Role of diacylglycerol kinase in cellular regulatory processes: a new regulator for cardiomyocyte hypertrophy

Diacylglycerol (DAG) kinase (DGK) phosphorylates and converts DAG to phosphatidic acid. DGK regulates cellular DAG levels and attenuates DAG signaling. The 10 mammalian DGK isoforms have been identified to date. In cardiac myocytes, DGKalpha, epsilon, and zeta are expressed, and DGKzeta is the predominant isoform. DGKzeta inhibits protein kinase C (PKC) activation and subsequent hypertrophic programs in response to endothelin-1 (ET-1) in neonatal rat cardiomyocytes. DGKzeta blocks cardiac hypertrophy induced by G protein-coupled receptor agonists and pressure overload in vivo. DGKzeta attenuates ventricular remodeling and improves survival after myocardial infarction. These data provide a novel insight for subcellular mechanisms of cardiac hypertrophy and heart failure, and DGKzeta may be a new therapeutic target to prevent cardiac hypertrophy and progression to heart failure.     

Caveolae, caveolin, and cavins: potential targets for the treatment of cardiac disease

Caveolae are omega-shaped membrane invaginations present in essentially all cell types of the cardiovascular system, including endothelial cells, smooth muscle cells, macrophages, cardiac myocytes, and fibroblasts. Numerous functions have been ascribed to this omega-shaped structure. Caveolae are enriched with different signaling molecules and ion channel regulatory proteins and function both in protein trafficking and signal transduction in these cell types. Caveolins are the structural proteins that are necessary for the formation of caveola membrane domains. Mechanistically, caveolins interact with a variety of downstream signaling molecules, as, for example, Src-family tyrosine kinase, p42/44 mitogen-activated protein (MAP) kinase, and endothelial nitric oxide synthase (eNOS) and hold the signal transducers in the inactive condition until activated with proper stimulus. Caveolae are gradually acquiring increasing attention as cellular organelles contributing to the pathogenesis of several structural and functional processes including cardiac hypertrophy, atherosclerosis, and heart failure. At present, very little is known about the role of caveolae in cardiac function and dysfunction, although recent studies with caveolin knock-out mouse have shown that caveolae and caveolins play a pivotal role in various human pathobiological conditions. This review will discuss the possible role and mechanism of action of caveolae and caveolins in different cardiac diseases.     

Impaired degranulation but enhanced cytokine production after Fc epsilonRI stimulation of diacylglycerol kinase zeta-deficient mast cells

Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcepsilonRI. Diacylglycerol kinase (DGK)zeta is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKzeta-/- mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKzeta-/- bone marrow-derived mast cells (BMMCs) demonstrate impaired degranulation after Fc epsilonRI cross-linking, associated with diminished phospholipase Cgamma activity, calcium flux, and protein kinase C-betaII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of Fc epsilonRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of Fc epsilonRI signals.     

Caged regulators of signaling pathways.

Although inhibitors can implicate individual protein kinases in specific signal transduction pathways, simple reversible inhibitors are unable to define both the time of initiation and the duration of protein kinase activity. How long must a specific protein kinase remain active in a particular transduction cascade to ensure that the message results in the appropriate cellular response? What is the precise temporal assembly order of individual components of an intracellular signaling complex? What, if any, temporal crosstalk is there between individual members of bisecting signaling pathways? Caged protein kinase inhibitors, as well as caged protein kinases, should serve as useful tools for temporal studies of signal transduction. We have prepared both a caged cyclic AMP-dependent protein kinase and a caged inhibitor of this enzyme. Upon photolysis, the functional activities of both species are dramatically unleashed in vitro. We have also explored both the activation and the inhibition of the cyclic AMP-dependent protein kinase-driven signaling pathway in rat embryo fibroblasts with these caged species via microinjection, and have now demonstrated that the activity of signal transduction pathways can be controlled by light in living cells.     

Dual inhibition of tumor energy pathway by 2-deoxyglucose and metformin is effective against a broad spectrum of preclinical cancer models

Tumor cell proliferation requires both growth signals and sufficient cellular bioenergetics. The AMP-activated protein kinase (AMPK) pathway seems dominant over the oncogenic signaling pathway suppressing cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway using a glycolysis inhibitor 2-deoxyglucose (2DG) and AMPK agonists, AICAR and metformin. We evaluated the in vitro antitumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not sufficient to promote tumor cell death, reflecting the limited efficacy showed in clinical trials. A combined use of 2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments mitochondrial energy transduction (OXPHOS) whereas metformin compromises OXPHOS. Importantly, forced energy restoration with methyl pyruvate reversed the cell death induced by 2DG and metformin, suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination of 2DG and metformin inhibited tumor growth in mouse xenograft models. Deprivation of tumor bioenergetics by dual inhibition of energy pathways might be an effective novel therapeutic approach for a broad spectrum of human tumors.     

Protein phosphorylation and signal transduction.

It is now generally accepted that protein phosphorylation-dephosphorylation has a role in the regulation of essentially all cellular functions. Thus, it is of interest that this process is involved in signal transduction. Nonetheless, the extent to which protein phosphorylation participates in signaling is truly remarkable. Almost every known signaling pathway eventually impinges on a protein kinase, or in some instances, a protein phosphatase. The diversity of these enzymes is noteworthy, and it is of interest that many biotechnology companies are eyeing them as potentially important targets for drugs. Such drugs may have important therapeutic applications, and in any event, they certainly will be useful to investigators who study signal transduction. Indeed, this already has been proven to be true.     

Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation

Perifosine is a novel p.o. bioavailable alkylphospholipid. Perifosine has displayed significant antiproliferative activity in vitro and in vivo in several human tumor model systems and has recently entered phase I clinical trials. Recent studies have identified that perifosine could cause cell cycle arrest with induction of p21(WAF1/CIP1) in a p53-independent fashion; however, the basis for that effect is not known. Structurally, perifosine resembles naturally occurring phospholipids. Therefore, we hypothesized that perifosine might perturb pathways related to phospholipids modulated by growth factor action. We demonstrate here that perifosine causes dose-dependent inhibition of protein kinase B/Akt phosphorylation and thus activation at concentrations causing growth inhibition of PC-3 prostate carcinoma cells. Only the myristoylated form of Akt (MYR-Akt), which bypasses the requirement for pleckstrin homology (PH) domain-mediated membrane recruitment, abrogated perifosine-mediated decrease of Akt phosphorylation and cell growth inhibition by perifosine. We demonstrate further that perifosine decreases the plasma membrane localization of Akt, and this is substantially relieved by MYR-Akt along with relief of downstream drug effect on induction of p21(WAF1/CIP1). Perifosine does not directly affect phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent kinase 1, or Akt activity at concentrations inhibiting Akt phosphorylation and membrane localization. Our results demonstrate that Akt is an important cellular target of perifosine action. In addition, these studies show that the membrane translocation of certain PH domain-containing molecules can be greatly perturbed by the alkylphospholipid class of drugs and imply further that the PI3K/Akt pathway contributes to regulation of p21(WAF1/CIP1) expression.     

Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis

Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit-expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collagen-induced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-alpha release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-alpha production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases.     

Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA- ligand interaction

Induced activation of protein tyrosine kinase(s) is a central event in signal transduction mediated via the low affinity receptor for IgG (Fc gamma RIIIA, CD16) in natural killer (NK) cells. Tyrosine phosphorylation may affect the function of several protein directly, or indirectly by inducing their association with other tyrosine phosphorylated proteins. Here, we report that Fc gamma RIII stimulation induces activation of phosphatidylinositol (PI)-3 kinase in NK cells. Phosphotyrosine immunoprecipitates from Fc gamma RIII-stimulated NK cells contain PI-kinase activity and PI-3 kinase can be directly precipitated from them. Conversely, a series of tyrosine-phosphorylated proteins is coprecipitated with PI-3 kinase from the stimulated, but not from control cells. Analogous results obtained using Jurkat T cells expressing transfected Fc gamma RIIIA alpha ligand binding chain in association with gamma 2 or zeta 2 homodimers indicate that both complexes transduce this effect, although the Fc gamma RIIIA-zeta 2 complexes do so with greater efficiency. Accumulation of phosphoinositide D3 phosphorylated products in stimulated cells confirms PI-3 kinase activation, indicating the participation of this enzyme in Fc gamma RIIIA-mediated signal transduction.     

Effects of treatment with haloperidol, chlorpromazine, and clozapine on protein kinase C (PKC) and phosphoinositide-specific phospholipase C (PI-PLC) activity and on mRNA and protein expression of PKC and PLC isozymes in rat brain.

The effects of acute (single) and chronic (21-day) administration of haloperidol (HAL), chlorpromazine (CPZ), or clozapine (CLOZ) on components of the phosphoinositide (PI)-signaling pathway were studied in rat brain. Chronic administration of HAL decreased protein kinase C (PKC) activity and mRNA and protein levels of PKC alpha and epsilon isozymes in both membrane and cytosol fractions of cortex, hippocampus, and striatum. Chronic administration of CPZ, however, decreased PKC activity only in the membrane fraction of cortex, hippocampus, and striatum, and had no effect on the levels of any PKC isozymes. On the other hand, chronic administration of CLOZ decreased PKC activity and mRNA and protein levels of PKC alpha, gamma, and epsilon isozymes in membrane and cytosol fractions of cortex, hippocampus, and cerebellum. Studies of the effects on phospholipase C (PLC) revealed that only chronic administration of CPZ significantly decreased PI-PLC activity and mRNA and protein levels of the specific PLC beta(1) isozyme in membrane and cytosol fractions of cortex, hippocampus, cerebellum, and striatum. Acute-treatment data suggest that CPZ or CLOZ had no significant effects on PI-PLC or PKC; however, HAL translocated PKC, as evidenced from increased PKC activity and protein levels of PKC alpha and epsilon isozymes in the membrane fraction and the decrease in these parameters in the cytosol fraction of cortex, hippocampus, and striatum. Our results thus suggest that the interaction of antipsychotic drugs with PKC and PLC may be associated with their mechanisms of action.     

Endocrine tumors and growth factors

A number of studies have revealed that various growth factors are produced in the malignant progression of endocrine tumors. Growth factors, classified based on their signal transduction pathways, exert the functions by binding to cell surface specific receptors, and promote the growth of tumors by autocrine and paracrine mechanisms. Growth factors such as VEGF and bFGF are also detected in endocrine tumors, and play an important role in the growth and metastasis of tumors via induction of angiogenesis. In the signal transduction pathways growth factors activate various kinases such as mitogen-activated protein kinase and/or phosphatidylinositol 3-kinase/Akt. Signaling cross-talk of growth factors, cytokines and steroid hormones has been observed in various endocrine tumors.     

Thyrotropin receptor--structure, autoantibody and signal transduction

Thyrotropin receptor (TSHR) belongs to type A G protein-coupled receptor family, which possesses a large entracellular domain (ED). Recent studies have revealed the importance of leucine rich repeats domain in the ED and the formation of oligomer in its TSH binding and its signal transduction. TSHR binds to Gs and Gq. It might also activate JAK/STAT kinase system. To further understanding the pathophysiology of Graves' disease, it will be needed to clarify the epitopes of thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody(TSBAb) and thyroid growth-stimulating IgG (TGI) and their relation to signal transduction pathway.     

Taming the PI3K team to hold inflammation and cancer at bay

Recent progress in understanding the molecular mechanisms of receptor signal transduction is continuously highlighting new unforeseen potential drug targets for yet unmet therapeutic needs. While the large number of different cell surface receptors challenge the concept of antagonists development, the finding of signal transduction platforms common to multiple receptor families has boosted the development of new therapeutic approaches. The identification of the role of phosphoinositide 3-kinase family members downstream receptors as directors of multiple cellular responses ranging from cell proliferation and survival to immunity and cardiovascular control, is an example of successful drug target validation studies. This review will focus on these findings and on the ongoing efforts to tame this family of enzymes to beat inflammation and cancer.     

Transforming growth factor-beta signal transduction in epithelial cells.

Transforming growth factor (TGF)-beta is a natural and potent growth inhibitor of a variety of cell types, including epithelial, endothelial, and hematopoietic cells. The ability of TGF-beta to potently inhibit the growth of many solid tumors of epithelial origin, including breast and colon carcinomas, is of particular interest. However, many solid tumor cells become refractory to the growth inhibitory effects of TGF-beta due to defects in TGF-beta signaling pathways. In addition, TGF-beta may stimulate the invasiveness of tumor cells via the paracrine effects of TGF-beta. Accordingly, in order to develop more effective anticancer therapeutics, it is necessary to determine the TGF-beta signal transduction pathways underlying the growth inhibitory effects and other cellular effects of TGF-beta in normal epithelial cells. Thus far, two primary signaling cascades downstream of the TGF-beta receptors have been elucidated, the Sma and mothers against decapentaplegic homologues and the Ras/mitogen-activated protein kinase pathways. The major objective of this review is to summarize TGF-beta signaling in epithelial cells, focusing on recent advances involving the Sma and mothers against decapentaplegic homologues and Ras/mitogen-activated protein kinase pathways. This review is particularly timely in that it provides a comprehensive summary of both signal transduction mechanisms and the cell cycle effects of TGF-beta.     

Essential role of the T cell-specific adapter protein in the activation of LCK in peripheral T cells

T cell-specific adapter protein (TSAd) is a SRC-homology-2 (SH2) domain-containing intracellular signaling molecule that is required for T cell antigen receptor (TCR)-induced cytokine synthesis in T cells. How TSAd functions in TCR signal transduction is not clear. Previous work has suggested a nuclear role for this adapter. However, other evidence suggests that TSAd also functions in the cytoplasm. Using T cells from TSAd-deficient mice, we now show that the major role of TSAd in the cytoplasm is in activation of the LCK protein tyrosine kinase at the outset of TCR signal transduction. Consequently, TSAd regulates several downstream signaling events, including intracellular calcium mobilization and activation of the Ras-extracellular signal-regulated kinase signaling pathway. TSAd regulates LCK activity directly through physical interaction with LCK SH3 and SH2 domains. These studies reveal TSAd as a positive regulator of proximal TCR signal transduction and provide important new information on the mechanism of TCR-induced LCK activation.     

Compensatory pathways induced by MEK inhibition are effective drug targets for combination therapy against castration-resistant prostate cancer

Targeted therapies have often given disappointing results when used as single agents in solid tumors, suggesting the importance of devising rational combinations of targeted drugs. We hypothesized that construction of such combinations could be guided by identification of growth and survival pathways whose activity or expression become upregulated in response to single-agent drug treatment. We mapped alterations in signaling pathways assessed by gene array and protein phosphorylation to identify compensatory signal transduction pathways in prostate cancer xenografts treated with a MAP/ERK kinase (MEK) inhibitor PD325901. In addition to numerous components of the extracellular signal-regulated kinase (ERK) signaling pathway, components of the IKK, hedgehog, and phosphoinositide 3-kinase/Akt/mTOR pathways were upregulated following treatment with PD325901. Combinations of PD325901 with inhibitors of any one of these upregulated pathways provided synergistically greater growth inhibition of in vitro cell growth and survival than the individual drugs alone. Thus, the identification of compensatory signal transduction pathways paves the way for rational combinatorial therapies for the effective treatment of prostate cancer.     

Signal transduction and drug development]

Extracellular information is translated into the cellular responses by various signal transduction systems such as G protein-coupled receptor-linked and protein tyrosine kinase receptor-mediated pathways. The changes or modifications of theses signaling proteins cause uncontrolled transformation or cell death. One of the features of the signaling molecules is that these proteins contain the domains or motifs for protein-protein or protein-lipid interactions governed by several amino acids. Therefore, these amino acids may become promising targets for the drug design. In contrast with intracellular molecules, the receptors located on cellular surface are still the best candidate for the drugs. The future analysis of cellular signal transduction system allows us to develop new drugs that cannot cause serious side effects in future.