Jeroen P. Roose

XTcf-3 Transcription Factor Mediates β-Catenin-Induced Axis Formation in Xenopus Embryos

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Drosophila Tcf and Groucho interact to repress wingless signalling activity

Wingless/Wnt signalling directs cell-fate choices during embryonic development,. Inappropriate reactivation of the pathway causes cancer. In Drosophila, signal transduction from Wingless stabilizes cytosolic Armadillo, which then forms a bipartite transcription factor with the HMG-box protein Drosophila Tcf (dTcf) and activates expression of Wingless-responsive genes. Here we report that in the absence of Armadillo, dTcf acts as a transcriptional repressor of Wingless-responsive genes, and we show that Groucho acts as a corepressor in this process. Reduction of dTcf activity partially suppresses wingless and armadillo mutant phenotypes, leading to derepression of Wingless-responsive genes. Furthermore, overexpression of wild-type dTcf enhances the phenotype of a weak wingless allele. Finally, mutations in the Drosophila groucho gene also suppress wingless and armadillo mutant phenotypes as Groucho physically interacts with dTcf and is required for its full repressor activity.

The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors

Tcf/Lef transcription factors mediate signalling from Wingless/Wnt proteins by recruiting Armadillo/β-catenin as a transcriptional co-activator. However, studies of Drosophila, Xenopus and Caenorhabditis elegans have indicated that Tcf factors may also be transcriptional repressors,. Here we show that Tcf factors physically interact with members of the Groucho family of transcriptional repressors. In transient transfection assays, the Xenopus Groucho homologue XGrg-4 inhibited activation of transcription of synthetic Tcf reporter genes. In contrast, the naturally truncated Groucho-family member XGrg-5 enhanced transcriptional activation. Injection of XGrg-4 into Xenopus embryos repressed transcription of Siamois and Xnr-3, endogenous targets of β-catenin–Tcf. Dorsal injection of XGrg-4 had a ventralizing effect on Xenopus embryos. Secondary-axis formation induced by a dominant-positive Armadillo–Tcf fusion protein was inhibited by XGrg-4 and enhanced by XGrg-5. These data indicate that expression of Tcf target genes is regulated by a balance between Armadillo and Groucho.

TCF transcription factors: molecular switches in carcinogenesis.

Although originally cloned as lymphoid transcription factors, members of the T-cell factor (Tcf) family are now well recognized as key activators/repressors in many developmental processes. Transcriptionally inert Tcf factors become potent transactivators upon interaction with the Wnt signaling product beta-catenin or its Drosophila counterpart Armadillo. In contrast, Tcf proteins mediate repression when bound to members of the Groucho family of transcriptional repressors, CBP and CtBP. Recently, Tcf factors have been reported as tumor inducers, aberrantly activating their target genes as a result of elevated beta-catenin levels in many types of cancer. These abnormal beta-catenin levels are usually caused by stabilizing mutations in beta-catenin itself or truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. In this review, we will give a chronological overview of the Tcf factors and the phenotypes of Tcf mutant mice, as well as Tcf-binding partners. We will discuss Tcf signaling upon interaction with different partners, resulting in activator and repressor roles of Tcf factors in the light of carcinogenic events.

A Diacylglycerol-Protein Kinase C-RasGRP1 Pathway Directs Ras Activation upon Antigen Receptor Stimulation of T Cells

ABSTRACT Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase Cθ (PKCθ) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear. We generated T-cell clones deficient in phorbol myristate acetate (a surrogate for DAG)-induced Ras activation. Analysis of a RasGRP1-deficient Jurkat T-cell clone and RasGRP1 RNA interference in wild-type cells revealed that RasGRP1 is required for optimal, antigen receptor-triggered Ras-Erk activation. RasGRP1 relies on its DAG-binding domain to selectively activate Erk kinases. Activation of Erk correlates with the phosphorylation of threonine residue 184 in RasGRP1. This phosphorylation event requires the activities of novel PKC kinases. Conversely, active PKCθ depends on RasGRP1 sufficiency to effectively trigger downstream events. Last, DAG-PKC-RasGRP1-driven Ras-Erk activation in T cells is a unique signaling event, not simply compensated for by SOS activity.

Intramolecular Regulatory Switch in ZAP-70: Analogy with Receptor Tyrosine Kinases

ABSTRACT ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras–GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38α. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

T Cell Receptor-Independent Basal Signaling via Erk and Abl Kinases Suppresses RAG Gene Expression

Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

Differential expression of the HMG box transcription factors XTcf-3 and XLef-1 during early Xenopus development

The recent discovery that the HMG box transcription factor XTCF-3 is involved in early axis specification in Xenopus laevis (Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J. Godsave, S., Korinek, V., Roose, J., Destree, O., Clevers, H., 1996. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399) led us to search for other members of the TCF/LEF family in this species. A newly identified HMG box factor was cloned with highest homology to human LEF-1, called XLEF-1. Unlike XTcf-3, XLef-1 is not expressed maternally, but its transcripts become detectable directly after the mid blastula transition (MBT). At later stages, both genes are expressed in the central nervous system (CNS), eyes, otic vesicles, head mesenchyme, neural crest and derivatives, branchial arches, developing heart, tailbud and limb buds. The expression pattern of Lef-1 during later stages of development is evolutionarily conserved.

STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development

Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca2+-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide–exchange factor RasGRP and depended on the concentration of the Ca2+ sensor STIM1, which controls the magnitude of Ca2+ entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca2+-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells.