Journal of Molecular Signaling最新文献

The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.

Background: Recent studies on the involvement of the G12 family of heterotrimeric G proteins (Gα12 and Gα13, the products of the GNA12 and GNA13 genes, respectively) in oncogenic pathways have uncovered a link between G12 signaling and cancer progression. However, despite a well characterized role of Rho GTPases, the potential role of secreted factors in the capacity of G12 signaling to promote invasion of cancer cells is just beginning to be addressed.

Methods: MDA-MB-231 and MCF10A breast cancer cell lines were employed as a model system to explore the involvement of secreted factors in G12-stimulated cell invasion. Factors secreted by cells expressing dominant-active Gα12 were identified by protein array, and their involvement in breast cancer cell invasion was assessed through both RNAi-mediated knockdown and antibody neutralization approaches. Bioinformatics analysis of the promoter elements of the identified factors suggested NF-κB elements played a role in their enhanced expression, which was tested by chromatin immunoprecipitation.

Results: We found that signaling through the Gα12 in MDA-MB-231 and MCF10A breast cancer cell lines enhances expression of interleukins (IL)-6 and -8, and matrix metalloproteinase (MMP)-2, and that these secreted factors play a role in G12-stimulated cell invasion. Furthermore, the enhanced expression of these secreted factors was found to be facilitated by the activation of their corresponding promoters, where NF-κB seems to be one of the major regulators. Inhibition of IL-6 and IL-8, or MMP-2 activity significantly decreased Gα12-mediated cell invasion.

Conclusions: These studies confirm and extend findings that secreted factors contribute to the oncogenic potential of G12 signaling, and suggest potential therapeutic targets to control this process.

Background: The periodontal ligament (PDL), connective tissue located between the cementum of teeth and alveolar bone of the mandibula, plays an important role in the maintenance and regeneration of periodontal tissues. We reported previously that endoglin was involved in the BMP-2-induced osteogenic differentiation of mouse PDL cells, which is associated with Smad-2 phosphorylation but not Smad-1/5/8 phosphorylation. In this study, to elucidate the detailed mechanism underlying the BMP-2 signalling pathway unique to PDL cells, we performed a microarray analysis to identify BMP-2-inducible genes in PDL-L2 cells, a mouse PDL-derived cell line, with or without endoglin knockdown.

Findings: Sixty-four genes were upregulated more than twofold by BMP-2 in PDL-L2 cells. Of these genes, 11 were endoglin-dependent, including Id4, which encodes ID4, a helix-loop-helix transcription factor closely associated with TGF-β signaling and osteoblast differentiation. The endoglin-dependent induction of ID4 by BMP-2 was also verified at a protein level.

Conclusion: Our findings indicate that ID4 could be a signal mediator involved in the BMP-2-induced endoglin-dependent osteogenic differentiation of PDL cells.

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR's function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR's discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs' effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.

Background: Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.

Method: We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.

Results: Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.

Conclusions: Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

Background: p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.

Methods: Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25rodent/27human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.

Results: No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.

Conclusions: The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1-GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.

Cancer remains a major health issue in the world and the effectiveness of current therapies is limited resulting in disease recurrence and resistance to therapy. Therefore to overcome disease recurrence and have improved treatment efficacy there is a continued effort to develop and test new anticancer drugs that are natural or synthetic - (conventional chemotherapeutics, small molecule inhibitors) and biologic (antibody, tumor suppressor genes, oligonucleotide) product. In parallel, efforts for identifying molecular targets and signaling pathways to which cancer cells are "addicted" are underway. By inhibiting critical signaling pathways that is crucial for cancer cell survival, it is expected that the cancer cells will undergo a withdrawal symptom akin to "de-addiction" resulting in cell death. Thus, the key for having an improved and greater control on tumor growth and metastasis is to develop a therapeutic that is able to kill tumor cells efficiently by modulating critical signaling pathways on which cancer cells rely for their survival.Currently several small molecule inhibitors targeted towards unique molecular signaling pathways have been developed and tested in the clinic. Few of these inhibitors have shown efficacy while others have failed. Thus, targeting a single molecule or pathway may be insufficient to completely block cancer cell proliferation and survival. It is therefore important to identify and test an anticancer drug that can inhibit multiple signaling pathways in a cancer cell, control growth of both primary and metastatic tumors and is safe.One biologic agent that has the characteristics of serving as a potent anticancer drug is interleukin (IL)-24. IL-24 suppresses multiple signaling pathways in a broad-spectrum of human cancer cells leading to tumor cell death, inhibition of tumor angiogenesis and metastasis. Additionally, combining IL-24 with other therapies demonstrated additive to synergistic antitumor activity. Clinical testing of IL-24 as a gene-based therapeutic for the treatment of solid tumors demonstrated that IL-24 is efficacious and is safe. The unique features of IL-24 support its further development as an anticancer drug for cancer treatment.In this review we summarize the current understanding on the molecular targets and signaling pathways regulated by IL-24 in mediating its anticancer activity.

The heterogeneity and multigenetic nature of nervous system aging make modeling of it a formidable task in mammalian species. The powerful genetics, simple anatomy and short life span of the nematode Caenorhabditis elegans offer unique advantages in unraveling the molecular genetic network that regulates the integrity of neuronal structures and functions during aging. In this review, we first summarize recent breakthroughs in the morphological and functional characterization of C. elegans neuronal aging. Age-associated morphological changes include age-dependent neurite branching, axon beading or swelling, axon defasciculation, progressive distortion of the neuronal soma, and early decline in presynaptic release function. We then discuss genetic pathways that modulate the speed of neuronal aging concordant with alteration in life span, such as insulin signaling, as well as cell-autonomous factors that promote neuronal integrity during senescence, including membrane activity and JNK/MAPK signaling. As a robust genetic model for aging, insights from C. elegans neuronal aging studies will contribute to our mechanistic understanding of human brain aging.

The importance of the neurotransmitter dopamine (DA) in the nervous system is underscored by its role in a wide variety of physiological and neural functions in both vertebrates and invertebrates. Binding of dopamine to its membrane receptors initiates precise signaling cascades that result in specific cellular responses. Dopamine receptors belong to a super-family of G-protein coupled receptors (GPCRs) that are characterized by seven trans-membrane domains. In mammals, five dopamine receptors have been identified which are grouped into two different categories D1- and D2-like receptors. The interactions of DA receptors with other proteins including specific Gα subunits are critical in deciding the fate of downstream molecular events carried out by effector proteins. In this mini-review we provide a synopsis of known protein-protein interactions of DA receptors and a perspective on the potential synergistic utility of Caenorhabditis elegans as a model eukaryote with a comparatively simpler nervous system to gain insight on the neuronal and behavioral consequences of the receptor interactions.