Tumor-promoting Phorbol Esters Research Articles

Two Sides of the Same Coin: Protein Kinase C γ in Cancer and Neurodegeneration.

Protein kinase C (PKC) isozymes transduce myriad signals within the cell in response to the generation of second messengers from membrane phospholipids. The conventional isozyme PKCγ reversibly binds Ca2+ and diacylglycerol, which leads to an open, active conformation. PKCγ expression is typically restricted to neurons, but evidence for its expression in certain cancers has emerged. PKC isozymes have been labeled as oncogenes since the discovery that they bind tumor-promoting phorbol esters, however, studies of cancer-associated PKC mutations and clinical trial data showing that PKC inhibitors have worsened patient survival have reframed PKC as a tumor suppressor. Aberrant expression of PKCγ in certain cancers suggests a role outside the brain, although whether PKCγ also acts as a tumor suppressor remains to be established. On the other hand, PKCγ variants associated with spinocerebellar ataxia type 14 (SCA14), a neurodegenerative disorder characterized by Purkinje cell degeneration, enhance basal activity while preventing phorbol ester-mediated degradation. Although the basis for SCA14 Purkinje cell degeneration remains unknown, studies have revealed how altered PKCγ activity rewires cerebellar signaling to drive SCA14. Importantly, enhanced basal activity of SCA14-associated mutants inversely correlates with age of onset, supporting that enhanced PKCγ activity drives SCA14. Thus, PKCγ activity should likely be inhibited in SCA14, whereas restoring PKC activity should be the goal in cancer therapies. This review describes how PKCγ activity can be lost or gained in disease and the overarching need for a PKC structure as a powerful tool to predict the effect of PKCγ mutations in disease.

Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC

Diacylglycerol (DAG)‐sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High‐resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane‐mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high‐resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH‐836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor‐promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC‐mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG‐sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can “cage” chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.

Abstract PS17-46: Pkc agonism restricts immune suppression and promotes antigen cross-presentation in triple negative breast cancer

Abstract Immunotherapy has revolutionized cancer treatment showing unprecedented long-term antitumor responses. However, most patients do not respond to immunotherapies due at least partly to immune suppression. Immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an immunosuppressive innate cell population that suppresses both innate and adaptive immunity. Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancers with poor responses to conventional therapies. TNBC patients harbor higher levels of MDSC populations compared to non-TNBC breast cancer patients. Consequently, TNBC and other solid tumor patients who have high levels of circulating MDSCs respond poorly to immunotherapy. On the other hand, cross-presenting dendritic cells (DCs) are essential to generate an antitumor immune response. Breast cancer patients who harbor higher numbers of these DCs have a better prognosis than patients with lower DC numbers. Several strategies aiming at achieving an effective combination with immunotherapy are under active investigation. The central dogma of these strategies consists of inducing T cells into “immunologically cold tumors” which are defined by having low neoantigen burden and a paucity of T cells and DCs. Hence, strategies that enhance cross-presenting DCs and T cell antitumor potential while altering MDSC’s suppressive function are likely to be effectively combined with immunotherapy for a maximum therapeutic benefit. Protein Kinase C (PKC) is a family of enzymes that play a critical role in cell signaling controlling the balance between survival and cell death. With the discovery in 1980s that PKC is a receptor for the tumor-promoting phorbol esters, the dogma that PKC is an oncoprotein was fueled. This led to more than three decades of failed clinical trials trying to inhibit PKC in cancer. Recent evidence suggests that PKC isozymes are generally inactivated in cancer and that most mutations affecting PKC isozymes are in fact loss of function mutations. This suggests that PKC is a tumor suppressor rather than an oncoprotein and that strategies in cancer treatment should focus on restoring PKC, rather than inhibiting it. To date, the role of PKC isozymes in antitumor immunity is unknown. Herein, our novel data suggest that PKC agonism using established agonists reduced MDSC generation from bone marrow (BM) progenitors specifically via activation of the PKC delta (PKCδ) isoform. PKC agonism induced MDSC differentiation to cross-presenting CD103+ DCs both ex-vivo and in vivo. Additionally, PKC agonist-treated purified MDSCs lost their suppressive capacity on CD8+ T cells in both in vitro and in vivo suppression assays. In contrast, PKC agonism significantly increased the generation of cross-presenting DCs (cDC1) from BM progenitors. Treatment of TNBC-bearing C57BL/6J mice with PKC agonist PEP005 markedly reduced tumor burden by decreasing the frequencies of M-MDSCs in tumor, spleen, and bone marrow while increasing cDC1 frequencies in tumor and spleen. These findings propose PKC as a common pathway in myeloid cells to tip the balance from immune suppression to effective antitumor immunity. Citation Format: Mehdi Chaib, Jeremiah Holt, Laura Sipe, Ajeeth Pingili, Deidre Daria, Liza Makowski. Pkc agonism restricts immune suppression and promotes antigen cross-presentation in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-46.

PHLPPing the balance: restoration of protein kinase C in cancer.

Protein kinase signalling, which transduces external messages to mediate cellular growth and metabolism, is frequently deregulated in human disease, and specifically in cancer. As such, there are 77 kinase inhibitors currently approved for the treatment of human disease by the FDA. Due to their historical association as the receptors for the tumour-promoting phorbol esters, PKC isozymes were initially targeted as oncogenes in cancer. However, a meta-analysis of clinical trials with PKC inhibitors in combination with chemotherapy revealed that these treatments were not advantageous, and instead resulted in poorer outcomes and greater adverse effects. More recent studies suggest that instead of inhibiting PKC, therapies should aim to restore PKC function in cancer: cancer-associated PKC mutations are generally loss-of-function and high PKC protein is protective in many cancers, including most notably KRAS-driven cancers. These recent findings have reframed PKC as having a tumour suppressive function. This review focusses on a potential new mechanism of restoring PKC function in cancer - through targeting of its negative regulator, the Ser/Thr protein phosphatase PHLPP. This phosphatase regulates PKC steady-state levels by regulating the phosphorylation of a key site, the hydrophobic motif, whose phosphorylation is necessary for the stability of the enzyme. We also consider whether the phosphorylation of the potent oncogene KRAS provides a mechanism by which high PKC expression may be protective in KRAS-driven human cancers.

Investigations of mouse models during tumorigenesis revealed essential but distinct in vivo functions among the PKC family.

PKC isozymes have been put in place as oncoproteins since the discovery that they can function as receptors for potent tumor-promoting phorbol esters in the 1980s. Despite nearly two decades of research, a clear in vivo proof of that concept was missing. The availability of so-called knock out mouse lines of individual PKC genes provided a tool to investigate isozyme specific in vivo functions in the context of tumor initiation, development and progression. This review aims to provide a limited overview of how the application of these mouse lines in combination with a cancer mouse model helped to understand PKC's in vivo function during tumorigenesis. The focus of this review will be on skin, colon and lung cancer.

Identification of a truncated β1-chimaerin variant that inactivates nuclear Rac1.

β1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the CHN2 gene, which also encodes the β2- and β3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of β1-chimaerin, β1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that β1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, β1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that β1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in β1-chimaerin that is absent in β1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that β1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.

Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models.

The story of PKC: A discovery marked by unexpected twists and turns.

Protein kinase C (PKC) is activated by 1,2-diacylglycerol as a second messenger in the signaling mechanism coupled with the hydrolysis of membrane inositol phospholipids, although it was not found by screening for a 1,2-diacylglycerol-dependent enzyme. PKC is also a receptor for the tumor-promoting phorbol esters, but it was not identified by its property of binding phorbol esters, either. Instead, the discovery and characterization of PKC, now known to comprise a family with multiple isoforms, was through a circuitous voyage filled with unexpected twists and turns. This review summarizes the discovery and the initial experiments of PKC as a historical perspective of the enzyme family in the context of the progress in the studies on protein phosphorylation. © 2018 IUBMB Life, 71(6):697-705, 2019.

Crosstalk between PKCα and PI3K/AKT Signaling Is Tumor Suppressive in the Endometrium

SUMMARYProtein kinase C (PKC) isozymes are commonly recognized as oncoproteins based on their activation by tumor-promoting phorbol esters. However, accumulating evidence indicates that PKCs can be inhibitory in some cancers, with recent findings propelling a shift in focus to understanding tumor suppressive functions of these enzymes. Here, we report that PKCα acts as a tumor suppressor in PI3K/AKT-driven endometrial cancer. Transcriptional suppression of PKCα is observed in human endometrial tumors in association with aggressive disease and poor prognosis. In murine models, loss of PKCα is rate limiting for endometrial tumor initiation. PKCα tumor suppression involves PP2A-family-dependent inactivation of AKT, which can occur even in the context of genetic hyperactivation of PI3K/AKT signaling by coincident mutations in PTEN, PIK3CA, and/or PIK3R1. Together, our data point to PKCα as a crucial tumor suppressor in the endometrium, with deregulation of a PKCα→PP2A/PP2A-like phosphatase signaling axis contributing to robust AKT activation and enhanced endometrial tumorigenesis.

Terpenes and Biological Activities of Euphorbia tortilis

Background: Euphorbia species are known for the diversity of terpenes, and the presence of toxic diterpenoids in Euphorbia species have raised occupational and eco-toxicological concerns. The present study reports the terpene profile of Euphorbia tortilis Rottler ex Ainslie, along with cytotoxicity assay. Method: Triterpenes were isolated from the hexane extract of E. tortilis through column chromatography and were characterized by various spectroscopic techniques. Phorbol esters were detected from the methanol extract of the latex through high performance liquid chromatography-electrospray ionizationquadrupole time of flight tandem mass spectrometry (HPLC-QTOF- MS/MS). Cytotoxicity of the extract was tested against human cervical cancer cell line HeLa and normal cardiac myoblasts cell line H9C2 using MTT assay. Results: The pentacyclic triterpenoids 3β-friedelinol, 3α-friedelinol, friedelin, epi-friedelinyl acetate and taraxerol were isolated and characterized from E. tortilis. Ten phorbol esters belonging to the tigliane family of diterpenoids were detected through HPLC-ESI-QTOF-MS/MS method. The major tumor promoting phorbol ester TPA (phorbol-12-myristate-13-acetate) reported from Euphorbiaceae family could not be detected in E. tortilis, whereas the potential anti HIV and cytotoxic phorbol ester prostratin has been detected. Latex extract was inactive on the normal cell line H9C2 within the concentration range of 10-100μg/ml, while the cell growth inhibition of the extract at 100μg/ml on Hela cell line was negligible (3.06%). Conclusion: Pentacyclic triterpenes were the major compounds isolated and characterized from E. tortilis. HPLC-ESI-QTOF-MS/MS method has been found as an ideal tool for the detection of minor phorbol esters. The terpenoid profile revealed the absence of toxic TPA. Furthermore, the latex extract was non toxic towards normal cell line H9C2. The findings are relevant in the context of toxicological concerns related to Euphorbia members. Keywords: Euphorbia tortilis, triterpene, tigliane diterpenene, phorbol ester, HPLC-ESI-QTOF-MS, cytotoxicity.

Abstract 2373: Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta

Abstract PKC isozymes, the major receptors for tumor-promoting phorbol esters, play a central role in cellular signal transduction downstream of the second messenger diacylglycerol. The PKCθ isozyme has been extensively studied particularly in the context of hematopoiesis and immunity, where is required for the development and maturation of specific T cell subsets. PKCθ also plays a role in Notch-driven T cell leukemia and in a subset of gastrointestinal stromal tumors, melanoma and breast cancers. The C1 domains represent the regulatory motif in the PKCs responsible for their recognition of diacylglycerol / phorbol esters. PKCθ is unique among the conventional and novel PKC isoforms in that one of the two C1 domains of PKCθ, the C1a domain, has been reported to show little or no affinity for diacylglycerol or phorbol ester, unlike the C1b domain. Likewise, the PKCθ C1b domain has been reported to play the predominant role in the membrane translocation and activation process of PKCθ. We observed that the C1a domain in fact has appreciable binding activity for [3H]phorbol 12,13-dibutyrate (PDBu), with a Kd of 254 nM, albeit much less than that of the C1b domain, 1.58 nM. Replacing individually in the C1a domain ten of the amino acid residues that differed between the C1a and C1b domains, using the corresponding residue from the C1b domain, we found that only the P9K replacement restored [3H]phorbol 12,13-dibutyrate binding activity to the C1a domain, yielding a Kd of 3.60 nM, close to that of the C1b domain. Strikingly, the reciprocal replacement in the C1b domain of K9P still preserved appreciable binding affinity, 11.7 nM. We conclude that other features in the C1a domain contribute to the extent to which the P9 residue negatively influences binding. We further explored the role of the C1 domains of PKCθ on translocation using confocal microscopy with a variety of full length constructs in which the C1 domains were manipulated. Consistent with the binding studies, we showed that the C1b domain was the main contributor to translocation, that the full length PKCθ with the C1b K9P retained response, albeit more weakly, and that deletion of the C1a domain or its replacement with a second C1b domain enhanced the translocation to the plasma membrane. Exploitation of the structural differences between the C1 domains in PKCθ and those of other PKC isoforms, both at the level of the direct ligand interactions and the more complicated interplay between C1 domain, ligand, and phospholipid bilayer, may help to afford specific ligands for this member of the PKC family. Citation Format: Agnes Czikora, Satyabrata Pany, Youngki You, Amandeep Saini, Noemi Kedei, Nancy E. Lewin, Adelle Abramovitz, Joydip Das, Peter M. Blumberg. Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2373. doi:10.1158/1538-7445.AM2017-2373

The tumor promoter-activated protein kinase Cs are a system for regulating filopodia.

Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, β, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter‐stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/β pseudosubstrate sequence, and an inhibitor of T‐cell protein tyrosine phosphatase (TC‐PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter‐treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ‐mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady‐state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long‐term “memory” of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

Protein kinase C as a tumor suppressor.

Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by tumor-promoting phorbol esters. Yet three decades of clinical trials using PKC inhibitors in cancer therapies not only failed, but in some cases worsened patient outcome. Why has targeting PKC in cancer eluded successful therapies? Recent studies looking at the disease for insight provide an explanation: cancer-associated mutations in PKC are generally loss-of-function (LOF), supporting an unexpected function as tumor suppressors. And, contrasting with LOF mutations in cancer, germline mutations that enhance the activity of some PKC isozymes are associated with degenerative diseases such as Alzheimer's disease. This review provides a background on the diverse mechanisms that ensure PKC is only active when, where, and for the appropriate duration needed and summarizes recent findings converging on a paradigm reversal: PKC family members generally function by suppressing, rather than promoting, survival signaling.

Protein kinase C in cancer: The top five unanswered questions.

Few kinases have been studied as extensively as protein kinase C (PKC), particularly in the context of cancer. As major cellular targets for the phorbol ester tumor promoters and diacylglycerol (DAG), a second messenger generated by stimulation of membrane receptors, PKC isozymes play major roles in the control of signaling pathways associated with proliferation, migration, invasion, tumorigenesis, and metastasis. However, despite decades of research, fundamental questions remain to be answered or are the subject of intense controversy. Primary among these unresolved issues are the role of PKC isozymes as either tumor promoter or tumor suppressor kinases and the incomplete understanding on isozyme-specific substrates and effectors. The involvement of PKC isozymes in cancer progression needs to be reassessed in the context of specific oncogenic and tumor suppressing alterations. In addition, there are still major hurdles in addressing isozyme-specific function due to the limited specificity of most pharmacological PKC modulators and the lack of validated predictive biomarkers for response, which impacts the translation of these agents to the clinic. In this review we focus on key controversial issues and upcoming challenges, with the expectation that understanding the intricacies of PKC function will help fulfill the yet unsuccessful promise of targeting PKCs for cancer therapeutics.

Reversing The Paradigm: Protein Kinase C As A Tumor Suppressor

Protein kinase C (PKC) isozymes transduce the myriad of signals resulting from receptor mediated hydrolysis of phospholipids, playing critical roles in diverse cellular functions. Its activity must be precisely tuned for cellular homeostasis and too much, or not enough, activity results in pathophysiologies. The discovery in the 1980s that PKCs are receptors for the potent tumor promoting phorbol esters led to the dogma that activation of PKC by phorbol esters promotes tumorigenesis. However, clinical trials with PKC inhibitors have not have not only failed but, in some cases, resulted in worsened outcome. Analysis of approximately 10% of the >500 mutations identified in human cancers reveals that most mutations are loss‐of‐function and none are activating. Loss‐of‐function mutations occur in all PKC subgroups and occur by disrupting diverse mechanisms: they impede second‐messenger binding, prevent phosphorylation, or inhibit catalysis. Thus, PKC functions as a tumor suppressor and its loss of confers a survival advantage. In contrast, we show that gain‐of‐function mutations accompany degenerative diseases such as Alzheimer's disease. Our results reveal that therapeutic strategies should focus on restoring PKC activity in cancer and inhibiting its activity in neurodegenerative diseases.Support or Funding InformationNIH GM43154

Spatio-Temporal Regulation of PKC Isoforms Imparts Signaling Specificity.

Nishizuka and colleagues discovered protein kinase C (PKC) as a calcium-dependent, lipid cofactor-sensitive protein kinase (1). Initially known as the only physiological effector for tumor-promoting phorbol esters, this ubiquitous enzyme eventually took center stage in cellular signaling. Part of the AGC kinase branch of the kinome, PKC is a family of serine/threonine kinases comprising 11 isoforms encoded by 9 genes and grouped into 4 classes – classical (cPKCs-α, βI, βII, γ), novel (nPKCs-δ, ϵ, η, θ), atypical (aPKCs-ζ, ι/λ), and PKCμ (a form between novel and atypical isoforms) (Box 1). Their considerable structural homology, overlapping substrate specificities, and biochemical properties indicated at least partial enzymatic redundancy and rendered the task of identifying isoform-specific functions challenging. The initial phase of PKC research correlated the unique structural features of the isoforms with their functions (Box 1) (2). But, with their structural overlaps and hugely varying functions in different models, specificity of the isoforms became a confounding puzzle demanding stringent isoform-specific regulation to avoid functional redundancy. To define this stringency, subsequent PKC research focused on the upstream and downstream regulatory mechanisms (3, 4), highlighting the importance of subcellular distribution as a function of time (5). As simultaneous activation of all PKC isoforms would be energetically and spatially conflicting for decoding the message received by the cell surface receptor, cell type, and stimulus-specific selective and sequential activation deemed justified (6). The recent phase of PKC research propounds a PKC-signaling module (7, 8) wherein an inter-isoform network regulates the PKC isoforms’ activity (Box 2; Figure ​Figure1B).1B). Here, we propose activator- and cofactor-specific sequential activation of PKC isoforms in a spatio-temporal model wherein selective subcellular compartmentalization quantitatively determines the isoform-specific effector functions. Box 1 Structural features of PKC isoforms defining functional specificity. PKC serve as a paradigm for the reversible regulation of membrane localization by the concerted action of two membrane-targeting modules. C1: a cysteine-rich region of approximately 50 residues present in all PKC isozymes. In cPKCs and nPKCs, it is present as a tandem repeat C1A and C1B. aPKCs contain a single copy of the domain termed atypical because it does not bind phorbol esters. The domain contains two pulled apart β-sheet forming the ligand-binding pocket. Two zinc atoms are coordinated by two histidines and six cysteines at opposite ends of the primary sequence stabilizing the domain. In aPKCs, one face of the ligand-binding pocket is compromised, so that the module cannot bind phorbol esters or DAG. Ligand binding dramatically alters the surface properties of the module (C1B). The ligand caps the hydrophilic ligand-binding pocket, so that the top-third of the C1 domain presents a continuous hydrophobic surface thus achieving membrane targeting by simply altering membrane properties. C2: an independent membrane-targeting module that binds calcium in cPKCs, but not in nPKCs. C2 domain is a β-strand rich globular domain with loops formed by sequences at opposite ends of the primary structure. Two topological variants exist – Type I for domains that follow the C1 domain (cPKCs) and Type II for domains that precede C1 (nPKCs). In the calcium-responsive C2 domains of cPKCs, the pocket is lined by multiple aspartic acid residues that coordinate two to three calcium ions that act as a bridge between the C2 domain and the phospholipid head groups of the membrane. CR, cysteine rich; PS, pseudosubstrate; DAG, diacylglycerol; PMA, phorbol-12-myristate 13-acetate; PB1, Phox–Bem 1; C, constant regions; V, variable regions.

Growth factor signaling to mTORC1 by amino acid-laden macropinosomes.

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid-dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow-derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor-mediated signaling by amino acids. Therefore, the amino acid-laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.

Dynamics and Membrane Interactions of Protein Kinase C.

Protein kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. Significant research efforts have been directed at understanding the structure, function, and regulatory modes of the enzyme since its discovery and identification as the first receptor for tumor-promoting phorbol esters. The activation of PKC involves a transition from the cytosolic autoinhibited latent form to the membrane-associated active form. The membrane recruitment step is accompanied by the conformational rearrangement of the enzyme, which relieves autoinhibitory interactions and thereby allows PKC to phosphorylate its targets. The multidomain structure and intrinsic flexibility of PKC present remarkable challenges and opportunities for the biophysical and structural biology studies of this class of enzymes and their interactions with membranes, the major focus of this Current Topic. I will highlight the recent advances in the field, outline the current challenges, and identify areas where biophysics and structural biology approaches can provide insight into the isoenzyme-specific regulation of PKC activity.

Targeted deletion of RasGRP1 impairs skin tumorigenesis

Ras is frequently activated in cutaneous squamous cell carcinoma, a prevalent form of skin cancer. However, the pathways that contribute to Ras-induced transformation have not been entirely elucidated. We have previously demonstrated that in transgenic mice, overexpression of the Ras activator RasGRP1 promotes the formation of spontaneous skin tumors and enhances malignant progression in the multistage carcinogenesis skin model that relies on the oncogenic activation of H-Ras. Utilizing a RasGRP1 knockout mouse model (RasGRP1 KO), we now show that lack of RasGRP1 reduced the susceptibility to skin tumorigenesis. The dependency on RasGRP1 was associated with a diminished response to the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Specifically, we found impairment of epidermal hyperplasia induced by TPA through keratinocyte proliferation. Using a keratinocyte cell line that carries a ras oncogenic mutation, we also demonstrated that RasGRP1 could further activate Ras in response to TPA. Thus, we propose that RasGRP1 upregulates signaling from Ras and contributes to epidermal tumorigenesis by increasing the total dosage of active Ras.

RasGRPs Are Targets of the Anti-Cancer Agent Ingenol-3-Angelate

Ingenol-3–angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A’s effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP’s C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin’s lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.