉진시켰다. 이러한 촉진 효과는 PKC의 억제제인 staurosporine (STA)에 의해 상쇄되었다. 이 결과는 굴중성 반응이 신호전달 체계에 관여하는 protein kinase C를 통하여 조절될 가능성을 제시하고 있다. 식물호르몬인 에틸렌도 뿌리 생장과 굴중성 반응에 중요한 역할을 한다고 알려져 있다. 에틸렌 생성은 <TEX>$10^{-6}$</TEX> M과 <TEX>$10^{-4}$</TEX> M PMA에 의하여 각각 26%와 37% 증가하였다. PMA는 또한 ACC synthase (ACS) 활성을 촉진시켰다. 또한 이 증가 효과는 STA에 의하여 상쇄되었다. 이 결과는 옥수수 뿌리에서 굴중성 반응은 에틸렌 생성을 거쳐 protein kinase를 통하여 조절될 가능성을 제시하고 있다. Phorbol 12-myristate 13-acetate (PMA), a known tumor-promoting phorbol ester, activates the signal transduction enzyme protein kinase C (PKC) in animal cells. We investigated the effect of PMA on the regulation of gravitropism via ethylene production in primary roots of maize. PMA stimulated root growth and the gravitropic response in a concentration-dependent manner at <TEX>$10^{-6}$</TEX> M and <TEX>$10^{-4}$</TEX> M over 8 hrs. These effects were prevented by treatment with staurosporine (STA), a potent inhibitor of PKC. These results support the possibility that the gravitropic response might be regulated through protein kinases that are involved in the signal transduction system. Ethylene is known to play a role in the regulation of root growth and gravitropism. Ethylene production was increased by about 26% and 37% of the control rate in response to <TEX>$10^{-6}$</TEX> M and <TEX>$10^{-4}$</TEX> M PMA, respectively. PMA also stimulated the activity of ACC synthase (ACS), which converts the S-adenosyl-L-methionine (AdoMet) to 1-aminocyclopropane-1-carboxylic acid (ACC) in the ethylene production pathway. These effects on ethylene production were also prevented by STA treatment. These results suggest that the root gravitropic response in maize is regulated through protein kinases via ethylene production.

Contribution of individual PKC isoforms to breast cancer progression

The protein kinase C (PKC) family of serine/threonine kinases has been intensively studied in cancer since their discovery as major receptors for the tumor-promoting phorbol esters. The contribution of each individual PKC isozyme to malignant transformation is only partially understood, but it is clear that each PKC plays different role in cancer progression. PKC deregulation is a common phenomenon observed in breast cancer, and PKC expression and localization are usually dynamically regulated during mammary gland differentiation and involution. In fact, the overexpression of several PKCs has been reported in malignant human breast tissue and breast cancer cell lines. In this review, we summarize the knowledge available on the specific roles of PKC isoforms in the development, progression, and metastatic dissemination of mammary cancer. We also discuss the role of PKC isoforms as therapeutic targets, and their potential as markers for prognosis or treatment response.

Membrane Depolarization Increases Membrane PtdIns(4,5)P2 Levels through Mechanisms Involving PKC βII and PI4 Kinase

In a previous study, we showed that membrane depolarization induced elevation of membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P(2), also known as PIP(2)) and subsequently increased the KCNQ2/Q3 currents expressed in Xenopus oocytes through increased PI4 kinase activity. In this study, the underlying mechanism for this depolarization-induced enhancement of PIP(2) synthesis was further investigated. Our results indicate that activation of protein kinase C (PKC) isozyme βII was responsible for the enhanced PIP(2) synthesis. We found that phorbol-12-myristate, 13-acetate (PMA), an activator of PKC, mimicked the effects of the membrane depolarization by increasing KCNQ2/Q3 activity, elevating membrane PIP(2) levels and increasing activity of PI4 kinase β. Furthermore, membrane depolarization enhanced PKC activity. The effects of both depolarization and PMA were blocked by a PKC inhibitor or PI4 kinase β RNA interference. Further results demonstrate that the depolarization selectively activated the PKC βII isoform and enhanced its interaction with PI4 kinase β. These results reveal that the depolarization-induced elevation of membrane PIP(2) is through activation of PKC and the subsequent increased activity of PI4 kinase β.

Pharmacological and Genetic Evaluation of Proposed Roles of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK), Extracellular Signal-regulated Kinase (ERK), and p90RSK in the Control of mTORC1 Protein Signaling by Phorbol Esters

Pharmacological and Genetic Evaluation of Proposed Roles of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK), Extracellular Signal-regulated Kinase (ERK), and p90RSK in the Control of mTORC1 Protein Signaling by Phorbol Esters

Investigation of the Reciprocal Relationship between the Expression of Two Gap Junction Connexin Proteins, Connexin46 and Connexin43

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.

The potential use of protein kinase D inhibitors for prevention/treatment of epidermal tumors

The serine/threonine kinase protein kinase D (PKD) has been proposed to be a pro-proliferative, anti-differentiative signal in epidermal keratinocytes. Indeed, the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces biphasic PKD activation, which mirrors the biphasic response of initial differentiation followed by proliferation and tumor promotion seen in TPA-treated keratinocytes in vitro and epidermis in vivo. Our objective was to test the idea that PKD's pro-proliferative and/or anti-differentiative effects in keratinocytes contribute to TPA-induced tumorigenesis. Using western analysis and assays of keratinocyte proliferation and differentiation, we investigated the effect of inhibitors of PKD on keratinocyte function. We found that overexpression of a constitutively active PKD mutant increased, and of a dominant-negative PKD mutant decreased, keratinocyte proliferation. A recently described selective PKD inhibitor showed low potency to inhibit keratinocyte proliferation or PKD activation. Therefore, we tested the ability of known only relatively selective PKD inhibitors on keratinocyte function and protein kinase activation. H89 {N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline-sulfonamide}, a reported inhibitor of PKD and cAMP-dependent protein kinase, enhanced the effect of a differentiating agent on a marker of keratinocyte differentiation. Another reported non-selective PKD inhibitor, resveratrol stimulated differentiation and inhibited proliferation. The protein kinase C/PKD inhibitor Gö6976 blocked the increase in proliferation (as measured by DNA specific activity) induced by chronic TPA without affecting the initial TPA-elicited differentiation. Our results support the idea that relatively selective PKD inhibitors, such as Gö6976, H89 and resveratrol, might be useful for preventing/treating epidermal tumorigenesis without affecting keratinocyte differentiation.

Bryostatin 1 Inhibits Phorbol Ester-Induced Apoptosis in Prostate Cancer Cells by Differentially Modulating Protein Kinase C (PKC) δ Translocation and Preventing PKCδ-Mediated Release of Tumor Necrosis Factor-α

Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCdelta-mediated release of TNFalpha, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFalpha release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCdelta to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCdelta peripheral translocation. Studies using a membrane-targeted PKCdelta construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCdelta may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy.

RasGRP1 Is Essential for Ras Activation by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate in Epidermal Keratinocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

Abstract 315: RasGRP1 is a critical link to Ras activation by phorbol esters in mouse epidermal keratinocytes

Abstract RasGRP1 is a member of the guanine nucleotide exchange factor family for Ras that binds with high affinity to ultrapotent diacylglycerol analogs like the phorbol esters. Recent work from our lab has demonstrated expression of RasGRP1 in epidermal keratinocytes and a role of this exchange factor in skin carcinogenesis. Further, the studies suggested that RasGRP1 participates in the action of tumor promoting phorbol esters like 12 - tetradecanoylphorbol - O - 13 - acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in epidermal carcinogenesis, we sought to discern if RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout mouse model (KO) to examine the response of primary keratinocytes to TPA. In contrast to the response of wild type (Wt) keratinocytes, which showed a rapid and sustained stimulation of Ras up to 30 minutes in response to TPA (Ras-GTP/Total Ras arbitrary units, 0 min TPA= 0.14 ± 0.06, 30 min TPA= 0.47 ± 0.06; n=5, **p&amp;lt;0.01), Ras-GTP levels in the KO cells were barely detected after TPA treatment (0 min TPA= 0.05 ± 0.02, 30 min TPA= 0.04 ± 0.02; n=7, ns). The lack of response was rescued by enforced expression of RasGRP1 in the KO keratinocytes, suggesting that the effect on Ras was specific to RasGRP1. To further confirm a RasGRP1-specific effect and rule out any indirect developmental effects that arise form early loss of RasGRP1 in the mouse mutant, we used an RNAi approach to silence RasGRP1 in Wt keratinocytes. This approach induced more than 90% depletion of RasGRP1 from the cells and led to a significant reduction in the ability of TPA to stimulate Ras, similar to the one observed in the KO keratinocytes. Analysis of specific Ras isoforms demonstrated that both H- and N-Ras depended on RasGRP1 for Ras activation by TPA, while activation of K-Ras could not be detected under our experimental conditions. Interestingly, while RasGRP1 was dispensable for ERK phosphorylation in response to TPA, phosphorylation of JNK was reduced in the KO keratinocytes. Notably, phosphorylation levels of the JNK2 isoform were significantly lower in the KO cells exposed to TPA compared to the levels observed in the Wt controls (p-JNK2/Total JNK2 arbitrary units, 30 min TPA, Wt= 0.15 ± 0.03, KO=0.05 ± 0.01; n=6-7, **p&amp;lt;0.01). Moreover, enforced expression of RasGRP1 was able to rescue the reduction in phopho-JNK2 levels observed in the RasGRP1 KO cells. Taken together, our data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes, and suggest JNK2 as one of the relevant downstream targets. These findings raise questions about the events taking place during TPA-induced tumor promotion in the epidermis -so far primarily attributed to PKC modulation-, and support further investigation on the role of RasGRP1 in keratinocyte transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 315.

CID755673 enhances mitogenic signaling by phorbol esters, bombesin and EGF through a protein kinase D-independent pathway

Recently, CID755673 was reported to act as a highly selective inhibitor of protein kinase D (PKD). In the course of experiments using CID755673, we noticed that it exerted unexpected stimulatory effects on [3H]thymidine incorporation and cell cycle progression in Swiss 3T3 cells stimulated by bombesin, a Gq-coupled receptor agonist, phorbol 12,13-dibutyrate (PDBu), a biologically active tumor promoting phorbol ester and epidermal growth factor (EGF). These stimulatory effects could be dissociated from the inhibitory effect of CID755673 on PKD activity, since enhancement of DNA synthesis was still evident in cells with severely down-regulated PKD1 after transfection of siRNA targeting PKD1. A major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with great caution in experiments attempting to elucidate the role of PKD family members in cellular regulation, particularly cell cycle progression from G1/Go to S phase.

12-O-Tetradecanoylphorbol-13-acetate Inhibits Melanoma Growth by Inactivation of STAT3 through Protein Kinase C-activated Tyrosine Phosphatase(s)

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).

Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS–PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.

Estrogen receptor α is a target of mTOR/S6K1 signaling in control of breast cancer cell proliferation.

Abstract Abstract #4062 Background: The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types, and the identity of breast cell-specific S6K1 targets is not well understood. In this study, we report that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen.&amp;#x2028; Materials and Methods: Using immunoblot analysis we determined expression levels of S6K1 in breast cancer cell lines, and the phosphorylation status of ERα on Ser167 after serum or mitogen stimulation. We compared the effect of low-serum media, and rapamycin and/or 4-hydroxytamoxifen (4-HT) addition on breast cancer cell proliferation using neutral red uptake assay.&amp;#x2028; Results: We determined that overexpression of S6K1 in breast cancer cells confers proliferative advantage in low serum, contributing to neoplastic transformation. Next, we wished to identify targets of S6K1 in control of proliferation of breast cancer cells. One of the proteins we identified as a target of S6K1 is ERα. ERα is frequently co-overexpressed with S6K1 in breast cancer cells, which could be an indication of co-selection, and ERα contains a putative S6K1 phosphorylation motif RERLAS167. We confirmed that ERα phosphorylation may be mediated by S6K1. We shown that in S6K1-overexpressing MCF7 cells, serum-stimulated ERα phosphorylation is rapamycin-sensitive. Moreover, ERα phosphorylation stimulated by insulin and tumor-promoting phorbol esters is also rapamycin-sensitive. S6K1 can phosphorylate ERα in an in vitro kinase assay. Lastly, shRNA-mediated suppression of S6K1 expression in MCF7 cells reduces ERα phosphorylation. Subsequently, we examined whether co-overexpression of S6K1 and ERα in breast cancer cells sensitizes them to combination therapy with rapamycin and tamoxifen. We analyzed proliferation of cells treated with rapamycin and/or 4-HT. We observed that in S6K1- and ERα-overexpressing cells, treatment with rapamycin and 4-HT resulted in greater inhibition of cell growth.&amp;#x2028; Discussion: Our data suggest that one of the targets of S6K1 signaling in breast cancer cells is ERα, and together, these proteins promote breast cancer cell proliferation. The ultimate objective would be to determine whether combined inhibition of S6K1 and the estrogen receptor could be used as targeted therapy for patients with double positive expression of S6K1 and ER. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4062.

Signal transduction of constitutively active protein kinase C epsilon

The protein kinase C (PKC) family is the most prominent target of tumor-promoting phorbol esters. For the PKCε isozyme, different intracellular localizations and oncogenic potential in several but not all experimental systems have been reported. To obtain information about PKCε-signaling, we investigated the effects of constitutively active rat PKCε (PKCεA/E, alanine 159 is replaced by glutamic acid) in HeLa cells in a doxycycline-inducible vector. Upon induction of PKCεA/E expression by doxycycline, the major part of PKCεA/E was localized to the Golgi. This led (i) to phosphorylations of PKCε S729, Elk-1 S383, PDK1 S241 and Rb S807/S811, (ii) to elevated expression of receptor of activated C kinase 2 (RACK2) after 12 h, and (iii) increased colony formation in soft agar, increased cell migration and invasion, but not to decreased doubling time. Following induction of PKCεA/E-expression by doxycycline for 24 h and additional short-term treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA), PKCεA/E translocated to the plasma membrane and increased phosphorylation of MARCKS S152/156. Treatment with doxycycline/TPA or TPA alone increased phosphorylations of Elk-1 S383, PDK1 S241, Rb S807/S811, PKCδ T505, p38MAPK T180/Y182, MEK1/2 S217/S221 and ERK2 T185/T187. MARCKS was not phosphorylated after treatment with TPA alone, demonstrating that in this system it is phosphorylated only by PKCε localized to the plasma membrane but not by PKCα or δ, the other TPA-responsive PKC isozymes in HeLa cells. These results demonstrate that PKCε can induce distinctly different signaling from the Golgi and from the plasma membrane.

Enzymological Analysis of Mutant Protein Kinase Cγ Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca2+ Homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cgamma (PKCgamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCgamma kinase activity. To determine the effect of these mutations on PKCgamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCgammas. We found that wild-type PKCgamma, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCgamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCgamma. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCgammas was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.