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The loss of glypican-3 induces alterations in Wnt signaling.

Loss-of-function mutations of the GPC3 gene are the cause of the human Simpson-Golabi-Behmel syndrome. Based on the overgrowth phenotype of the Simpson-Golabi-Behmel syndrome patients and the key role played by the insulin-like growth factor (IGF) signaling system in regulating embryonic growth, it was speculated that GPC3 regulates IGF signaling. In order to test the validity of this hypothesis, we mated GPC3 knockout mice with insulin receptor substrate-1 (IRS-1) nullizygous mice. We found that GPC3 regulates organism growth independent of IRS-1, suggesting that GPC3 does not modulate IGF signaling. Instead, we found that GPC3 knockout mice exhibit alterations in the Wnt signaling pathway, which is also associated with the regulation of cell proliferation. In particular, the loss of GPC3 led to the inhibition of the non-canonical Wnt/JNK signaling pathway, while concomitantly causing the activation of canonical Wnt/beta-catenin signaling. These in vivo findings were confirmed in vitro upon the ectopic overexpression of GPC3 in mesothelioma cells. In these cells, the GPC3-induced increase in JNK activity was associated with an enhanced response to Wnt5a. Most interestingly, the heparan sulfate chains of GPC3 were not required for its stimulatory activity on Wnt5a signaling and for the formation of GPC3-Wnt5a complexes. We propose that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling, by potentiating non-canonical Wnt signaling, while inhibiting the canonical Wnt signaling pathway.

Animals↗

Molecular dissection reveals decreased activity and not dominant negative effect in human OTX2 mutants.

The paired-type homeodomain transcription factor Otx2 is essential for forebrain and eye development. Severe ocular malformations in humans have recently been associated with heterozygous OTX2 mutations. To document the molecular defects in human mutants, Otx2 structural characterization was carried out. A collection of deletion and point mutants was created to perform transactivation, DNA binding, and subcellular localization analyses. Transactivation was ascribed to both N- and C-termini of the protein, and DNA binding to the minimal homeodomain, where critical amino acid residues were identified. Acute nuclear localization appeared controlled by a nuclear localization sequence located within the homeodomain which acts in conjunction with a novel nuclear retention domain that we unraveled located in the central part of the protein. This region, which is poorly conserved among Otx proteins, was also endowed with dominant negative activity suggesting that it might confer unique properties to Otx2. Molecular diagnostic of human mutant OTX2 proteins discriminates hypomorphic and loss of function mutations from other mutations that may not be relevant to ocular pathology.

Active Transport, Cell Nucleus↗

Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway.

Animals↗

Altered renal distal tubule structure and renal Na(+) and Ca(2+) handling in a mouse model for Gitelman's syndrome.

Gitelman's syndrome, an autosomal recessive renal tubulopathy caused by loss-of-function mutations in the thiazide-sensitive NaCl co-transporter (NCC) of the distal convoluted tubule (DCT), is characterized by mild renal Na(+) wasting, hypocalciuria, hypomagnesemia, and hypokalemic alkalosis. For gaining further insights into the pathophysiology of Gitelman's syndrome, the impact of NCC ablation on the morphology of the distal tubule, on the distribution and abundance of ion transport proteins along its length, and on renal tubular Na(+) and Ca(2+) handling in a gene-targeted mouse model was studied. NCC-deficient mice had significantly elevated plasma aldosterone levels and exhibited hypocalciuria, hypomagnesemia, and compensated alkalosis. Immunofluorescent detection of distal tubule marker proteins and ultrastructural analysis revealed that the early DCT, which physiologically lacks epithelial Na(+) (ENaC) and Ca(2+) (TRPV5) channels, was virtually absent in NCC-deficient mice. In contrast, the late DCT seemed intact and retained expression of the apical ENaC and TRPV5 as well as basolateral Na(+)-Ca(2+) exchanger. The connecting tubule exhibited a marked epithelial hypertrophy accompanied by an increased apical abundance of ENaC. Ca(2+) reabsorption seemed unaltered in the distal convolution (i.e., the DCT and connecting tubule) as indicated by real-time reverse transcription-PCR, Western blotting, and immunohistochemistry for TRPV5 and Na(+)-Ca(2+) exchanger and micropuncture experiments. The last experiments further indicated that reduced glomerular filtration and enhanced fractional reabsorption of Na(+) and Ca(2+) upstream and of Na(+) downstream of the DCT provide some compensation for the Na(+) transport defect in the DCT and contribute to the hypocalciuria. Thus, loss of NCC leads to major structural remodeling of the renal distal tubule that goes along with marked changes in glomerular and tubular function, which may explain some of the clinical features of Gitelman's syndrome.

Alkalosis↗

Interstitial deletion 9q22.32-q33.2 associated with additional familial translocation t(9;17)(q34.11;p11.2) in a patient with Gorlin-Goltz syndrome and features of Nail-Patella syndrome.

The phenotype of Gorlin-Goltz syndrome or basal cell nevus syndrome (BCNS, #109400, OMIM), a Mendelian trait due to PTCH mutations has been reported in a few cases of interstitial deletion of chromosome 9q. We present an 11-year-old girl with clinical features consistent with BCNS including bridging of sella turcica, biparietal bossing, downward slanting palpebral fissures, mandible prognathism, pectus excavatum, thumb abnormalities, occult spina bifida at L5-S4, numerous basal cell nevi, and single basal cell carcinoma. Cytogenetic analysis using high-resolution banding techniques and fluorescence in situ hybridization (FISH) revealed interstitial chromosome deletion 9q22.32-q33.2 involving the PTCH gene as a secondary breakage event to a chromosome translocation t(9;17)(q34.1;p11.2)mat. Further FISH studies showed the translocation breakpoint on 9q34.11 maps proximal to ABL, between the BAC clone RP11-88G17 and the LMX1B gene. The latter gene encodes a transcription factor, in which loss of function mutations are responsible for the nail-patella syndrome (NPS, #161200 OMIM). Interestingly, some features of our proband (e.g., bilateral patellar dysplasia and abnormal clavicular shape), as well as her healthy sister who carries the same translocation, are also found in patients with NPS. The chromosome 17p11.2 breakpoint maps in the Smith-Magenis syndrome common deletion region, within two overlapping BAC clones, CTD-2354J3 and RP11-311F12.

Basal Cell Nevus Syndrome↗

Undulated short-tail deletion mutation in the mouse ablates Pax1 and leads to ectopic activation of neighboring Nkx2-2 in domains that normally express Pax1.

Previous studies have indicated that the Undulated short-tail deletion mutation in mouse Pax1 (Pax1(Un-s)) not only ablates Pax1, but also disturbs a gene or genes nearby Pax1. However, which gene(s) is involved and how the Pax1(Un-s) phenotype is confined to the Pax1-positive tissues remain unknown. In the present study, we determined the Pax1(Un-s) deletion interval to be 125 kb and characterized genes around Pax1. We show that the Pax1(Un-s) mutation affects four physically linked genes within or near the deletion, including Pax1, Nkx2-2, and their potential antisense genes. Remarkably, Nkx2-2 is ectopically activated in the sclerotome and limb buds of Pax1(Un-s) embryos, both of which normally express Pax1. This result suggests that the Pax1(Un-s) deletion leads to an illegitimate interaction between remotely located Pax1 enhancers and the Nkx2-2 promoter by disrupting an insulation mechanism between Pax1 and Nkx2-2. Furthermore, we show that expression of Bapx1, a downstream target of Pax1, is more strongly affected in Pax1(Un-s) mutants than in Pax1-null mutants, suggesting that the ectopic expression of Nkx2-2 interferes with the Pax1-Bapx1 pathway. Taken together, we propose that a combination of a loss-of-function mutation of Pax1 and a gain-of-function mutation of Nkx2-2 is the molecular basis of the Pax1(Un-s) mutation.

Animals↗

Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region.

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 was previously reported to have two or more common deletion regions where tumor suppressor genes may be located. In this study, we performed deletion mapping of 44 malignant astrocytomas using 12 microsatellite markers on chromosome 10q and demonstrated that the minimal common region of loss of heterozygosity (LOH) was present between D10S192 and D10S566 localized at 10q25.1. Subsequently, we have identified a novel gene, termed h-neu, within the region frequently deleted and found that h-neu encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. Northern blot and RT-PCR analyses revealed that h-neu mRNA was expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains expressed h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript revealed one of the glioma cell lines, U251MG, had a single nucleotide substitution which resulted in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. The D-neu gene is known to serve a critical function in neurogenesis in Drosophila, and loss-of-function mutations produce hyperplasia of primitive neuronal cells. These observations led us to hypothesize that h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors.

Amino Acid Sequence↗

Incomplete penetrance and variable expressivity of a growth defect as a consequence of knocking out two K(+) transporters in the euascomycete fungus Podospora anserina.

We describe an example of incomplete penetrance and variable expressivity in the filamentous fungus Podospora anserina, two genetic properties classically associated with mutations in more complex organisms, such as green plants and animals. We show that the knockouts of two TRK-related K(+) transporters of this ascomycete present variability in their phenotype that cannot be attributed to fluctuations of the genetic background or the environment. Thalli of the knockout strains derived from independent monokaryotic ascospores or from a single monokaryotic ascospore and cultivated under standard growth conditions may or may not present impaired growth. When impaired, thalli exhibit a range of phenotypes. Environmental conditions control expressivity to a large extent and penetrance to a low extent. Restoration of functional potassium transport by heterologous expression of K(+) transporters from Neurospora crassa abolishes or strongly diminishes the growth impairment. These data show that incomplete penetrance and variable expressivity can be an intrinsic property of a single Mendelian loss-of-function mutation. They also show that such variability in the expression of a mutant phenotype can be promoted by a phenomenon not obviously related to the well-known chromatin structure modifications, i.e., potassium transport. They provide a framework to understand human channelopathies with similar properties.

Base Sequence↗

RET(Men2B)-transgene produces sympathoadrenal tumors but does not prevent intestinal aganglionosis in gdnf-/- or gfr alpha-1(-/-) mice.

Multiple endocrine neoplasia type 2B (MEN2B) syndrome is caused by a missense mutation in the RET gene, which replaces Met918 by Thr in the intracellular kinase domain of the protein. This single amino acid substitution transforms the receptor into a constitutively active monomeric kinase (RET(Men2B)) and produces an autosomal dominant syndrome characterized by medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies, and mucosal ganglioneuromas. The ligand, GDNF, stimulates RET activity through a co-receptor, GFR alpha-1. In vitro studies have shown that the kinase and mitogenic properties of RET(Men2B) are enhanced by GDNF/GFR alpha-1 stimulation. A relevant clinical question is whether ablation of either GDNF or GFR alpha-1 could alter penetrance or severity of the MEN2B syndrome. We report that ganglioneuromatous tumors caused by a RET(Men2B) transgene in mice are not affected grossly or microscopically by the absence of gdnf or gfr alpha-1. Loss-of-function mutations in ret, gdnf, or gfr alpha-1 cause pan-intestinal aganglionosis in mice. We find that expression of the RET(Men2B) transgene in enteric neural progenitors, after they colonize the gut, does not prevent intestinal aganglionosis associated with gdnf or gfr alpha-1 deficiency.

Acetylcholinesterase↗

Differential expression of the actin-binding proteins, alpha-actinin-2 and -3, in different species: implications for the evolution of functional redundancy.

The alpha-actinins are a multigene family of four actin-binding proteins related to dystrophin. The two skeletal muscle isoforms of alpha-actinin (ACTN2 and ACTN3) are major structural components of the Z-line involved in anchoring the actin-containing thin filaments. In humans, ACTN2 is expressed in all muscle fibres, while ACTN3 expression is restricted to a subset of type 2 fibres. We have recently demonstrated that alpha-actinin-3 is absent in approximately 18% of individuals in a range of human populations, and that homozygosity for a premature stop codon (577X) accounts for most cases of true alpha-actinin-3 deficiency. Absence of alpha-actinin-3 is not associated with an obvious disease phenotype, raising the possibility that ACTN3 is functionally redundant in humans, and that alpha-actinin-2 is able to compensate for alpha-actinin-3 deficiency. We now present data concerning the expression of ACTN3 in other species. Genotyping of non-human primates indicates that the 577X null mutation has likely arisen in humans. The mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Murine Actn2 and Actn3 are differentially expressed, spatially and temporally, during embryonic development and, in contrast to humans, alpha-actinin-2 expression does not completely overlap alpha-actinin-3 in postnatal skeletal muscle, suggesting independent function. Furthermore, sequence comparison of human, mouse and chicken alpha-actinin genes demonstrates that ACTN3 has been conserved over a long period of evolutionary time, implying a constraint on evolutionary rate imposed by continued function of the gene. These observations provide a real framework in which to test theoretical models of genetic redundancy as they apply to human populations. In addition we highlight the need for caution in making conclusions about gene function from the phenotypic consequences of loss-of-function mutations in animal knockout models.

Actinin↗

A novel mouse model of hypogonadotrophic hypogonadism: N-ethyl-N-nitrosourea-induced gonadotropin-releasing hormone receptor gene mutation.

An autosomal-recessive mutation that causes hypogonadotrophic hypogonadism was isolated during an N-ethyl-N-nitrosourea mutagenesis screen in mice. Affected males had micropenis and small, undescended testes with spermatogenesis arrested at the pachytene stage of meiosis, leading to sterility. Androgen-sensitive organs were small and immature. Affected females were externally normal but sterile with small ovaries due to an arrest at the secondary stage of folliculogenesis, and the uterus and oviducts were thin and immature. Circulating reproductive hormones were significantly decreased in affected males and females. There was also a dramatic reduction in the numbers of FSH- and LH-producing gonadotrophs. Meiotic mapping of the mutation and candidate gene sequencing determined that the N-ethyl-N-nitrosourea-induced lesion is in the third transmembrane domain of the GnRH receptor gene (Gnrhr). In vitro studies indicate that the mutant receptor is not coupled to the plasma membrane signal transduction system. Moreover, this mutant cannot be rescued with defined GnRH receptor pharmacoperones (pharmacological chaperones), an approach that rescues many other misfolded mutants. The mutant GnRH receptor was also shown to exert a dominant-negative effect on wild-type receptor function, indicating that the mutant receptor is unable to fold properly and likely misrouted within the cell, not reaching the plasma membrane. Surprisingly, Gnrhr mutant transcripts were significantly up-regulated in the pituitaries of Gnrhr mutants, revealing a previously unknown autoregulatory feedback loop. This is the first report of a mouse with a Gnrhr loss of function mutation. These GnRH-insensitive mice provide a novel animal model for the study of human idiopathic hypogonadotrophic hypogonadism.

Animals↗

The LQT syndromes--current status of molecular mechanisms.

Our knowledge on the molecular genetics of inherited cardiac arrhythmias is very recent in comparison to the advances of genetics achieved in other inherited cardiac disorders. This is related to the high mortality and early disease onset of these arrhythmias resulting in mostly small nucleus families. Thus, traditional genetic linkage studies that are based on the genetic information obtained from large multi-generation families were made difficult. In 1991, the first chromosomal locus for congenital long-QT (LQT) syndrome was identified on chromosome 11p15.5 (LQT1 locus) by linkage analysis. Meanwhile, the disease-causing gene at the LQT1 locus (KCNQ1), a gene encoding a K+ channel subunit of the IKs channel, and three other, major genes, all encoding cardiac ion channel components, have been identified. Taken together, LQT syndrome turned out to be a heterogeneous channelopathy. Moreover, the power of linkage studies to reveal the genetic causes of the LQT syndrome was also important to identify unknown but fundamental channel components that contribute to the ion currents tuning ventricular repolarization. In-vitro expression of the altered ion channel genes demonstrated in each case that the altered ion channel function produces prolongation of the action potential and thus the increasing propensity to ventricular tachyarrhythmias. Since these ion channels are pharmacological targets of many antiarrhythmic (and other) drugs, individual and potentially deleterious drug responses may be related to genetic variation in ion channel genes. Very recently, also in acquired LQT syndrome, which is a frequent clinical disorder in cardiology a genetic basis has been proposed in part since mutations in LQT genes have been specifically found. The discovery of ion channel defects in LQT syndrome represents the major achievement in our understanding and implies potential therapeutic options. The knowledge of the genomic structure of the LQT genes now offers the possibility to detect the underlying genetic defect in 80-90% of all patients. With this specific information, containing the type of ion channel (Na+ versus K+ channel) and electrophysiological alteration by the mutation (loss-of-function versus change-of-function mutation), gene-directed, elective drug therapies have been initiated in genotyped LQT patients. Based on preliminary data, that were supported by in vitro studies, this approach may be useful in recompensating the characteristic phenotypes in some LQT patients. Mutation detection is a new diagnostic tool which may become of more increasing importance in patients with a normal QTc or just a borderline prolongation of the QTc interval at presentation. These patients represent approximately 40% of all familial cases. Moreover, LQT3 syndrome and idiopathic ventricular fibrillation are allelic disorders and genetically overlap. In both mutations in the LQT3 gene SCN5A encoding the Na+ channel alpha-subunit for INa have been reported. Thus, the clinical nosology of inherited arrhythmias may be reconsidered after elucidation of the underlying molecular bases. Meanwhile, genotype-phenotype correlations in large families are on the way to evaluate intergene, interfamilial, and intrafamilial differences in the clinical phenotype reflecting gene specific, gene-site specific, and individual consequences of a given mutation. LQT syndrome is phenotypically heterogeneous due to the reduced penetrance and variable expressivity associated with the mutations. This paper discusses the current data on molecular genetics and genotype-phenotype correlations and the implications for diagnosis and treatment.

Chromosome Mapping↗

Retroviral integration at the Epi1 locus cooperates with Nf1 gene loss in the progression to acute myeloid leukemia.

Juvenile myelomonocytic leukemia (JMML) is a disease that occurs in young children and is associated with a high mortality rate. In most patients, JMML has a progressive course leading to death by virtue of infection, bleeding, or progression to acute myeloid leukemia (AML). As it is known that children with neurofibromatosis type 1 syndrome have a markedly increased risk of developing JMML, we have previously developed a mouse model of JMML through reconstitution of lethally irradiated mice with hematopoietic stem cells homozygous for a loss-of-function mutation in the Nf1 gene (D. L. Largaespada, C. I. Brannan, N. A. Jenkins, and N. G. Copeland, Nat. Genet. 12:137-143, 1996). In the course of these experiments, we found that all these genetically identical reconstituted mice developed a JMML-like disorder, but only a subset went on to develop more acute disease. This result strongly suggests that additional genetic lesions are responsible for disease progression to AML. Here, we describe the production of a unique tumor panel, created using the BXH-2 genetic background, for identification of these additional genetic lesions. Using this tumor panel, we have identified a locus, Epi1, which maps 30 to 40 kb downstream of the Myb gene and appears to be the most common site of somatic viral integration in BXH-2 mice. Our findings suggest that proviral integrations at Epi1 cooperate with loss of Nf1 to cause AML.

Acute Disease↗

The PKCalpha-D294G mutant found in pituitary and thyroid tumors fails to transduce extracellular signals.

Protein kinase C (PKC) is a key regulator of cell proliferation, differentiation, and apoptosis and is one of the drug targets of anticancer therapy. Recently, a single point mutation (D294G) in PKCalpha has been found in pituitary and thyroid tumors with more invasive phenotype. Although the PKCalpha-D294G mutant is implicated in the progression of endocrine tumors, no apparent biochemical/cell biological abnormalities underlying tumorigenesis with this mutant have been found. We report here that the PKCalpha-D294G mutant is unable to bind to cellular membranes tightly despite the fact that it translocates to the membrane as efficiently as the wild-type PKCalpha upon treatment of phorbol ester. The impaired membrane binding is associated with this mutant's inability to transduce several antitumorigenic signals as it fails to mediate phorbol ester-stimulated translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS), to activate mitogen-activated protein kinase and to augment melatonin-stimulated neurite outgrowth. Thus, the PKCalpha-D294G is a loss-of-function mutation. We propose that the wild-type PKCalpha may play important antitumorigenic roles in the progression of endocrine tumors. Therefore, developing selective activators instead of inhibitors of PKCalpha might provide effective pharmacological interventions for the treatment of certain endocrine tumors.

Animals↗

Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. Several lines of evidence strongly implicate mitochondrial dysfunction as a major causative factor in PD, although the molecular mechanisms responsible for mitochondrial dysfunction are poorly understood. Recently, loss-of-function mutations in the parkin gene, which encodes a ubiquitin-protein ligase, were found to underlie a familial form of PD known as autosomal recessive juvenile parkinsonism (AR-JP). To gain insight into the molecular mechanism responsible for selective cell death in AR-JP, we have created a Drosophila model of this disorder. Drosophila parkin null mutants exhibit reduced lifespan, locomotor defects, and male sterility. The locomotor defects derive from apoptotic cell death of muscle subsets, whereas the male sterile phenotype derives from a spermatid individualization defect at a late stage of spermatogenesis. Mitochondrial pathology is the earliest manifestation of muscle degeneration and a prominent characteristic of individualizing spermatids in parkin mutants. These results indicate that the tissue-specific phenotypes observed in Drosophila parkin mutants result from mitochondrial dysfunction and raise the possibility that similar mitochondrial impairment triggers the selective cell loss observed in AR-JP.

Amino Acid Sequence↗

Cbfa1/RUNX2 directs specific expression of the sclerosteosis gene (SOST).

Loss-of-function mutations in the sclerosteosis gene (SOST) cause a rare sclerosing bone dysplasia characterized by skeletal overgrowth. Cbfa1/RUNX2 is a key transcriptional regulator of osteoblast function. Here we link these two pathways by demonstrating, via gel shift and transient transfection analyses, that Cbfa1 binding to the proximal SOST promoter contributes to differential SOST expression in two osteosarcoma cell lines. Additionally, an E-box binding motif in the 1.8-kb proximal SOST promoter appears to be functional in SAOS-2 cells, but does not account for SAOS-specific expression of SOST. The regulation of SOST expression by Cbfa1 suggests a potential role for the sclerosteosis gene in homeostatic regulation of osteoblast differentiation and function. Furthermore, the juxtaposition of Cbfa1, E-box, and C/EBP binding sites in the SOST proximal promoter bears an intriguing resemblance to the promoter for osteocalcin, another osteoblast-specific gene with a loss-of-function phenotype of bone overgrowth.

Adaptor Proteins, Signal Transducing↗

DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

Adenovirus E1A Proteins↗

Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family.

Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila. Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse. Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis. The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood. Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family. This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains. In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3. These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice. Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types.

Adaptor Proteins, Signal Transducing↗