Upstream Stimuli Research Articles

A-kinase Anchoring Proteins: Temporal and Spatial Regulation of Intracellular Signal Transduction in the Cardiovascular System

The prototypical intracellular second messenger cAMP is locally controlled by multimolecular protein complexes organized by A-kinase anchoring proteins (AKAPs). AKAPs serve as scaffolds for different sets of cAMP-metabolizing enzymes that control cAMP gradients and regulate cellular responses. In addition to adenylyl cyclases and phosphodiesterases, AKAPs bind signaling enzymes and ion channels that are important regulators of cardiac contractility and pathophysiological myocyte remodeling and hypertrophy. Compartmentation increases the local concentration of cAMP signaling components, providing faster, higher fidelity regulation of cellular processes. This review series highlights the contribution of AKAPs in the heart as scaffold proteins integrating protein kinases, phosphatases and other effector molecules involved in cAMP-dependent signaling.

Mammalian Son of Sevenless Guanine Nucleotide Exchange Factors: Old Concepts and New Perspectives

The Son of Sevenless (Sos) factors were originally discovered 2 decades ago as specialized Ras activators in signaling pathways controlling the process of R7 cell development in the eye of Drosophila melanogaster. The 2 known members of the mammalian Sos family (Sos1 and Sos2) code for ubiquitously expressed, highly homologous (69% overall) proteins involved in coupling signals originated by cell surface receptor tyrosine kinases (RTKs) to downstream, Ras-dependent mitogenic signaling pathways. Mechanistically, the Sos proteins function as enzymatic factors interacting with Ras proteins in response to upstream stimuli to promote guanine nucleotide exchange (GDP/GTP) and subsequent formation of the active Ras-GTP complex. In this review, we summarize current knowledge on structural, regulatory, and functional aspects of the Sos family, focusing on specific aspects of Sos biology such as structure-function relationship, crosstalk with different signaling pathways, and in vivo functional significance as deduced from phenotypic characterization of Sos knockout mice and human genetic syndromes caused by germline hSos1 mutations.

Pulmonary vascular disease related to hemodynamic stress in the pulmonary circulation.

Hemodynamic stress in the pulmonary vessel is directly linked to the development of vascular remodeling and dysfunction, ultimately leading to pulmonary hypertension. Recently, some advances have been made in our molecular understanding of the exogenous upstream stimuli that initiate hemodynamic pertubations as well as the downstream vasoactive effectors that control these responses. However, much still remains unknown regarding how these complex signaling pathways connect in order to result in these characteristic pathophysiological changes. This chapter will describe our current understanding of and needed areas of research into the clinical, physiological, and molecular changes associated with pressure/volume overload in the pulmonary circulation.

Stressin' Sestrins take an aging fight

Sestrins (Sesns) are a family of highly conserved stress-responsive proteins, transcriptionally regulated by p53 and forkhead transcription factor that exhibit oxidoreductase activity in vitro and can protect cells from oxidative stress. However, their major biochemical and physiological function does not appear to depend on their redox (reduction and oxidation) activity. Sesns promote activation of adenosine-5′-monophosphate (AMP)-dependent protein kinase in both mammals and flies. Stress-induced Sesn expression results in inhibition of the target of rapamycin complex 1 (TORC1) and the physiological and pathological implications of disrupting the Sesns-TORC1 crosstalk are now being unravelled. Detailing their mechanism of action and exploring their roles in human physiology point to exciting new insights to topics as diverse as stress, cancer, metabolism and aging.

Differential roles for CaM kinases in mediating excitation–morphogenesis coupling during formation and maturation of neuronal circuits

Ca(2+) -regulated reorganization of actin cytoskeleton is one of the key cell biological events that critically regulate neuronal morphogenesis during circuit formation, spinogenesis during synapse development, and activity-dependent structural plasticity at mature synapses. However, it remains unclear as to what extent the underlying Ca(2+) signaling processes are shared or segregated. Here, we present evidence from the literature that collectively begins to suggest that distinct calmodulin-dependent protein kinase (CaMK) isoforms are differentially expressed in time and in subcellular space, and thus may be selectively activated and engaged by distinct upstream stimuli; each CaMK isoform, in turn, couples to related, but separate, cytoskeletal and transcriptional regulatory pathways, dependent on its abundance or physical proximity with either the upstream or downstream signaling complexes. These signal transduction characteristics provide the basis for better understanding the role of excitation-morphogenesis coupling via multiple CaMKs during neuronal circuit and synapse formation.

Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a

RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.

Abstract B88: A selective Raf kinase inhibitor induces cell death and tumor regression of human cancer cell lines encoding B-RafV600E mutation

Abstract Activation of the Ras-Raf-MEK-ERK pathway has been implicated in a large range of human cancers. Growth factor receptor stimulation by extracellular ligands activates Ras, which then sets in motion a signal transduction cascade through the Raf, MEK and ERK serine/threonine kinases. Mutation of the B-Raf kinase constitutively activates MAPK signalling, thus bypassing the need for upstream stimuli. This has been genetically associated with several human cancers, especially occurrence of the B-RafV600E mutant and its high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. The ability to selectively and potently inhibit B-Raf should provide a potential therapy for patients with mutant B-Raf tumors, for which addictive dependency on this pathway is observed. We have identified a novel, potent, and selective Raf kinase inhibitor that is capable of inhibiting the kinase activity of wild-type B-Raf, B-RafV600E and c-Raf with IC50 values of 3.2, 0.8, and 5.0 nM, respectively. Kinase panel screening for over 270 kinases has indicated that this inhibitor is selective for Raf kinase, with ∼400 fold selectivity towards B-Raf over 91% of the other kinases tested. Specific cellular inhibition of B-RafV600E kinase by this inhibitor leads to decreased ERK phosphorylation and inhibition of cell proliferation by an initial arrest in the G1 phase of the cell cycle, followed by cell death. This inhibition is selective for cancer cells that specifically encode the mutation for B-RafV600E. Oral compound administration inhibits the growth of B-RafV600E mutant melanoma (A375P) and colon cancer (Colo205) human tumor xenografts, growing subcutaneously in immuno-compromised mice. This cell-specific B-RafV600E inhibitor is currently being evaluated in a human Phase I clinical trial. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B88.

Aberrations of Genes Regulating NF Kappa B Pathway in B-Cell Malignant Lymphoma.

Abstract 971NFκB is a tightly regulated transcription factor of lymphocyte activation, proliferation and development. Controlled activity of NF κ B signaling pathway plays critical roles in coordination of immune and inflammatory response. Constitutive NFκB activation is recognized as a key pathological feature in several subsets of B-cell malignant lymphoma, and it is well known that lymphoma frequently occurred in association with chronic inflammation. Recently, our group showed frequent inactivation of A20, a negative regulator of NF κ B, in B-lineage malignant lymphomas. However, the molecular mechanism underlying the aberrant NF κ B activation in lymphomagenesis has not fully understood.In this study, to clarify the genetic basis of the aberrant NFκB activation, we performed genome-wide analysis of copy number alterations as well as allelic imbalances of primary B-lineage lymphoma specimens using Affymetrix GeneChip 250K genomic microarray with the CNAG/AsCNAR algorithm. We also searched for possible mutations in CARD11, CYLD, IKK and TRAF family genes and IκB genes, to obtain comprehensive registry of lesions in genes regulating NFκB pathway. This study included 238 primary lymphoma samples, including 64 samples of diffuse large B-cell lymphomas (DLBCL), 52 of follicular lymphomas (FL), 35 of mantle cell lymphomas (MCL), and 87 of mucosa-associated tissue (MALT) lymphomas. Five Hodgkin lymphoma-derived cell line (KM-H2, L1236, HDLM2, RPMI1666, L540) was also analyzed.Through a genome-wide analysis, we identified that each histology type had a unique genomic signature, suggesting a distinctive underlying molecular pathogenesis for different histology types. In contrast to the fact that A20 mutation was highly associated with loss of heterozygosity at 6q23.3, mutations of CARD11 (5 cases of DLBCL, 2 cases of MALT lymphoma) and IκB family genes (2 cases of DLBCL and 1 cases of MALT lymphoma) had no association with copy number abnormality at the locus of the genes. In total, mutations and copy number alterations in genes regulating NFκB pathway were found in more than 40% of B-cell lymphomas, which underpinned the importance of aberrant NFκB activation in lymphomagenesis.To also assess the role of uncontrolled signaling of NFκB pathway in lymphomagenesis, we re-expressed wild-type A20 in two lymphoma-derived cell lines without normal functional A20 alleles (KM-H2 and L1236). In both cells, re-expression of wild-type A20 resulted in suppression of cell growth and induction of apoptosis, accompanied by down-regulation of NFκB activation. The A20-deficient KM-H2 stably generated tumors in immunodeficient mice, whereas the tumorigenicity was effectively suppressed by re-expression of A20. We further investigated the role of A20 inactivation during clonal expansion of lymphoma by competitive proliferation assays using A20-deficient lymphoma-derived cell lines with or without re-expression of A20. The proportion of A20-expressing cells gradually decreased during competitive cell culture, and A20-expressing cells outgrew control cells in NOG mice, indicating the importance of A20 inactivation in clonal evolution of lymphoma.We demonstrated that uncontrolled NFκB signaling caused by alterations of multiple genes is a common feature of B-lineage lymphomas. Considering the physiological function of NFκB activation induced upon a variety of upstream stimuli, our observations provide an intriguing insight into and the pathogenesis of lymphoma. Our study also indicated that NFκB inhibition may have a role in lymphoma therapeutics. Disclosures:No relevant conflicts of interest to declare.

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK*Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phosphorylated tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phosphorylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phosphorylation-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phosphorylation-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phosphorylation of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK*Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK*Grb7 complexes and Grb7 phosphorylation by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK*Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK*Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.

Frequent inactivation of A20 in B-cell lymphomas

A20 is a negative regulator of the NF-kappaB pathway and was initially identified as being rapidly induced after tumour-necrosis factor-alpha stimulation. It has a pivotal role in regulation of the immune response and prevents excessive activation of NF-kappaB in response to a variety of external stimuli; recent genetic studies have disclosed putative associations of polymorphic A20 (also called TNFAIP3) alleles with autoimmune disease risk. However, the involvement of A20 in the development of human cancers is unknown. Here we show, using a genome-wide analysis of genetic lesions in 238 B-cell lymphomas, that A20 is a common genetic target in B-lineage lymphomas. A20 is frequently inactivated by somatic mutations and/or deletions in mucosa-associated tissue lymphoma (18 out of 87; 21.8%) and Hodgkin's lymphoma of nodular sclerosis histology (5 out of 15; 33.3%), and, to a lesser extent, in other B-lineage lymphomas. When re-expressed in a lymphoma-derived cell line with no functional A20 alleles, wild-type A20, but not mutant A20, resulted in suppression of cell growth and induction of apoptosis, accompanied by downregulation of NF-kappaB activation. The A20-deficient cells stably generated tumours in immunodeficient mice, whereas the tumorigenicity was effectively suppressed by re-expression of A20. In A20-deficient cells, suppression of both cell growth and NF-kappaB activity due to re-expression of A20 depended, at least partly, on cell-surface-receptor signalling, including the tumour-necrosis factor receptor. Considering the physiological function of A20 in the negative modulation of NF-kappaB activation induced by multiple upstream stimuli, our findings indicate that uncontrolled signalling of NF-kappaB caused by loss of A20 function is involved in the pathogenesis of subsets of B-lineage lymphomas.

Oxidative Stress and Autophagy in the Regulation of Lysosome-Dependent Neuron Death

Lysosomes critically regulate the pH-dependent catabolism of extracellular and intracellular macromolecules delivered from the endocytic/heterophagy and autophagy pathways, respectively. The importance of lysosomes to cell survival is underscored not only by their unique ability effectively to degrade metalloproteins and oxidatively damaged macromolecules, but also by the distinct potential for induction of both caspase-dependent and -independent cell death with a compromise in the integrity of lysosome function. Oxidative stress and free radical damage play a principal role in cell death induced by lysosome dysfunction and may be linked to several upstream and downstream stimuli, including alterations in the autophagy degradation pathway, inhibition of lysosome enzyme function, and lysosome membrane damage. Neurons are sensitive to lysosome dysfunction, and the contribution of oxidative stress and free radical damage to lysosome dysfunction may contribute to the etiology of neurodegenerative disease. This review provides a broad overview of lysosome function and explores the contribution of oxidative stress and autophagy to lysosome dysfunction-induced neuron death. Putative signaling pathways that either induce lysosome dysfunction or result from lysosome dysfunction or both, and the role of oxidative stress, free radical damage, and lysosome dysfunction in pediatric lysosomal storage disorders (neuronal ceroid lipofuscinoses or NCL/Batten disease) and in Alzheimer's disease are emphasized.

Increased temperature and protein oxidation lead to HSP72 mRNA and protein accumulation in the in vivo exercised rat heart

Expression of myocardial heat shock protein 72 (HSP72), mediated by its transcription factor, heat shock factor 1 (HSF1), increases following exercise. However, the upstream stimuli governing exercise-induced HSF1 activation and subsequent Hsp72 gene expression in the whole animal remain unclear. Exercise-induced increases in body temperature may promote myocardial radical production, leading to protein oxidation. Conceivably, myocardial protein oxidation during exercise may serve as an important signal to promote nuclear HSF1 migration and activation of Hsp72 expression. Therefore, these experiments tested the hypothesis that prevention of exercise-induced increases in body temperature attenuates cardiac protein oxidation, diminishes HSF1 activation and decreases HSP72 expression in vivo. To test this hypothesis, in vivo exercise-induced changes in body temperature were manipulated by exercising male rats in either cold (4 degrees C) or warm ambient conditions (22 degrees C). Warm exercise increased both body temperature (+3 degrees C) and myocardial protein oxidation, whereas these changes were attenuated by cold exercise. Interestingly, exercise in both conditions did not significantly increase myocardial nuclear localized phosphorylated HSF1. Nonetheless, warm exercise elevated left-ventricular HSP72 mRNA by ninefold and increased myocardial HSP72 protein levels by threefold compared with cold-exercised animals. Collectively, these data indicate that elevated body temperature and myocardial protein oxidation promoted exercise-induced cardiac HSP72 mRNA expression and protein accumulation following in vivo exercise. However, these results suggest that exercise-induced myocardial HSP72 protein accumulation is not a result of nuclear-localized, phosphorylated HSF1, indicating that other transcriptional or post-transcriptional regulatory mechanisms are involved in exercise-induced HSP72 expression.

Role of mitogen-activated protein kinase kinase kinases in signal integration

Mitogen-activated protein kinases (MAPKs) are members of a dynamic protein kinase network through which diverse stimuli regulate the spatio-temporal activities of complex biological systems. MAPKs regulate critical cellular functions required for homeostasis such as the expression of cytokines and proteases, cell cycle progression, cell adherence, motility and metabolism. MAPKs therefore influence cell proliferation, differentiation, survival, apoptosis and development. In vertebrates, five MAPK families are regulated by MAPK kinase kinase-MAPK kinase-MAPK (MKKK-MKK-MAPK) phosphorelay systems. There are at least 20 MKKKs that selectively phosphorylate and activate different combinations of the seven MKKs, resulting in a specific activation profile of members within the five MAPK families. MKKKs are differentially activated by upstream stimuli including cytokines, antigens, toxins and stress insults providing a mechanism to integrate the activation of different MAPKs with the cellular response to each stimulus. Thus, MKKKs can be considered as 'signaling hubs' that regulate the specificity of MAPK activation. In this review, we describe how the MKKK 'hub' function regulates the specificity of MAPK activation, highlighting MKKKs as targets for therapeutic intervention in cancer and other diseases.

Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.

Investigation of the Iron Responsive Elements of the Hepcidin Promoter by In Vivo Imaging of Mice.

Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. It performs this function by modulating iron transfer by both the intestinal epithelial cells and macrophages. Low or high hepcidin levels result in increased or decreased iron absorption respectively. Hepcidin is mainly liver-derived and regulated at least, in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation have all been shown to influence hepcidin mRNA expression in intact animals. Regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating hepatocytes or mixtures of hepatocytes and macrophages or marrow cells with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains a mystery. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we took advantage of the fact that 2 hepcidin genes exist in mice. Hepcidin 1 is homologous to human hepcidin while hepcidin 2 is the result of a tandem duplication of murine hepcidin 1. In mice hepcidin 1 mRNA expression responds to iron and to inflammation, but hepcidin 2 responds only to iron. We established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals' liver after transfecting hydrodynamically hepcidin promoter/luciferase constructs into mice. Murine hepcidin 1, murine hepcidin 2 and human hepcidin promoters were all shown to have basal luciferase activity. Luciferase expression was lower in HFE knockout mice than in wild type controls. When transfected mice were stimulated with iron or endotoxin, the murine hepcidin 1 and human hepcidin constructs were found to respond to both of these stimuli. The murine hepcidin 2 transfected mice showed a weak response to iron stimulation while there was no stimulation by endotoxin. These findings validate, in an in vivo system, what is known about these promoters from other studies. Subsequent experiments have focused on making shorter hepcidin promoter constructs to identify the key regions of the hepcidin promoter involved in iron induction of hepcidin expression. This strategy will allow us to map the iron response site of the hepcidin promoter and this should aid in identifying transcription factors and upstream stimuli involved in regulating expression of hepcidin by iron.

Epsilon protein kinase C as a potential therapeutic target for the ischemic heart

Ischemic heart disease is the leading cause of morbidity and mortality in the western world. Ischemic damage can occur by acute myocardial infarction, stable angina, cardiac stunning, and myocardial hibernation. In addition, 'scheduled' ischemic events, occurring during cardiac surgery, heart transplantation, and elective angioplasty, can also result in cardiac damage. Ischemic or pharmacological preconditioning can decrease the extent of damage to the myocardium. Although the mechanism of preconditioning-mediated cardioprotection is not fully understood, epsilonPKC has been implicated as a critical mediator of this process in animal studies. The use of isozyme-specific pharmacological tools has permitted a better elucidation of the upstream stimuli and the downstream transducers of epsilonPKC in the pathways leading to cardioprotection. While little is known about the role of epsilonPKC in these pathways in humans, animal studies suggest a potential therapeutic role of epsilonPKC. This review will focus on the role of epsilonPKC in cardiac protection and on the signal transduction cascades that have been implicated in this protection.

Atrophic Remodeling of the Transplanted Rat Heart

We have previously shown that the common feature of both pressure overload-induced hypertrophy and atrophy is a reactivation of the fetal gene program. Although gene expression profiles and signal transduction pathways in pressure overload hypertrophy have been well studied, little is known about the mechanisms underlying atrophic remodeling of the unloaded heart. Here, we induced atrophic remodeling by heterotopic transplantation of the rat heart. The activity parameters of three signal transduction pathways important in hypertrophy, i.e. mitogen-activated protein (MAP) kinase, mammalian target of rapamycin (mTOR), and Janus kinase/signal transducers and activators of transcription (JAK/STAT), were interrogated. Gene expression of upstream stimuli – insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) – and metabolic correlates, i.e. peroxisome proliferator-activated receptor-α (PPARα) and PPARα-regulated genes, of these pathways were also measured. In addition, we measured transcript levels of genes known to regulate skeletal muscle atrophy, all of which are negatively regulated by IGF-1 (Mafbx/Atrogin-1, MuRF-1). Atrophic remodeling of the heart was associated with increased expression of IGF-1 and FGF-2. Transcript levels of the nuclear receptor PPARα were decreased, as were the levels of PPARα-regulated genes. Furthermore, there was phosphorylation of ERK1, STAT3, and p70S6K with unloading. Consistent with the increase in IGF-1, we found a decrease in Mafbx/Atrogin-1 and MuRF-1 transcript levels. Rapamycin administration at 0.8 mg/kg/day for 7 days resulted in enhanced atrophy and attenuated the phosphorylation of ERK1, STAT3, and p70S6K without altering gene expression. We conclude that there is significant crosstalk between the mTOR, MAP kinase, and JAK/STAT signaling cascades. Furthermore, ubiquitin ligases, known to be essential for skeletal muscle atrophy, decrease in unloading-induced cardiac atrophy.

B-Raf and C-Raf are required for Ras-stimulated p42 MAP kinase activation in Xenopus egg extracts

During mitosis, a select pool of MEK1 and p42/p44 MAPK becomes activated at the kinetochores and spindle poles, without substantial activation of the bulk of the cytoplasmic p42/p44 MAPK. Recently, we set out to identify the MAP kinase kinase kinase (MAPKKK) responsible for this mitotic activation, using cyclin-treated Xenopus egg extracts as a model system, and presented evidence that Mos was the relevant MAPKKK . However, a second MAPKKK distinct from Mos was readily detectable as well. Here, we partially purify this second MAPKKK and identify it as B-Raf. No changes in the activity of B-Raf were detectable during progesterone-induced oocyte maturation, after egg fertilization, or during the early embryonic cell cycle, arguing against a role for B-Raf in the mitotic activation of MEK1 and p42 MAPK. Ras proteins can bring about activation of MEK1 and p42 MAPK in extracts, and Ras may contribute to signaling from the classical progesterone receptor during oocyte maturation and from receptor tyrosine kinases during early embryogenesis. We found that both B-Raf and C-Raf, but not Mos, are required for Ras-induced MEK1 and p42 MAPK activation. These data indicate that two upstream stimuli, active Ras and active Cdc2, utilize different MAPKKKs to activate MEK1 and p42 MAPK.

Molecular Functions of BCL10 and MALT1 in Normal and Neoplastic Lymphocytes

Antigen receptors on B and T lymphocytes utilize specific adapter and scaffolding proteins to relay antigen recognition at the cell surface to the activation of downstream signaling pathways. One central pathway that is induced upon TCR and BCR ligation cumulates in the activation of NF-κB. This cascade is essential for the elicitation of proper immune responses but when aberrantly activated can also promote diseases including malignancy. Recent work identified the CARD protein BCL10 and the paracaspase MALT1 as key regulators of antigen receptor mediated NF-κB induction. The genes encoding these proteins were originally identified from recurrent chromosomal translocations in mucosa associated lymphoid tissue (MALT) lymphoma associated with advanced disease. In physiological adaptive immune responses antigen receptor proximal signaling and PKC activation induces recruitment of BCL10 and MALT1 to the scaffold protein CARMA1 at the immunoreceptor. Here BCL10 and MALT1 cooperate directly to assemble a multi protein complex that mediates lysine-63-linked ubiquitination of NEMO, the regulatory subunit of the I B kinase (IKK). This mechanism induces signal specific IKK activation, leading to phosphorylation and degradation of the NF-κB inhibitor IκB-α and finally results in controlled activation of NF-κB. However chromosomal translocations in MALT lymphoma uncouple BCL10 or MALT1 from upstream stimuli and promote antigen independent lymphoma growth with tumor progression. This review discusses the mechanisms of BCL10 and MALT1 signal transduction and their critical role in lymphocyte physiology and neoplasia. Keywords: bcl, malt1, nf-κb, cell survival, signal transduction, lymphoma

Impairment of oxidative burst in porcine neutrophils induced by pseudorabies virus.

Industrial swine production is affected by several serious viral diseases, such as pseudorabies, hog cholera, porcine reproductive and respiratory syndrome, which are frequently complicated with the increased incidence of bacterial complications such as Actinobacillus pleuropneumoniae (APP). This clinical observation is suggestive of a virus-bacteria synergism on the pathogenesis. One hypothesis is that viruses induce polymorphonuclear cell (PMNs, primarily neutrophils) dysfunction resulting in defective antibacterial resistance. The purpose of this study was to use the pseudorabies virus (PrV) as a model to explore the possibility of virus-induced PMN dysfunctions in pigs. The goals were to evaluate, in ex vivo settings, the oxidative burst (OB) function of pig PMNs, and to evaluate whether PrV could affect these responses to APP. We found that PrV served as a mild OB stimulant (2-fold) to pig PMNs, which also launched a significant burst to phorbol 12-myristate 13-diacetate (PMA; 61-fold), to non-opsonized, heat-killed and formaldehyde-fixed APP (8-fold), and to normal pig serum-opsonized APP (34-fold). Interestingly, the PMA-induced OB could be reduced 50-70% by preincubating PMNs with PrV, and the critical target was not likely the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase itself. Inactivated PrV was as efficient as viable PrV at exerting the inhibitory effect. On the other hand, PrV exerted a primarily additive effect on APP-induced OB, when the cytotoxic effect of APP on PMNs was avoided. The current finding suggests the possibility that activated PMNs are susceptible to PrV-induced dysfunction, and that the PrV-APP synergism may require upstream stimuli of PMNs to be initiated.