κB-Ras proteins are fast-exchanging GTPases and function via nucleotide-independent binding of Ral GTPase-activating protein complexes.

κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion.

A pan-cancer analysis implicates human NKIRAS1 as a tumor-suppressor gene.

The NF-κB family of transcription factors and the Ras family of small GTPases are important mediators of proproliferative signaling that drives tumorigenesis and carcinogenesis. The κB-Ras proteins were previously shown to inhibit both NF-κB and Ras activation through independent mechanisms, implicating them as tumor suppressors with potentially broad relevance to human cancers. In this study, we have used two mouse models to establish the relevance of the κB-Ras proteins for tumorigenesis. Additionally, we have utilized a pan-cancer bioinformatics analysis to explore the role of the κB-Ras proteins in human cancers. Surprisingly, we find that the genes encoding κB-Ras 1 (NKIRAS1) and κB-Ras 2 (NKIRAS2) are rarely down-regulated in tumor samples with oncogenic Ras mutations. Reduced expression of human NKIRAS1 alone is associated with worse prognosis in at least four cancer types and linked to a network of genes implicated in tumorigenesis. Our findings provide direct evidence that loss of NKIRAS1 in human tumors that do not carry oncogenic RAS mutations is associated with worse clinical outcomes.

Acid-base homeostasis orchestrated by NHE1 defines the pancreatic stellate cell phenotype in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.

Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells.

Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.

Impact of the Nuclear Envelope on Malignant Transformation, Motility, and Survival of Lung Cancer Cells.

Nuclear pore complexes (NPCs) selectively mediate all nucleocytoplasmic transport and engage in fundamental cell-physiological processes. It is hypothesized that NPCs are critical for malignant transformation and survival of lung cancer cells, and test the hypothesis in lowly and highly metastatic non-small human lung cancer cells (NSCLCs). It is shown that malignant transformation is paralleled by an increased NPCs density, and a balanced pathological weakening of the physiological stringency of the NPC barrier. Pharmacological interference using barrier-breaking compounds collapses the stringency. Concomitantly, it induces drastic overall structural changes of NSCLCs, terminating their migration. Moreover, the degree of malignancy is found to be paralleled by substantially decreased lamin A/C levels. The latter provides crucial structural and mechanical stability to the nucleus, and interacts with NPCs, cytoskeleton, and nucleoskeleton for cell maintenance, survival, and motility. The recent study reveals the physiological importance of the NPC barrier stringency for mechanical and structural resilience of normal cell nuclei. Hence, reduced lamin A/C levels in conjunction with controlled pathological weakening of the NPC barrier stringency may facilitate deformability of NSCLCs during the metastasis steps. Modulation of the NPC barrier presents a potential strategy for suppressing the malignant phenotype or enhancing the effectiveness of currently existing chemotherapeutics.

The RAL signaling network: Cancer and beyond.

The RAL proteins RALA and RALB belong to the superfamily of small RAS-like GTPases (guanosine triphosphatases). RAL GTPases function as molecular switches in cells by cycling through GDP- and GTP-bound states, a process which is regulated by several guanine exchange factors (GEFs) and two heterodimeric GTPase activating proteins (GAPs). Since their discovery in the 1980s, RALA and RALB have been established to exert isoform-specific functions in central cellular processes such as exocytosis, endocytosis, actin organization and gene expression. Consequently, it is not surprising that an increasing number of physiological functions are discovered to be controlled by RAL, including neuronal plasticity, immune response, and glucose and lipid homeostasis. The critical importance of RAL GTPases for oncogenic RAS-driven cellular transformation and tumorigenesis still attracts most research interest. Here, RAL proteins are key drivers of cell migration, metastasis, anchorage-independent proliferation, and survival. This chapter provides an overview of normal and pathological functions of RAL GTPases and summarizes the current knowledge on the involvement of RAL in human disease as well as current therapeutic targeting strategies. In particular, molecular mechanisms that specifically control RAL activity and RAL effector usage in different scenarios are outlined, putting a spotlight on the complexity of the RAL GTPase signaling network and the emerging theme of RAS-independent regulation and relevance of RAL.

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation.

The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.

κB-Ras and Ral GTPases regulate acinar to ductal metaplasia during pancreatic adenocarcinoma development and pancreatitis.

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

Structure of the TSC2 GAP Domain: Mechanistic Insight into Catalysis and Pathogenic Mutations.

The TSC complex is the cognate GTPase-activating protein (GAP) for the small GTPase Rheb and a crucial regulator of the mechanistic target of rapamycin complex 1 (mTORC1). Mutations in the TSC1 and TSC2 subunits of the complex cause tuberous sclerosis complex (TSC). We present the crystal structure of the catalytic asparagine-thumb GAP domain of TSC2. A model of the TSC2-Rheb complex and molecular dynamics simulations suggest that TSC2 Asn1643 and Rheb Tyr35 are key active site residues, while Rheb Arg15 and Asp65, previously proposed as catalytic residues, contribute to the TSC2-Rheb interface and indirectly aid catalysis. The TSC2 GAP domain is further stabilized by interactions with other TSC2 domains. We characterize TSC2 variants that partially affect TSC2 functionality and are associated with atypical symptoms in patients, suggesting that mutations in TSC1 and TSC2 might predispose to neurological and vascular disorders without fulfilling the clinical criteria for TSC.

Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus Provides Insight into Complex Assembly and Tuberous Sclerosis Pathogenesis.

Tuberous sclerosis complex (TSC) is caused by mutations in the TSC1 and TSC2 tumor suppressor genes. The gene products hamartin and tuberin form the TSC complex that acts as GTPase-activating protein for Rheb and negatively regulates the mammalian target of rapamycin complex 1 (mTORC1). Tuberin contains a RapGAP homology domain responsible for inactivation of Rheb, but functions of other protein domains remain elusive. Here we show that the TSC2 N terminus interacts with the TSC1 C terminus to mediate complex formation. The structure of the TSC2 N-terminal domain from Chaetomium thermophilum and a homology model of the human tuberin N terminus are presented. We characterize the molecular requirements for TSC1-TSC2 interactions and analyze pathological point mutations in tuberin. Many mutations are structural and produce improperly folded protein, explaining their effect in pathology, but we identify one point mutant that abrogates complex formation without affecting protein structure. We provide the first structural information on TSC2/tuberin with novel insight into the molecular function.

Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells.

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

κB-Ras proteins regulate both NF-κB-dependent inflammation and Ral-dependent proliferation.

The transformation of cells generally involves multiple genetic lesions that undermine control of both cell death and proliferation. We now report that κB-Ras proteins act as regulators of NF-κB and Ral pathways, which control inflammation/cell death and proliferation, respectively. Cells lacking κB-Ras therefore not only show increased NF-κB activity, which results in increased expression of inflammatory mediators, but also exhibit elevated Ral activity, which leads to enhanced anchorage-independent proliferation (AIP). κB-Ras deficiency consequently leads to significantly increased tumor growth that can be dampened by inhibiting either Ral or NF-κB pathways, revealing the unique tumor-suppressive potential of κB-Ras proteins. Remarkably, numerous human tumors show reduced levels of κB-Ras, and increasing the level of κB-Ras in these tumor cells impairs their ability to undergo AIP, thereby implicating κB-Ras proteins in human disease.

Crosstalk in NF-κB signaling pathways.

NF-κB transcription factors are critical regulators of immunity, stress responses, apoptosis and differentiation. A variety of stimuli coalesce on NF-κB activation, which can in turn mediate varied transcriptional programs. Consequently, NF-κB-dependent transcription is not only tightly controlled by positive and negative regulatory mechanisms but also closely coordinated with other signaling pathways. This intricate crosstalk is crucial to shaping the diverse biological functions of NF-κB into cell type- and context-specific responses.

IkappaBbeta acts to inhibit and activate gene expression during the inflammatory response.

The activation of pro-inflammatory gene programs by nuclear factor-kappaB (NF-kappaB) is primarily regulated through cytoplasmic sequestration of NF-kappaB by the inhibitor of kappaB (IkappaB) family of proteins. IkappaBbeta, a major isoform of IkappaB, can sequester NF-kappaB in the cytoplasm, although its biological role remains unclear. Although cells lacking IkappaBbeta have been reported, in vivo studies have been limited and suggested redundancy between IkappaBalpha and IkappaBbeta. Like IkappaBalpha, IkappaBbeta is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus. The crystal structure of IkappaBbeta bound to p65 suggested this complex might bind DNA. In vitro, hypophosphorylated IkappaBbeta can bind DNA with p65 and c-Rel, and the DNA-bound NF-kappaB:IkappaBbeta complexes are resistant to IkappaBalpha, suggesting hypophosphorylated, nuclear IkappaBbeta may prolong the expression of certain genes. Here we report that in vivo IkappaBbeta serves both to inhibit and facilitate the inflammatory response. IkappaBbeta degradation releases NF-kappaB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-alpha (TNF-alpha). Surprisingly, absence of IkappaBbeta results in a dramatic reduction of TNF-alpha in response to LPS even though activation of NF-kappaB is normal. The inhibition of TNF-alpha messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IkappaBbeta bound to p65:c-Rel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaBbeta acts through p65:c-Rel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaBbeta(-/-) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IkappaBbeta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha production during the inflammatory response.

A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.

The NF-kappaB family of transcription factors and its regulation.

Nuclear factor-kappaB (NF-kappaB) consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival. Inducible NF-kappaB activation depends on phosphorylation-induced proteosomal degradation of the inhibitor of NF-kappaB proteins (IkappaBs), which retain inactive NF-kappaB dimers in the cytosol in unstimulated cells. The majority of the diverse signaling pathways that lead to NF-kappaB activation converge on the IkappaB kinase (IKK) complex, which is responsible for IkappaB phosphorylation and is essential for signal transduction to NF-kappaB. Additional regulation of NF-kappaB activity is achieved through various post-translational modifications of the core components of the NF-kappaB signaling pathways. In addition to cytosolic modifications of IKK and IkappaB proteins, as well as other pathway-specific mediators, the transcription factors are themselves extensively modified. Tremendous progress has been made over the last two decades in unraveling the elaborate regulatory networks that control the NF-kappaB response. This has made the NF-kappaB pathway a paradigm for understanding general principles of signal transduction and gene regulation.

Distinct isocomplexes of the TRAPP trafficking factor coexist inside human cells.

The transport protein particle (TRAPP) complex is required for proper vesicular transport from the ER to the Golgi. The composition of yeast TRAPP is well characterized, but the organization of mammalian TRAPP complex remains elusive. Using a tandem affinity purification (TAP) approach, we provide first experimental proof for the association of NIBP (NIK/IKKbeta binding protein) with Bet3 and find two human paralogs of Trs33 (A and B) associated with Bet3. Interaction studies and gel filtration analysis reveal that both proteins are part of human TRAPP and might mark two distinct isocomplexes that exert different functions in the regulation of ER-to-Golgi traffic.

Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation.

Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.

Essential role for IkappaB kinase beta in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation.

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.