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1.  Genetic Models of Apoptosis-Induced Proliferation Decipher Activation of JNK and Identify a Requirement of EGFR Signaling for Tissue Regenerative Responses in Drosophila 
PLoS Genetics  2014;10(1):e1004131.
Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation.
Author Summary
Work in recent years has revealed that apoptotic caspases not only induce apoptosis, but also have non-apoptotic functions. One of these functions is apoptosis-induced proliferation, a relatively recently discovered phenomenon by which apoptotic cells induce proliferation of surviving neighboring cells. This phenomenon may have important implications for stem cell activity, tissue regeneration and tumorigenesis. Here, we describe the development of a genetic model of apoptosis-induced proliferation and the use of this model for convenient and unbiased genetic screening to identify genes involved in the process. We tested mutants of our RNAi transgenic lines targeting the core components of the apoptotic pathway and of JNK signaling, a known mediator of apoptosis-induced proliferation. These assays demonstrate the feasibility of the system for systematic genetic screening and identified several new genes upstream of JNK that are involved in apoptosis-induced proliferation. Finally, we tested the model in a pilot screen for chromosome arm 2L and identified spi, the EGF ligand in flies, as important for apoptosis-induced proliferation. We confirmed the involvement of EGF in a genuine apoptosis-induced regeneration system. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation and regeneration.
PMCID: PMC3907308  PMID: 24497843
2.  Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome 
First systematic analysis of the evolutionary conserved InR/TOR pathway interaction proteome in Drosophila.Quantitative mass spectrometry revealed that 22% of identified protein interactions are regulated by the growth hormone insulin affecting membrane proximal as well as intracellular signaling complexes.Systematic RNA interference linked a significant fraction of network components to the control of dTOR kinase activity.Combined biochemical and genetic data suggest dTTT, a dTOR-containing complex required for cell growth control by dTORC1 and dTORC2 in vivo.
Cellular growth is a fundamental process that requires constant adaptations to changing environmental conditions, like growth factor and nutrient availability, energy levels and more. Over the years, the insulin receptor/target of rapamycin pathway (InR/TOR) emerged as a key signaling system for the control of metazoan cell growth. Genetic screens carried out in the fruit fly Drosophila melanogaster identified key InR/TOR pathway components and their relationships. Phenotypes such as altered cell growth are likely to emerge from perturbed dynamic networks containing InR/TOR pathway components, which stably or transiently interact with other cellular proteins to form complexes and networks thereof. Systematic studies on the topology and dynamics of protein interaction networks become therefore highly relevant to gain systems level understanding of deregulated cell growth. Despite much progress in genetic analysis only few systematic protein interaction studies have been reported for Drosophila, which in most cases lack quantitative information representing the dynamic nature of such networks. Here, we present the first quantitative affinity purification mass spectrometry (AP–MS/MS) analysis on the evolutionary conserved InR/TOR signaling network in Drosophila. Systematic RNAi-based functional analysis of identified network components revealed key components linked to the regulation of the central effector kinase dTOR. This includes also dTTT, a novel dTOR-containing complex required for the control of dTORC1 and dTORC2 in vivo.
For systematic AP–MS analysis, we generated Drosophila Kc167 cell lines inducibly expressing affinity-tagged bait proteins previously linked to InR/TOR signaling. Bait expressing Kc167 cell lines were harvested before and after insulin stimulation for subsequent affinity purification. Following LC–MS/MS analysis and probabilistic data filtering using SAINT (Choi et al, 2010), we generated a quantitative network model from 97 high confidence protein–protein interactions and 58 network components (Figure 2). The presented network displayed a high degree of orthologous interactions conserved also in human cells and identified a number of novel molecular interactions with InR/TOR signaling components for future hypothesis driven analysis.
To measure insulin-induced changes within the InR/TOR interaction proteome, we applied a recently introduced label-free quantitative MS approach (Rinner et al, 2007). The obtained quantitative data suggest that 22% of all interactions in the network are regulated by insulin. Major changes could be observed within the membrane proximal InR/chico/PI3K signaling complexes, and also in 14-3-3 protein containing signaling complexes and dTORC1, a complex that contains besides dTOR all major orthologous proteins found also in human mTORC1 including the two dTORC1 substrates d4E-BP (Thor) and S6 Kinase (S6K). Insulin triggered both, dissociation and association of dTORC1 proteins. Among the proteins that showed enhanced binding to dTORC1 upon insulin stimulation we found Unkempt, a RING-finger protein with a proposed role in ubiquitin-mediated protein degradation (Lores et al, 2010). Besides dTORC1 our systematic AP–MS analysis also revealed the presence of dTORC2, the second major TOR complex in Drosophila. dTORC2 contains the Drosophila orthologous of human mTORC2 proteins, but in contrast to dTORC1 was not affected upon insulin stimulation. Interestingly, we also found a specific set of proteins that were not linked to the canonical TOR complexes TORC1 and TORC2 in dTOR purifications. These include LqfR (liquid facets related), Pontin, Reptin, Spaghetti and the gene product of CG16908. We found the same set of proteins when we used CG16908 as a bait, suggesting complex formation among the identified proteins. None of the dTORC1/2 components besides dTOR was identified in CG16908 purifications, indicating that these proteins form dTOR complexes distinct from dTORC1 and dTORC2. Based on known interaction information from other species and data obtained from this study we refer to this complex as dTTT (Drosophila TOR, TELO2, TTI1) (Horejsi et al, 2010; [18]Hurov et al, 2010; [20]Kaizuka et al, 2010). A directed quantitative MS analysis of dTOR complex components suggests that dTORC1 is the most abundant dTOR complex we identified in Kc167 cells.
We next studied the potential roles of the identified network components for controlling the activity of the dInR/TOR pathway using systematic RNAi depletion and quantitative western blotting to measure the changes in abundance of phosphorylated substrates of dTORC1 (Thor/d4E-BP, dS6K) and dTORC2 (dPKB) in RNAi-treated cells (Figure 5). Overall, we could identify 16 proteins (out of 58) whose depletion caused an at least 50% increase or decrease in the levels of phosphorylated d4E-BP, S6K and/or PKB compared with control GFP RNAi. Besides established pathway components, we found several novel regulators within the dInR/TOR interaction network. For example, RNAi against the novel insulin-regulated dTORC1 component Unkempt resulted in enhanced phosphorylation of the dTORC1 substrate d4E-BP, which suggests a negative role for Unkempt on dTORC1 activity. In contrast, depletion of CG16908 and LqfR caused hypo-phosphorylation of all dTOR substrates similar to dTOR itself, suggesting a positive role for the dTTT complex on dTOR activity. Subsequently, we tested whether dTTT components also plays a role in dTOR-mediated cell growth in vivo. Depletion of both dTTT components, CG16908 and LqfR, in the Drosophila eye resulted in a substantial decrease in eye size. Likewise, FLP-FRT-mediated mitotic recombination resulted in CG16908 and LqfR mutant clones with a similar reduced growth phenotype as observed in dTOR mutant clones. Hence, the combined biochemical and genetic analysis revealed dTTT as a dTOR-containing complex required for the activity of both dTORC1 and dTORC2 and thus plays a critical role in controlling cell growth.
Taken together, these results illustrate how a systematic quantitative AP–MS approach when combined with systematic functional analysis in Drosophila can reveal novel insights into the dynamic organization of regulatory networks for cell growth control in metazoans.
Using quantitative mass spectrometry, this study reports how insulin affects the modularity of the interaction proteome of the Drosophila InR/TOR pathway, an evolutionary conserved signaling system for the control of metazoan cell growth. Systematic functional analysis linked a significant number of identified network components to the control of dTOR activity and revealed dTTT, a dTOR complex required for in vivo cell growth control by dTORC1 and dTORC2.
Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.
PMCID: PMC3261712  PMID: 22068330
cell growth; InR/TOR pathway; interaction proteome; quantitative mass spectrometry; signaling
3.  Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway 
Cell Cycle  2011;10(23):4110-4118.
Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs) that encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here, we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway, including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs and place Drosophila endocytic nTSGs into a network that involves two major signaling pathways implicated in oncogenesis.
PMCID: PMC3272291  PMID: 22101275
Drosophila; endocytic tumor suppressor; Yki; JNK; Tsg101; AP-2; Hippo
4.  c-Jun N-Terminal Kinase 1 Phosphorylates Myt1 To Prevent UVA-Induced Skin Cancer▿  
Molecular and Cellular Biology  2009;29(8):2168-2180.
The c-Jun N-terminal kinase (JNK) signaling pathway is known to mediate both survival and apoptosis of tumor cells. Although JNK1 and JNK2 have been shown to differentially regulate the development of skin cancer, the underlying mechanistic basis remains unclear. Here, we demonstrate that JNK1, but not JNK2, interacts with and phosphorylates Myt1 ex vivo and in vitro. UVA induces substantial apoptosis in JNK wild-type (JNK+/+) or JNK2-deficient (JNK2−/−) mouse embryonic fibroblasts but has no effect on JNK1-deficient (JNK1−/−) cells. In addition, UVA-induced caspase-3 cleavage and DNA fragmentation were suppressed by the knockdown of human Myt1 in skin cancer cells. JNK1 deficiency results in suppressed Myt1 phosphorylation and caspase-3 cleavage in skin exposed to UVA irradiation. In contrast, the absence of JNK2 induces Myt1 phosphorylation and caspase-3 cleavage in skin exposed to UVA. The overexpression of JNK1 with Myt1 promotes cellular apoptosis during the early embryonic development of Xenopus laevis, whereas the presence of JNK2 reduces the phenotype of Myt1-induced apoptotic cell death. Most importantly, JNK1−/− mice developed more UVA-induced papillomas than either JNK+/+ or JNK2−/− mice, which was associated with suppressed Myt1 phosphorylation and decreased caspase-3 cleavage. Taken together, these data provide mechanistic insights into the distinct roles of the different JNK isoforms, specifically suggesting that the JNK1-mediated phosphorylation of Myt1 plays an important role in UVA-induced apoptosis and the prevention of skin carcinogenesis.
PMCID: PMC2663297  PMID: 19204086
5.  c-Jun N-terminal Kinase 2 Regulates Multiple Receptor Tyrosine Kinase Pathways in Mouse Mammary Tumor Growth and Metastasis 
Genes & Cancer  2011;2(1):31-45.
c-Jun N-terminal kinase 2 (JNK2) isoforms are transcribed from the jnk2 gene and are highly homologous with jnk1 and jnk3 transcriptional products. JNK proteins mediate cell proliferation, stress response, and migration when activated by a variety of stimuli, including receptor tyrosine kinases (RTKs), but their ability to influence tumor metastasis is ill defined. To evaluate JNK2 in this manner, we used the highly metastatic 4T1.2 mammary tumor cells. Short hairpin RNA expression directed toward JNK2 (shJNK2) decreases tumor cell invasion. In vivo, shJNK2 expression slows tumor growth and inhibits lung metastasis. Subsequent analysis of tumors showed that shJNK2 tumors express lower GRB2-associated binding protein 2 (GAB2). In vitro, knockdown of JNK2 or GAB2 inhibits Akt activation by hepatocyte growth factor (HGF), insulin, and heregulin-1, while phosphorylation of ERK is constitutive and Src dependent. Knockdown of GAB2 phenocopies knockdown of JNK2 in vivo by reducing tumor growth and metastasis, supporting that JNK2 mediates tumor progression by regulating GAB2. The influence of jnk2 in the host or microenvironment was also evaluated using syngeneic jnk2–/– and jnk2+/+ mice. Jnk2–/– mice experience longer survival and less bone and lung metastasis compared to jnk2+/+ mice after intracardiac injection of 4T1.2 cells. GAB2 has previously been shown to mediate osteoclast differentiation, and osteoclasts are critical mediators of tumor-related osteolysis. Thus, studies focusing on the role of JNK2 on osteoclast differentiation were undertaken. ShJNK2 expression impairs osteoclast differentiation, independently of GAB2. Further, shJNK2 4T1.2 cells express less RANKL, a stimulant of osteoclast differentiation. Together, our data support that JNK2 conveys Src/phosphotidylinositol 3-kinase (PI3K) signals important for tumor growth and metastasis by enhancing GAB2 expression. In osteoclast progenitor cells, JNK2 promotes differentiation, which may contribute to the progression of bone metastasis. These studies identify JNK2 as a tumor and host target to inhibit breast cancer growth and metastasis.
PMCID: PMC3111003  PMID: 21779479
JNK2; metastasis; GAB2; receptor tyrosine kinases; osteoclast
6.  Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network 
PLoS ONE  2009;4(10):e7305.
The regulation of apoptosis under basal (non-stress) conditions is crucial for normal mammalian development and also for normal cellular turnover in different tissues throughout life. Deficient regulation of basal apoptosis, or its perturbation, can result in impaired development and/or disease states including cancer. In contrast to stress-induced apoptosis the regulation of apoptosis under basal conditions is poorly understood. To address this issue we have compared basal- and stress-induced apoptosis in human epithelial cells of normal and cancerous origins. For this purpose we focussed our study on the opposing pro-apoptotic JNK/anti-apoptotic NFκB pathways.
Methodology/Principal Findings
Combinatorial RNAi plus gene knockout were employed to access and map basal regulatory pathways of apoptosis. Follow-on, in-depth analyses included exogenous expression of phosphorylation mutants and chromatin immunoprecipitation. We demonstrate that basal apoptosis is constitutively suppressed by JNK2 in a range of human cancer cell lines. This effect was not observed in non-cancer cells. Silencing JNK2 by RNAi resulted in JNK1-dependent apoptosis of cancer cells via up-regulation of the AP-1 factor c-Jun. Unexpectedly we discovered that JNK1 and c-Jun promote basal apoptosis in the absence of “activating phosphorylations” typically induced by stress. Hypo-phosphorylated c-Jun accumulated to high levels following JNK2 silencing, auto-regulated its own expression and suppressed expression of Bcl-3, an unusual IκB protein and regulator of NFκB. Basal apoptosis was mediated by components of the TNFα response pathway but was mechanistically distinct from TNFα-induced apoptosis.
Our results demonstrate that mechanistically distinct pathways operate to regulate apoptosis in mammalian cells under basal (physiological) versus stress-induced conditions. We also describe a novel apoptotic network which governs the basal survival of cancer cells. Such information is crucial for understanding normal cellular turnover during mammalian development and subsequently throughout life. This information also opens new avenues for therapeutic intervention in human proliferative disease states including cancer.
PMCID: PMC2752166  PMID: 19806201
7.  JNK1 controls dendritic field size in L2/3 and L5 of the motor cortex, constrains soma size, and influences fine motor coordination 
Genetic anomalies on the JNK pathway confer susceptibility to autism spectrum disorders, schizophrenia, and intellectual disability. The mechanism whereby a gain or loss of function in JNK signaling predisposes to these prevalent dendrite disorders, with associated motor dysfunction, remains unclear. Here we find that JNK1 regulates the dendritic field of L2/3 and L5 pyramidal neurons of the mouse motor cortex (M1), the main excitatory pathway controlling voluntary movement. In Jnk1-/- mice, basal dendrite branching of L5 pyramidal neurons is increased in M1, as is cell soma size, whereas in L2/3, dendritic arborization is decreased. We show that JNK1 phosphorylates rat HMW-MAP2 on T1619, T1622, and T1625 (Uniprot P15146) corresponding to mouse T1617, T1620, T1623, to create a binding motif, that is critical for MAP2 interaction with and stabilization of microtubules, and dendrite growth control. Targeted expression in M1 of GFP-HMW-MAP2 that is pseudo-phosphorylated on T1619, T1622, and T1625 increases dendrite complexity in L2/3 indicating that JNK1 phosphorylation of HMW-MAP2 regulates the dendritic field. Consistent with the morphological changes observed in L2/3 and L5, Jnk1-/- mice exhibit deficits in limb placement and motor coordination, while stride length is reduced in older animals. In summary, JNK1 phosphorylates HMW-MAP2 to increase its stabilization of microtubules while at the same time controlling dendritic fields in the main excitatory pathway of M1. Moreover, JNK1 contributes to normal functioning of fine motor coordination. We report for the first time, a quantitative Sholl analysis of dendrite architecture, and of motor behavior in Jnk1-/- mice. Our results illustrate the molecular and behavioral consequences of interrupted JNK1 signaling and provide new ground for mechanistic understanding of those prevalent neuropyschiatric disorders where genetic disruption of the JNK pathway is central.
PMCID: PMC4162472  PMID: 25309320
JNK; dendrite; motor cortex; MAP2; microtubules; cytoskeleton; schizophrenia; behavior
8.  Synaptic and genomic responses to JNK and AP-1 signaling in Drosophila neurons 
BMC Neuroscience  2005;6:39.
The transcription factor AP-1 positively controls synaptic plasticity at the Drosophila neuromuscular junction. Although in motor neurons, JNK has been shown to activate AP-1, a positive regulator of growth and strength at the larval NMJ, the consequences of JNK activation are poorly studied. In addition, the downstream transcriptional targets of JNK and AP-1 signaling in the Drosophila nervous system have yet to be identified. Here, we further investigated the role of JNK signaling at this model synapse employing an activated form of JNK-kinase; and using Serial Analysis of Gene Expression and oligonucleotide microarrays, searched for candidate early targets of JNK or AP-1 dependent transcription in neurons.
Temporally-controlled JNK induction in postembryonic motor neurons triggers synaptic growth at the NMJ indicating a role in developmental plasticity rather than synaptogenesis. An unexpected observation that JNK activation also causes a reduction in transmitter release is inconsistent with JNK functioning solely through AP-1 and suggests an additional, yet-unidentified pathway for JNK signaling in motor neurons. SAGE profiling of mRNA expression helps define the neural transcriptome in Drosophila. Though many putative AP-1 and JNK target genes arose from the genomic screens, few were confirmed in subsequent validation experiments. One potentially important neuronal AP-1 target discovered, CG6044, was previously implicated in olfactory associative memory. In addition, 5 mRNAs regulated by RU486, a steroid used to trigger conditional gene expression were identified.
This study demonstrates a novel role for JNK signaling at the larval neuromuscular junction and provides a quantitative profile of gene transcription in Drosophila neurons. While identifying potential JNK/AP-1 targets it reveals the limitations of genome-wide analyses using complex tissues like the whole brain.
PMCID: PMC1175850  PMID: 15932641
9.  A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling 
PLoS Pathogens  2009;5(11):e1000655.
Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling.
Author Summary
Innate immunity is the sole immune response in the overwhelming majority of multicellular organisms and drives the sophisticated antigen-specific adaptive defenses of vertebrates. Defective regulation of immune signal transduction pathways has disastrous consequences for affected individuals and can result in life-threatening conditions that include cancer, autoimmune and neurological conditions. Thus, there is a major need to identify the regulatory circuits that govern activation of critical innate immune response pathways. The genetically accessible model organism Drosophila melanogaster is an ideal springboard for such studies, as core aspects of innate immune pathways are evolutionarily conserved and novel discoveries in Drosophila often inspire subsequent developments in the characterization of biomedically relevant mammalian pathways. Drosophila responses to certain bacterial invaders proceed through the IMD pathway, which contains partially overlapping signal transduction JNK and NF-κB arms. While substantial efforts have illuminated much of the NF-κB arm, there is a considerable paucity of information on the regulation of the JNK arm. We conducted a survey of the entire Drosophila genome for novel regulators the Imd/dJNK pathway. In this study, we uncovered a novel link between the proliferative Pvr pathway and the IMD pathway.
PMCID: PMC2766254  PMID: 19893628
10.  Phosphoproteomic Analyses Reveal Signaling Pathways That Facilitate Lytic Gammaherpesvirus Replication 
PLoS Pathogens  2013;9(9):e1003583.
Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides – a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication.
Author Summary
Systems-level evaluations of infection-related changes to host phosphoprotein networks are not currently available for any gammaherpesvirus (GHV). Here we describe a quantitative phosphoproteomic analysis of productive GHV replication that demonstrates alterations in the phosphorylation status of more than 80% of host phosphoproteins and identifies 18 viral phosphoproteins. Systematic bioinformatics analyses reveal a predominance of MAPK and CDK signaling events within infected cells and suggest a virus-induced reorganization of signal-transduction pathways within the host phosphoprotein network. Functional experiments confirmed that CDKs and ERK MAPKs facilitate efficient viral replication and identify transcription factor c-Jun as a potential downstream target contributing to MHV68 replication. Finally, we identify the viral cyclin D ortholog as a major pathogen-encoded factor contributing to the MAPK/CDK signature of the infected cell phosphoproteome. These data provide new insight into both viral and host factors that regulate phosphorylation-dependent signaling during lytic GHV replication and offer a new resource for better defining host-pathogen interactions in general.
PMCID: PMC3777873  PMID: 24068923
11.  dJun and Vri/dNFIL3 Are Major Regulators of Cardiac Aging in Drosophila 
PLoS Genetics  2012;8(11):e1003081.
Cardiac aging is a complex process, which is influenced by both environmental and genetic factors. Deciphering the mechanisms involved in heart senescence therefore requires identifying the molecular pathways that are affected by age in controlled environmental and genetic conditions. We describe a functional genomic investigation of the genetic control of cardiac senescence in Drosophila. Molecular signatures of heart aging were identified by differential transcriptome analysis followed by a detailed bio-informatic analysis. This approach implicated the JNK/dJun pathway and the transcription factor Vri/dNFIL3 in the transcription regulatory network involved in cardiac senescence and suggested the possible involvement of oxidative stress (OS) in the aging process. To validate these predictions, we developed a new in vivo assay to analyze heart performance in various contexts of adult heart-specific gene overexpression and inactivation. We demonstrate that, as in mammals, OS plays a central role in cardiac senescence, and we show that pharmacological interventions impinging on OS slow heart senescence. These observations strengthen the idea that cardiac aging is controlled by evolutionarily conserved mechanisms, further validating Drosophila as a model to study cardiac senescence. In addition, we demonstrate that Vri, the ortholog of the vertebrate NFIL3/E4B4 transcription factor, is a major genetic regulator of cardiac aging. Vri overexpression leads to major heart dysfunctions, but its loss of function significantly reduces age-related cardiac dysfunctions. Furthermore, we unambiguously show that the JNK/AP1 pathway, the role of which in cardiac aging in mammals is controversial, is activated during cardiac aging and has a detrimental effect on cardiac senescence. This data-driven functional genomic analysis therefore led to the identification of key components of the Gene Regulatory Network of cardiac aging in Drosophila and may prompt to investigate the involvement of their counterparts in the cardiac aging process in mammals.
Author Summary
Age-associated changes in cardiac structure and function have been implicated in the markedly increased risk of cardiovascular disease, but the molecular basis of these processes is ill-defined. It is difficult to study the genetics of heart aging in mammalian models because of their long life spans and their complexity, involving notably genetic redundancy. Here, we address this issue through identification of molecular signatures of cardiac aging in Drosophila, a model organism in which heart senescence occurs within 2 months. Tissue-specific transcriptome comparison of young and aging fly hearts were performed followed by in silico predictions of the regulatory networks involved. This analysis implicated oxidative stress (OS), the JNK/dJun pathway, and Vri/dNFIL3 in the gene regulatory network that drives cardiac senescence. Measuring heart variables in vivo following heart-specific genetic and pharmacological manipulations confirmed these predictions. We show that OS has a central role in the aging of the fly heart. Moreover, heart-specific partial knockdown of dJun and Vri prevented cardiac senescence, demonstrating that they are essential regulators of cardiac aging. Thus, our results uncover two major genetic determinants of Drosophila cardiac aging whose activities enhance heart senescence. It may therefore be valuable to investigate their involvement in the cardiac aging process in mammals.
PMCID: PMC3510041  PMID: 23209438
12.  Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites 
Molecular and Cellular Biology  2005;25(9):3784-3792.
To further understand how the mitogen-activated protein kinase (MAPK) signaling pathways regulate AP-1 activity, we have elucidated the physiological role of these cascades in the regulation of c-jun gene expression. c-Jun is a crucial component of AP-1 complexes and has been shown in vitro to be a point of integration of numerous signals that can differentially affect its expression as well as its transcriptional activity. Our strategy was based on the use of (i) genetically modified fibroblasts deficient in components of the MAPK cascades and (ii) pharmacological reagents. The results demonstrate that c-Jun NH2-terminal protein kinase (JNK) is essential for a basal level of c-Jun expression and for c-Jun phosphorylation in response to stress. In addition to JNK, p38 MAPK or ERK1/2 and ERK5 are required for mediating UV radiation- or epidermal growth factor (EGF)-induced c-Jun expression, respectively. Further studies indicate that p38 MAPK inhibits the activation of JNK in response to EGF, causing a down-regulation of c-Jun. Overall, these data provide important insights into the mechanisms that ultimately determine the function of c-Jun as a regulator of cell fate.
PMCID: PMC1084315  PMID: 15831482
13.  Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila 
Ma, X | Shao, Y | Zheng, H | Li, M | Li, W | Xue, L
Cell Death & Disease  2013;4(10):e864-.
Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, we have identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl−/− triggered tumor invasion and loss of cell polarity gene-induced cell migration. Our genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, we showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, we found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. Our data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a.
PMCID: PMC3920939  PMID: 24136228
Src42A; invasion; cell death; JNK; Bendless; dUev1a
14.  The Jun Kinase 2 Isoform Is Preferentially Required for Epidermal Growth Factor-Induced Transformation of Human A549 Lung Carcinoma Cells 
Molecular and Cellular Biology  1999;19(3):1938-1949.
We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathway in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Bost, R. McKay, N. Dean, and D. Mercola, J. Biol. Chem. 272:33422–33429, 1997). Several isoforms families of JNK, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative splicing of three different genes and have distinct substrate binding properties. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been screened, and two high-affinity and specific candidates have been identified. Antisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 μM but did not alter JNK2 mRNA or protein levels. Conversely, antisense JNK2 specifically eliminated JNK2 steady-state mRNA and protein expression with an EC50 of 0.1 μM. Antisense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-induced total JNK activity, whereas sense and scrambled-sequence control oligonucleotides had no effect. The elimination of mRNA, protein, and JNK activities lasted 48 and 72 h following a single Lipofectin treatment with antisense JNK1 and JNK2, respectively, indicating sufficient duration for examining the impact of specific elimination on the phenotype. Direct proliferation assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did. EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment but not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but not JNK1 is utilized for mediating the effects of EGF. This study represents the first demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.
PMCID: PMC83987  PMID: 10022881
15.  scribble mutants promote aPKC and JNK-dependent epithelial neoplasia independently of Crumbs 
BMC Biology  2009;7:62.
Metastatic neoplasias are characterized by excessive cell proliferation and disruptions to apico-basal cell polarity and tissue architecture. Understanding how alterations in cell polarity can impact upon tumour development is, therefore, a central issue in cancer biology. The Drosophila gene scribble (scrib) encodes a PDZ-domain scaffolding protein that regulates cell polarity and acts as a tumour suppressor in flies. Increasing evidence also implicates the loss of human Scrib in cancer. In this report, we investigate how loss of Scrib promotes epithelial tumourigenesis in Drosophila, both alone and in cooperation with oncogenic mutations.
We find that genetically distinct atypical protein kinase C (aPKC)-dependent and Jun N-terminal kinase (JNK)-dependent alterations in scrib mutants drive epithelial tumourigenesis. First, we show that over-expression of the apical cell polarity determinants Crumbs (Crb) or aPKC induces similar cell morphology defects and over-proliferation phenotypes as scrib loss-of-function. However, the morphological and proliferative defects in scrib mutants are independent of Crb function, and instead can be rescued by a dominant negative (kinase dead) aPKC transgene. Secondly, we demonstrate that loss of Scrib promotes oncogene-mediated transformation through both aPKC and JNK-dependent pathways. JNK normally promotes apoptosis of scrib mutant cells. However, in cooperation with oncogenic activated Ras or Notch signalling, JNK becomes an essential driver of tumour overgrowth and invasion. aPKC-dependent signalling in scrib mutants cooperates with JNK to significantly enhance oncogene-mediated tumour overgrowth.
These results demonstrate distinct aPKC and JNK-dependent pathways through which loss of Scrib promotes tumourigenesis in Drosophila. This is likely to have a direct relevance to the way in which human Scrib can similarly restrain an oncogene-mediated transformation and, more generally, on how the outcome of oncogenic signalling can be profoundly perturbed by defects in apico-basal epithelial cell polarity.
PMCID: PMC2760524  PMID: 19778415
16.  Inhibited Neurogenesis in JNK1-Deficient Embryonic Stem Cells 
Molecular and Cellular Biology  2005;25(24):10791-10802.
The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.
PMCID: PMC1316944  PMID: 16314504
17.  c-Jun N-terminal kinase 3 (JNK3) Mediates Paraquat- and Rotenone-Induced Dopaminergic Neuron Death 
Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment.
PMCID: PMC3061491  PMID: 20418776
Dopaminergic neuron; JNK3; Paraquat; Parkinson disease; Reactive oxygen species; Rotenone
18.  Targeting p53 via JNK Pathway: A Novel Role of RITA for Apoptotic Signaling in Multiple Myeloma 
PLoS ONE  2012;7(1):e30215.
The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK activators in the treatment of MM.
PMCID: PMC3262803  PMID: 22276160
19.  Ajuba Family Proteins Link JNK to Hippo Signaling 
Science signaling  2013;6(292):10.1126/scisignal.2004324.
Wounding, apoptosis, or infection can trigger a proliferative response in neighboring cells to replace damaged tissue. Studies in Drosophila have implicated Jun N-terminal kinase (JNK)-dependent activation of Yorkie (Yki) as essential to regeneration-associated growth, as well as growth associated with neoplastic tumors. Yki is a transcriptional co-activator that is inhibited by Hippo signaling, a conserved pathway that regulates growth. We found identified a conserved mechanism by which JNK regulated Hippo signaling. Genetic studies in Drosophila identified Jub (also known as Ajuba LIM protein) as required for JNK-mediated activation of Yki, and showed that Jub contributed to wing regeneration after wounding and to tumor growth. Biochemical studies revealed that JNK promoted the phosphorylation of Ajuba family proteins in both Drosophila and mammalian cells. Binding studies in mammalian cells indicated that JNK increased binding between the Ajuba family proteins LIMD1 or WTIP and LATS1, a kinase within the Hippo pathway that inhibits the Yki homolog YAP. Moreover, JNK promoted binding of LIMD1 and LATS1 through direct phosphorylation of LIMD1. These results identify Ajuba family proteins as a conserved link between JNK and Hippo signaling, and imply that JNK increases Yki and YAP activity by promoting the binding of Ajuba family proteins to Warts and LATS.
PMCID: PMC3830546  PMID: 24023255
20.  Involvement of Lgl and Mahjong/VprBP in Cell Competition 
PLoS Biology  2010;8(7):e1000422.
Mahjong is a novel Lethal giant larvae-binding protein that plays a vital role in cell competition in both flies and mammals.
During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby “loser cells” undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj−/−) do not have obvious patterning defects during embryonic or larval development. However, mahj−/− cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl−/− cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj−/− or lgl−/− mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl−/− cells strongly suppresses JNK activation and blocks apoptosis of lgl−/− cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system.
Author Summary
Cell transformation arises from the activation of oncoproteins and/or inactivation of tumor suppressor proteins. During the initial stage of carcinogenesis, transformation occurs in a single epithelial cell that grows within an epithelial monolayer. However, it remains unclear what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been shown that normal and transformed cells often compete with each other for survival in an epithelial tissue, in a process called “cell competition.” Lethal giant larvae (Lgl) is a tumor suppressor protein in flies and mammals. Using biochemical methods, we identified Mahjong as a novel binding partner of Lgl in flies and mammals. Furthermore, we have demonstrated that Mahjong is involved in cell competition in both flies and mammals. In particular, we found that canine kidney epithelial cells depleted for Mahjong undergo apoptosis, but only when surrounded by non-transformed cells. This represents the first evidence that cell competition can occur in a mammalian cell culture system. Although it is not clear at present what molecules/signaling pathways are regulated by Lgl/Mahjong during cell competition, future studies might reveal important pathway components that could be targeted therapeutically to prevent tumor cells from “winning” in their race against normal tissue cells.
PMCID: PMC2903597  PMID: 20644714
21.  JNK3 Maintains Expression of the Insulin Receptor Substrate 2 (IRS2) in Insulin-Secreting Cells: Functional Consequences for Insulin Signaling 
PLoS ONE  2012;7(5):e35997.
We have recently shown that silencing of the brain/islet specific c-Jun N-terminal Kinase3 (JNK3) isoform enhances both basal and cytokine-induced beta-cell apoptosis, whereas silencing of JNK1 or JNK2 has opposite effects. While it is known that JNK1 or JNK2 may promote apoptosis by inhibiting the activity of the pro-survival Akt pathway, the effect of JNK3 on Akt has not been documented. This study aims to determine the involvement of individual JNKs and specifically JNK3 in the regulation of the Akt signaling pathway in insulin-secreting cells. JNK3 silencing strongly decreases Insulin Receptor Substrate 2 (IRS2) protein expression, and blocks Akt2 but not Akt1 activation by insulin, while the silencing of JNK1 or JNK2 activates both Akt1 and Akt2. Concomitantly, the silencing of JNK1 or JNK2, but not of JNK3, potently phosphorylates the glycogen synthase kinase3 (GSK3β). JNK3 silencing also decreases the activity of the transcription factor Forkhead BoxO3A (FoxO3A) that is known to control IRS2 expression, in addition to increasing c-Jun levels that are known to inhibit insulin gene expression. In conclusion, we propose that JNK1/2 on one hand and JNK3 on the other hand, have opposite effects on insulin-signaling in insulin-secreting cells; JNK3 protects beta-cells from apoptosis and dysfunction mainly through maintenance of a normal IRS2 to Akt2 signaling pathway. It seems that JNK3 mediates its effects mainly at the transcriptional level, while JNK1 or JNK2 appear to mediate their pro-apoptotic effect in the cytoplasm.
PMCID: PMC3341388  PMID: 22563476
22.  The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal Kinase Kinases 
Molecular and Cellular Biology  1999;19(2):1569-1581.
The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.
PMCID: PMC116085  PMID: 9891090
23.  The JNK Pathway Is a Key Mediator of Anopheles gambiae Antiplasmodial Immunity 
PLoS Pathogens  2013;9(9):e1003622.
The innate immune system of Anopheles gambiae mosquitoes limits Plasmodium infection through multiple molecular mechanisms. For example, midgut invasion by the parasite triggers an epithelial nitration response that promotes activation of the complement-like system. We found that suppression of the JNK pathway, by silencing either Hep, JNK, Jun or Fos expression, greatly enhanced Plasmodium infection; while overactivating this cascade, by silencing the suppressor Puckered, had the opposite effect. The JNK pathway limits infection via two coordinated responses. It induces the expression of two enzymes (HPx2 and NOX5) that potentiate midgut epithelial nitration in response to Plasmodium infection and regulates expression of two key hemocyte-derived immune effectors (TEP1 and FBN9). Furthermore, the An. gambiae L3–5 strain that has been genetically selected to be refractory (R) to Plasmodium infection exhibits constitutive overexpression of genes from the JNK pathway, as well as midgut and hemocyte effector genes. Silencing experiments confirmed that this cascade mediates, to a large extent, the drastic parasite elimination phenotype characteristic of this mosquito strain. In sum, these studies revealed the JNK pathway as a key regulator of the ability of An. gambiae mosquitoes to limit Plasmodium infection and identified several effector genes mediating these responses.
Author Summary
The mosquito Anopheles gambiae is a major vector of human malaria, a disease caused by Plasmodium falciparum parasites that results in more than half a million deaths each year. Several signaling pathways in the mosquito have been shown to mediate the mosquito immune responses to Plasmodium infection. In this manuscript we investigated the participation of the Jun-N-terminal kinase (JNK) pathway in mosquito defense responses. We found that JNK signaling is required for mosquito midgut cells to induce expression of two enzymes, HPx2 and NOX5, that mediate epithelial nitration in response to parasite invasion. These reactions modify the parasites and promote activation of the mosquito complement-like system that results in parasite lysis. The JNK pathway also regulates the basal level of expression of TEP1 and FBN9, two key components of the complement-like system that are produced by hemocytes and secreted into the mosquito hemolymph. Our studies revealed that JNK signaling plays a key role for mosquitoes to limit Plasmodium infection, making it an important determinant of malaria transmission to humans.
PMCID: PMC3764222  PMID: 24039583
24.  Dynamic interaction networks in a hierarchically organized tissue 
We have integrated gene expression profiling with database and literature mining, mechanistic modeling, and cell culture experiments to identify intercellular and intracellular networks regulating blood stem cell self-renewal.Blood stem cell fate in vitro is regulated non-autonomously by a coupled positive–negative intercellular feedback circuit, composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9).The antagonistic signals converge in a core intracellular network focused around PI3K, Raf, PLC, and Akt.Model simulations enable functional classification of the novel endogenous ligands and signaling molecules.
Intercellular (between cell) communication networks are required to maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the recognized importance of intercellular networks in regulating adult stem and progenitor cell fate, the specific cell populations involved, and the underlying molecular mechanisms are largely undefined. Although a limited number of studies have applied novel bioinformatic approaches to unravel intercellular signaling in other cell systems (Frankenstein et al, 2006), a comprehensive analysis of intercellular communication in a stem cell-derived, hierarchical tissue network has yet to be reported.
As a model system to explore intercellular communication networks in a hierarchically organized tissue, we cultured human umbilical cord blood (UCB)-derived stem and progenitor cells in defined, minimal cytokine-supplemented liquid culture (Madlambayan et al, 2006). To systematically explore the molecular and cellular dynamics underlying primitive progenitor growth and differentiation, gene expression profiles of primitive (lineage negative; Lin−) and mature (lineage positive; Lin+) populations were generated during phases of stem cell expansion versus depletion. Parallel phenotypic and subproteomic experiments validated that mRNA expression correlated with complex measures of proteome activity (protein secretion and cell surface expression). Using a curated list of secreted ligand–receptor interactions and published expression profiles of purified mature blood populations, we implemented a novel algorithm to reconstruct the intercellular signaling networks established between stem cells and multi-lineage progeny in vitro. By correlating differential expression patterns with stem cell growth, we predict cell populations, pathways, and secreted ligands associated with stem cell self-renewal and differentiation (Figure 3A).
We then tested the correlative predictions in a series of cell culture experiments. UCB progenitor cell cultures were supplemented with saturating amounts of 18 putative regulatory ligands, or cocultured with purified mature blood lineages (megakaryocytes, monocytes, and erythrocytes), and analyzed for effects on total cell, progenitor, and primitive progenitor growth. At the primitive progenitor level, 3/5 novel predicted stimulatory ligands (EGF, PDGFB, and VEGF) displayed significant positive effects, 5/7 predicted inhibitory factors (CCL3, CCL4, CXCL10, TNFSF9, and TGFB2) displayed negative effects, whereas only 1/5 non-correlated ligand (CXCL7) displayed an effect. Also consistent with predictions from gene expression data, megakaryocytes and monocytes were found to stimulate and inhibit primitive progenitor growth, respectively, and these effects were attributable to differential secretome profiles of stimulatory versus inhibitory ligands.
Cellular responses to external stimuli, particularly in heterogeneous and dynamic cell populations, represent complex functions of multiple cell fate decisions acting both directly and indirectly on the target (stem cell) populations. Experimentally distinguishing the mode of action of cytokines is thus a difficult task. To address this we used our previously published interactive model of hematopoiesis (Kirouac et al, 2009) to classify experimentally identified regulatory ligands into one of four distinct functional categories based on their differential effects on cell population growth. TGFB2 was classified as a proliferation inhibitor, CCL4, CXCL10, SPARC, and TNFSF9 as self-renewal inhibitors, CCL3 a proliferation stimulator, and EGF, VEGF, and PDGFB as self-renewal stimulators.
Stem and progenitor cells exposed to combinatorial extracellular signals must propagate this information through intracellular molecular networks, and respond appropriately by modifying cell fate decisions. To explore how our experimentally identified positive and negative regulatory signals are integrated at the intracellular level, we constructed a blood stem cell self-renewal signaling network through extensive literature curation and protein–protein interaction (PPI) network mapping. We find that signal transduction pathways activated by the various stimulatory and inhibitory ligands converge on a limited set of molecular control nodes, forming a core subnetwork enriched for known regulators of self-renewal (Figure 6A). To experimentally test the intracellular signaling molecules computationally predicted as regulators of stem cell self-renewal, we obtained five small molecule antagonists against the kinases Phosphatidylinositol 3-kinase (PI3K), Raf, Akt, Phospholipase C (PLC), and MEK1. Liquid cultures were supplemented with the five molecules individually, and resultant cell population outputs compared against model simulations to deconvolute the functional effects on proliferation (and survival) versus self-renewal. This analysis classifies inhibition of PI3K and Raf activity as selectively targeting self-renewal, PLC as selectively targeting survival, and Akt as selectively targeting proliferation; MEK inhibition appears non-specific for these processes.
This represents the first systematic characterization of how cell fate decisions are regulated non-autonomously through lineage-specific interactions with differentiated progeny. The complex intercellular communication networks can be approximated as an antagonistic positive–negative feedback circuit, wherein progenitor expansion is modulated by a balance of megakaryocyte-derived stimulatory factors (EGF, PDGF, VEGF, and possibly serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). This complex milieu of endogenous regulatory signals is integrated and processed within a core intracellular signaling network, resulting in modulation of cell-level kinetic parameters (proliferation, survival, and self-renewal). We reconstruct a stem cell associated intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular and intracellular signaling. These findings lay the groundwork for novel strategies to control blood stem cell self-renewal in vitro and in vivo.
Intercellular (between cell) communication networks maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the importance of intercellular networks in stem cell biology, their rules, structure and molecular components are poorly understood. Herein, we describe the structure and dynamics of intercellular and intracellular networks in a stem cell derived, hierarchically organized tissue using experimental and theoretical analyses of cultured human umbilical cord blood progenitors. By integrating high-throughput molecular profiling, database and literature mining, mechanistic modeling, and cell culture experiments, we show that secreted factor-mediated intercellular communication networks regulate blood stem cell fate decisions. In particular, self-renewal is modulated by a coupled positive–negative intercellular feedback circuit composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). We reconstruct a stem cell intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular, and intracellular signaling. This represents the first systematic characterization of how stem cell fate decisions are regulated non-autonomously through lineage-specific interactions with differentiated progeny.
PMCID: PMC2990637  PMID: 20924352
cellular networks; hematopoiesis; intercellular signaling; self-renewal; stem cells
25.  Plasticity of the MAPK Signaling Network in Response to Mechanical Stress 
PLoS ONE  2014;9(7):e101963.
Cells display versatile responses to mechanical inputs and recent studies have identified the mitogen-activated protein kinase (MAPK) cascades mediating the biological effects observed upon mechanical stimulation. Although, MAPK pathways can act insulated from each other, several mechanisms facilitate the crosstalk between the components of these cascades. Yet, the combinatorial complexity of potential molecular interactions between these elements have prevented the understanding of their concerted functions. To analyze the plasticity of the MAPK signaling network in response to mechanical stress we performed a non-saturating epistatic screen in resting and stretched conditions employing as readout a JNK responsive dJun-FRET biosensor. By knocking down MAPKs, and JNK pathway regulators, singly or in pairs in Drosophila S2R+ cells, we have uncovered unexpected regulatory links between JNK cascade kinases, Rho GTPases, MAPKs and the JNK phosphatase Puc. These relationships have been integrated in a system network model at equilibrium accounting for all experimentally validated interactions. This model allows predicting the global reaction of the network to its modulation in response to mechanical stress. It also highlights its context-dependent sensitivity.
PMCID: PMC4099004  PMID: 25025279

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