Regulation of cell signaling by the cytoplasmic domains of the heat-stable enterotoxin receptor: identification of autoinhibitory and activating motifs.

Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.

Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.

p53-dependent growth arrest of REF52 cells containing newly amplified DNA.

The rat cell line REF52 is not permissive for gene amplification. Simian virus 40 tumor (T) antigen converts these cells to a permissive state, as do dominant negative mutants of p53, suggesting that the effect of T antigen is due mainly to its ability to bind to p53. To manipulate permissivity, we introduced a temperature-sensitive mutant of T antigen (tsA58) into REF52 cells and selected for resistance to N-(phosphonacetyl)-L-aspartate (PALA). Most freshly isolated PALA-resistant colonies, each of approximately 200 cells, selected at a permissive temperature, arrested when shifted to a nonpermissive temperature. Growth arrest was stable, with no evidence of apoptosis, as long as T antigen was absent but was reversed when T antigen was restored. In contrast, PALA-resistant clones grown to approximately 10(7) cells at a permissive temperature did not arrest when shifted to a nonpermissive temperature. All PALA-resistant clones examined had amplified carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) genes, present in structures consistent with a mechanism involving bridge-breakage-fusion (BBF) cycles. We propose that p53-mediated growth arrest operates only early during the complex process of gene amplification, when newly formed PALA-resistant cells contain broken DNA, generated in BBF cycles. During propagation under permissive conditions, the broken DNA ends are healed, and, even though the p53-mediated pathway is still intact at a nonpermissive temperature and the cells contain amplified DNA, they are not arrested in the absence of broken DNA. The data support the hypothesis that BBF cycles are an important mechanism of amplification and that the broken DNA generated in each cycle is a key signal that regulates permissivity for gene amplification.

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Insulin and Oleate Promote Translocation of Inosine-5' Monophosphate Dehydrogenase to Lipid Bodies

In the present study we identify inosine-5' monophosphate dehydrogenase (IMPDH), a key enzyme in de novo guanine nucleotide biosynthesis, as a novel lipid body-associated protein. To identify new targets of insulin we performed a comprehensive 2-DE analysis of P-32-labelled proteins isolated from 3T3-L1 adipocytes (Hill et al. J Biol Chem 2000; 275: 24313-24320). IMPDH was identified by liquid chromatography/tandem mass spectrometry as a protein which was phosphorylated in a phosphatidylinositol (PI) 3-kinase-dependent manner upon insulin treatment. Although insulin had no significant effect on IMPDH activity, we observed translocation of IMPDH to lipid bodies following insulin treatment. Induction of lipid body formation with oleic acid promoted dramatic redistribution of IMPDH to lipid bodies, which appeared to be in contact with the endoplasmic reticulum, the site of lipid body synthesis and recycling. Inhibition of PI 3-kinase blocked insulin- and oleate-induced translocation of IMPDH and reduced oleate-induced lipid accumulation. However, we found no evidence of oleate-induced IMPDH phosphorylation, suggesting phosphorylation and translocation may not be coupled events. These data support a role for IMPDH in the dynamic regulation of lipid bodies and fatty acid metabolism and regulation of its activity by subcellular redistribution in response to extracellular factors that modify lipid metabolism.

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120(ctn) interacts with E-cadherin, because p120(ctn) localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.

Heterozygote effects in mice with partial truncations in the growth hormone receptor cytoplasmic domain: Assessment of growth parameters and phenotype

The GH receptor (GHR) is essential for normal postnatal growth and development, and the molecular basis of GHR action has been studied intensively. Clinical case studies and more recently mouse models have revealed the extensive phenotype of impaired GH action. We recently reported two new mouse models, possessing cytoplasmic truncations at position 569 (plus Y539/545-F) and 391, which were created to identify functional subdomains within the cytoplasmic signaling domain. In the homozygous state, these animals show progressively impaired postnatal growth coupled with complex changes in gene expression. We describe here an extended phenotype analysis encompassing the heterozygote state to identify whether single copies of these mutant receptors bring about partial or dominant-negative phenotypes. It appears that the retention of the ubiquitin-dependent endocytosis motif the N-terminal cytoplasmic domain permits turnover of these mutant receptors because no dominant-negative phenotype is seen. Nonetheless, we do observe partial impairment of postnatal growth in heterozygotes supporting limited haploinsufficiency. Reproductive function is impaired in these models in a progressive manner, in parallel with loss of signal transducer and activator of transcription-5 activation ability. In summary, we describe a more comprehensive phenotypic analysis of these mouse models, encompassing overall and longitudinal body growth, reproductive function, and hormonal status in both the heterozygote and homozygote state. Our results suggest that patients expressing single copies of similarly mutated GHRs would not display an obvious clinical phenotype.

Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice

Sox7, Sox17 and Sox18 constitute group F of the Sox family of HMG box transcription factor genes. Dominant-negative mutations in Sox18 underlie the cardiovascular defects observed in ragged mutant mice. By contrast, Sox18(-/-) mice are viable and fertile, and display no appreciable anomaly in their vasculature, suggesting functional compensation by the two other SoxF genes. Here, we provide direct evidence for redundant function of Sox17 and Sox18 in postnatal neovascularization by generating Sox17(+/-)-Sox18(-/-) double mutant mice. Whereas Sox18(-/-) and Sox17(+/-)-Sox18(+/)-mice showed no vascular defects, approximately half of the Sox17(+/-)-Sox18(-/-) pups died before postnatal day 21 (P21). They showed reduced neovascularization in the liver sinusoids and kidney outer medulla vasa recta at P7, which most likely caused the ischemic necrosis observed by P14 in hepatocytes and renal tubular epithelia. Those that survived to adulthood showed similar, but milder, vascular anomalies in both liver and kidney, and females were infertile with varying degrees of vascular abnormalities in the reproductive organs. These anomalies corresponded with sites of expression of Sox7 and Sox17 in the developing postnatal vasculature. In vitro angiogenesis assays, using primary endothelial cells isolated from the P7 livers, showed that the Sox17(+/-)-Sox18(-/-)endothelial cells were defective in endothelial sprouting and remodeling of the vasculature in a phenotype-dependent manner. Therefore, our findings indicate that Sox17 and Sox18, and possibly all three SoxF genes, are cooperatively involved in mammalian vascular development.

GP130/OSMR is the only LIF/IL-6 family receptor complex to promote osteoblast differentiation of calvaria progenitors

Leukemia inhibitory factor (LIF) and its receptor (LIFR) are "twins" of Oncostatin M (OSM) and OSMR, respectively, likely having arisen through gene duplications. We compared their effects in a bone nodule-forming model of in vitro osteogenesis, rat calvaria (RC) cell cultures. Using a dominant-negative LIF mutant (hLIF-05), we showed that in RC cell cultures mouse OSM (mOSM) activates exclusively glycoprotein 130 (gp130)/OSMR. In treatments starting at early nodule formation stage, LIF, mOSM, IL-11, and IL-6 + sIL-6R inhibit bone nodule formation, that is, osteoprogenitor differentiation. Treatment with mOSM, and no other cytokine of the family, in early cultures (day 1-3 or 1-4) increases bone colony numbers. hLIF-05 also dose dependently stimulates bone nodule formation, confirming the inhibitory action of gp130/LIFR on osteogenesis. In pulse treatments at successive stages of bone nodule formation and maturation, LIF blocks osteocalcin (OCN) expression by differentiated osteoblasts, but has no effect on bonesialoprotein (BSP) expression. Mouse OSM inhibits OCN and BSP expression in preconfluent cultures with no or progressively reduced effects at later stages, reflecting the disruption of early nodules, possibly due to the strong apoptotic action of mOSM in RC cell cultures. In summary, LIFR and OSMR display differential effects on differentiation and phenotypic expression of osteogenic cells, most likely through different signal transduction pathways. In particular, gp130/OSMR is the only receptor complex of the family to stimulate osteoprogenitor differentiation in the RC cell culture model. © 2005 Wiley-Liss, Inc.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Rôle de la protéine DUSP5 dans l’autophagie des cardiomyocytes

Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes.

Regulation of prostate cancer cell function by activators of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.

Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA, The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis, To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes, Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes, Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas.

Chopper, a new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death

The cytoplasmic juxtamembrane region of the p75 neurotrophin receptor (p75(NTR)) has been found to be necessary and sufficient to initiate neural cell death. The region was named Chopper to distinguish it from CD95-like death domains. A 29-amino acid peptide corresponding to the Chopper region induced caspase- and calpain-mediated death in a variety of neural and nonneural cell types and was not inhibited by signaling through Trk (unlike killing by full-length p75(NTR)). Chopper triggered cell death only when bound to the plasma membrane by a lipid anchor, whereas non-anchored Chopper acted in a dominant-negative manner, blocking p75(NTR)-mediated death both in vitro and in vivo. Removal of the ectodomain of p75(NTR) increased the potency of Chopper activity, suggesting that it regulates the association of Chopper with downstream signaling proteins.