Danny N. Dhanasekaran

JNK Signaling in Apoptosis

Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation of pro-apoptotic genes through the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and antiapoptotic proteins through distinct phosphorylation events. This review analyses our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis.

Scaffold proteins of MAP-kinase modules

Mitogen-activated protein kinases (MAPKs) regulate critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Recent studies have shown that a novel class of scaffold proteins mediates the structural and functional organization of the three-tier MAPK module. By linking the MAP3K, MAP2K and MAPK into a multienzyme complex, these MAPK-specific scaffold proteins provide an insulated physical conduit through which signals from the respective MAPK can be transmitted to the appropriate spatiotemporal cellular loci. Scaffold proteins play a determinant role in modulating the signaling strength of their cognate MAPK module by regulating the signal amplitude and duration. The scaffold proteins themselves are finely regulated resulting in dynamic intra- and inter-molecular interactions that can modulate the signaling outputs of MAPK modules. This review focuses on defining the diverse mechanisms by which these scaffold proteins interact with their respective MAPK modules and the role of such interactions in the spatiotemporal organization as well as context-specific signaling of the different MAPK modules.

G Protein regulation of MAPK networks

G proteins provide signal-coupling mechanisms to heptahelical cell surface receptors and are critically involved in the regulation of different mitogen-activated protein kinase (MAPK) networks. The four classes of G proteins, defined by the Gs, Gi, Gq and G12 families, regulate ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by different mechanisms. The α- as well as βγ-subunits are involved in the regulation of these MAPK modules in a context-specific manner. While the α- and βγ-subunits primarily regulate the MAPK pathways via their respective effector-mediated signaling pathways, recent studies have unraveled several novel signaling intermediates including receptor tyrosine kinases and small GTPases through which these G-protein subunits positively as well as negatively regulate specific MAPK modules. Multiple mechanisms together with specific scaffold proteins that can link G-protein-coupled receptors or G proteins to distinct MAPK modules contribute to the context-specific and spatio-temporal regulation of mitogen-activated protein signaling networks by G proteins.

MAPKs: function, regulation, role in cancer and therapeutic targeting.

The mitogen-activated protein kinases (MAPKs) are generally expressed in all cell types, yet they function to regulate specific responses that differ from cell type to cell type. The MAPKs most intensely studied are extracellular signal-regulated kinase (ERK)1/2, the p38 kinase (p38a, b, g and d), c-Jun N-terminal kinase (JNK)1, 2, 3 and ERK5. There is less known about ERK7 function and regulation. To demonstrate the interest in MAPKs, one needs to simply search PubMed for numbers of publications citing the different MAPKs. As of December 22, 2006 PubMed listed 33 261 papers that cited ERK1/2 (12 314), JNK (8727), p38 (12 005), ERK5 (202) and ERK7 (13). The reason for such intense study of MAPKs is their involvement in the cellular response to almost all stimuli, that activate membrane, cytoplasmic and/or nuclear signaling networks. How are MAPKs activated by such a plethora of stimuli but yet have highly specific biological functions? The answer is in part related to the spatio-temporal regulation of MAPKs within cells. As part of a threekinase module, MAPKs are phosphorylated and activated by MAPK kinases (MKKs). MAPK kinase kinases (MKKKs) phosphorylate and activate MKKs. Whereas there are at least eleven MAPKs, there are only seven MKKs, but at least 20 MKKKs. The differing regulatory domains and motifs encoded in the different MKKKs selectively control localization, activation and inactivation of associated MKKs and MAPKs. In addition, many MKKKs can phosphorylate and activate more than one class of MKKs providing for the ability to coordinately activate two or more different classes MAPKs in response to the activation of a specific MKKK. For example, the MKKK, MEKK3, can activate both p38 and ERK5. In addition, scaffold proteins such as kinase suppressor of Ras, b-arrestin and the JNK-interacting proteins organize MAPK modules in complexes with other proteins, control trafficking and subcellular location, and duration of MAPK signaling. Thus, the combination of differential control of MKKK activation and the organization of signaling complexes by scaffolding proteins provides a combinatorial diversity for the integration of MAPK networks in the cellular response to stimuli such as cytokines, growth factors, antigens, toxins, pharmacological drugs and stresses such as temperature change and irradiation, and changes in cell shape, cell– cell interaction and extracellular matrix composition (Figure 1). Given the role of MAPKs in so many critical responses required for cellular homeostasis, it is no wonder that loss of fine control of MAPK regulation resulting from mutation or changes in expression of proteins regulating MAPK signaling, such as activating Ras or Raf mutations, epidermal growth factor receptor overexpression or inactivation of MKK4, contribute to cancer. Changes in MAPK regulation appears to also contribute to inflammation and neurodegeneration and many other diseases. For these reasons, the MAPKs have been a focus of intense drug discovery programs by both academic laboratories and the biotechnology/pharmaceutical industry. To date, there have been at least 25 small molecule inhibitors targeting specific MAPKs, MKKs or MKKKs that have reached earlyto late-stage clinical trials. Certainly for cancer, small molecule inhibitors targeting Raf or MKK1/2 are progressing in trials for many different tumor types. To truly understand the nature of human diseases such as cancer, it is imperative to define the behavior of signaling networks in the cell and how the dynamics of signaling controls phenotype. A necessary goal for the future is to understand the dynamic spatio-temporal control of MAPK signaling networks and how they contribute to physiological responses in cells, organs and organisms. This requires a detailed understanding of how MAPK signaling networks are integrated with upstream inputs and parallel signaling pathways for the control of physiological responses, and how this integration of signaling networks is dysregulated in disease. To define network control by MAPKs, it is imperative to study the regulation and function of all MAPKs and how each contributes to the integration and spatio-temporal control of physiological responses. Success in defining the critical elements of a signaling network involves large-scale measurements of a biological system and to use these measurements in computational models to predict network behavior and Correspondence: Dr GL Johnson, Department of Pharmacology Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7365, USA. E-mail: glj@med.unc.edu Oncogene (2007) 26, 3097–3099 & 2007 Nature Publishing Group All rights reserved 0950-9232/07

Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency

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Molecular and serum markers in hepatocellular carcinoma: Predictive tools for prognosis and recurrence

Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region -729/-542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations.

Role of m1 receptor-G protein coupling in cell proliferation in the prostate

With increased understanding of cancer biology, a multitude of pathological, genetic, and molecular events that drive hepatocarcinogenesis, including angiogenesis, invasion, and metastasis, has been identified. Lately, they are being aggressively evaluated due to challenges involved in establishing early diagnosis, optimizing therapy for cancer inducing hepatotrophic viruses, minimizing the emergence of new tumors, and preventing recurrence after surgical resection or liver transplantation. This comprehensive review examines and critiques the evidence from published manuscripts reporting various tissue and serum biomarkers involved in hepatocellular carcinoma. These markers not only help in prediction of prognosis or recurrence, but may also assist in deciding appropriate modality of therapy and represent novel targets for potential therapeutic agents.

Transducing the Signals: A G Protein Takes a New Identity

The prostate gland from several animal species contains variable levels of muscarinic subtypes, but only the human prostate expresses significant levels of the m1 subtype. We studied muscarinic receptor activity in human benign prostatic hypertrophy (BPH) as well as several cell lines derived from prostate cancer. The BPH we studied expresses approximately 75% of the m1 receptor and undetectable levels of the other receptor subtypes whereas PC3 cells express only the m3 receptor subtype. DU145 and LnCaP cells express approximately equal levels of m1 and m3 receptor subtypes. Only the PC3 cells responded to carbachol with an increase in turnover of polyphosphoinositides, and none of the cell lines responded with effects on cAMP metabolism. Co-precipitation of receptors with heterotrimeric guanine nucleotide-binding regulatory proteins demonstrated interactions of the m1 receptors with Gi, Gq and G16 in BPH tissue and of the m1 and m3 receptors with Gi, Gq and G12 in PC3 and DU145 cells. Mitogen activated protein kinase (ERK) activity was seen in response to carbachol in PC3 and DU145 but not LnCaP cells. Finally, carbachol promoted cell proliferation in all three cell lines. Thus, there appears to be no consistent relationship between ERK activity, cell proliferation, and the subtype mediating the proliferative response, amongst these prostate cancer cell lines.

Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells

The prevailing dogma is that heterotrimeric G proteins exclusively transduce signals from the seven-transmembrane motif–containing cell surface receptors, also known as G protein–coupled receptors (GPCRs). New evidence indicates that Gα13, the α subunit of the G protein G13, breaks away from this traditional exclusive signaling alliance with GPCRs to transmit signals from receptor tyrosine kinases (RTKs), such as platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptor (VEGFR). Gα13 is involved in cell migration in response to GPCRs activated by lysophosphatidic acid (LPA) or thrombin. A new report indicates that Gα13 is also required for cell migration induced by the growth factors, such as PDGF, EGF, or VEGF. GPCR coupling is not required for such RTK-to-Gα13 signaling. This new identity for Gα13 as a signal transducer for both GPCRs and RTKs may be a forerunner for similar findings involving other Gα subunits. This expanding role of G proteins in both GPCR signaling and RTK signaling is likely to have a great impact not only on our understanding of cell signaling in general, but also more specifically where the dysregulation of signaling by GPCRs, RTKs, and G proteins cause pathophysiological changes such as in the case of tumorigenesis, tumor progression and/or metastasis.

Original ArticlesCurcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells

Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells.