Human Molecular Genetics, 2001, Vol. 10, No. 11 1201-1213
© 2001 Oxford University Press
Ataxin-7 interacts with a Cbl-associated protein that it recruits into neuronal intranuclear inclusions
1INSERM U289, Hôpital de la Salpêtrière, 47 boulevard de l’Hôpital, 75651 Paris, Cedex 13, France, 2Laboratoire de Neuropathologie Escourolle, Hôpital de la Salpêtrière, Paris, France, 3INSERM U495, Hôpital de la Salpêtrière, Paris, France, 4CNRS URA 1968, Institut Pasteur, Paris, France and 5INSERM U528, Institut Curie, Paris, France
Received 21 February 2001; Revised and Accepted 17 March 2001.
DDBJ/EMBL/GenBank accession nos AF330623 and AF330624.
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ABSTRACT |
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Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat in the coding region of the SCA7 gene. The disease primarily affects the cerebellum and the retina, but also many other central nervous system (CNS) structures as the disease progresses. Ataxin-7, encoded by the SCA7 gene, is a protein of unknown function expressed in many tissues including the CNS. In normal brain, ataxin-7 is found in the cytoplasm and/or nucleus of neurons, but in SCA7 brain ataxin-7 accumulates in intranuclear inclusions. Ataxin-7 is expressed ubiquitously, but mutation leads to neuronal death in only certain areas of the brain. This selective pattern of degeneration might be explained by interaction with a partner that is specifically expressed in vulnerable cells. We used a two-hybrid approach to screen a human retina cDNA library for ataxin-7-binding proteins, and isolated R85, a splice variant of Cbl-associated protein (CAP). R85 and CAP are generated by alternative splicing of the gene SH3P12 which we localized on chromosome 10q23–q24. The interaction between ataxin-7 and the SH3P12 gene products (SH3P12GPs) was confirmed by pull-down and co-immunoprecipitation. SH3P12GPs are expressed in Purkinje cells in the cerebellum. Ataxin-7 colocalizes with full-length R85 (R85FL) in co-transfected Cos-7 cells and with one of the SH3P12GPs in neuronal intranuclear inclusions in brain from a SCA7 patient. We propose that this interaction is part of a physiological pathway related to the function or turnover of ataxin-7. Its role in the pathophysiological process of SCA7 disease is discussed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinocerebellar ataxia 7 (SCA7) is a progressive autosomal dominant neurodegenerative disorder characterized clinically by cerebellar ataxia associated with variable neurological signs and progressive macular dystrophy (1). Neuropathological examination reveals moderate to severe neuronal loss and gliosis in the cerebellum and associated structures (inferior olive, cerebellar cortex, dentate nucleus, pontine nuclei), in the basal ganglia (globus pallidus, substantia nigra, subthalamic nucleus, red nucleus) and the spinal cord (2–5). In the retina, photoreceptors, ganglion and bipolar cells degenerate, as do the visual pathways in the central nervous system (CNS) (3).
SCA7 is caused by expansion of a CAG repeat encoding a polyglutamine tract in a 892 amino acid protein of unknown function, ataxin-7 (6). Normal SCA7 alleles contain 4–35 CAG repeats, whereas pathological alleles contain from 36 to 306 CAG repeats (1). This type of mutation has been found previously in eight other diseases: SCA1, SCA2, SCA3/Machado–Joseph disease, SCA6, SCA15 (TATA binding protein), Huntington’s disease, spinal and bulbar muscular atrophy (SBMA) and dentatorubral-pallidoluysian atrophy (DRPLA) (7–9).
Neuronal intranuclear inclusions (NIIs) are pathological structures characteristic of polyglutamine disorders (7). They are observed in several brain regions in SCA7 patients (4,10) and in neurons from patients with other polyglutamine diseases (7), with the exception of SCA6, in which the inclusions are perinuclear and are only seen in the cytoplasm of Purkinje cells (11). Numerous proteins such as transcription factors (like TBP and CREB) or transcription factor binding proteins (like CBP) have been found in NIIs (12,13). Intranuclear inclusions have also been found in animal and cellular models of polyglutamine diseases (7,14).
The function of ataxin-7 is unknown, but it contains several known functional domains, such as polyalanine and polyglutamine repeats, a bipartite nuclear localization signal (NLS) and two serine-rich regions. Expression studies in Cos-1 cells recently confirmed that the NLS directs normal and pathological ataxin-7 to the nucleus (15). Downstream of the polyglutamine tract, ataxin-7 contains four polyproline sequences predicted to be SH3-binding domains (one of class I and the other three of class II). None of the polyproline domains are WW-binding domains. Mushegian et al. (16) identified a short motif in ataxin-7 homologous to a motif found in arrestins, which binds selectively to the phosphorylated activate forms of G protein-coupled receptors and quenches their signaling (16,17). The arrestin-like sequence in ataxin-7 binds phosphate in vitro (16). Binding to its protein partner(s) may thererefore be phosphorylation-dependent.
Normal ataxin-7 is widely expressed in brain, retina and peripheral tissues, including striated muscle, testis and thyroid gland (5). In the CNS, ataxin-7 is preferentially expressed in neurons (18), but the distributions of ataxin-7 mRNA and protein do not correlate with the selective neuronal cell loss in SCA7 patients. For example, Purkinje cells that are extremely vulnerable express low levels of ataxin-7 (5). In normal human brain, ataxin-7 is predominantly located in the cytoplasm of cell bodies and processes (5,18), but it is also reported to be found in the nucleus (5). Since expanded ataxin-7 accumulates in NIIs, the nuclear localization might be related to neuronal loss in SCA7 patients (5). In Cos-1 cells, part of the nuclear ataxin-7 was associated with the nuclear matrix, the promyelocytic leukemia protein (PML)-oncogenic domains (PODs) and nucleoli (15).
As in other polyglutamine diseases, the selective pattern of degeneration contrasts with the ubiquitous expression of ataxin-7, perhaps because of the presence of protein partners which are specific to vulnerable neurons. Numerous partners of polyglutamine disease proteins have been identified. In some cases, the binding affinities of these partners have been shown to either increase (HAP1, HYPA, HYPB, SH3GL3 and calmodulin with huntingtin, LANP with ataxin-1, RERE with atrophin-1 and PQBP-1 for any polyglutamine tract) or decrease (HIP1 with huntingtin) as a function of tract length (19–26).
The molecular basis of most of these diseases and the normal function of ataxin-7 still remain unknown. In this study, we describe a new protein that interacts with ataxin-7. Since SCA7 patients have retinal degeneration, we used a two-hybrid approach to screen a human retina cDNA library for ataxin-7-binding proteins, and have isolated R85, a splice variant of Cbl associated protein (CAP) (27). Full-length R85 (R85FL) cDNA contains a bipartite NLS, two sorbin-like domains and three SH3 domains, and interacts with normal and mutated ataxin-7. R85FL, CAP (27) and ponsin (28) are generated by alternative splicing from a single gene, SH3P12. The interaction between ataxin-7 and SH3P12 gene products (SH3P12GPs) was confirmed by pull-down and co-immunoprecipitation. Ataxin-7 colocalizes with R85FL in co-transfected Cos-7 cells, and with an SH3P12GP in NIIs in brain from an SCA7 patient. We propose that this interaction is part of a physiological pathway related to the function or turnover of ataxin-7, and may be involved in the pathophysiological process in SCA7.
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RESULTS |
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Identification of R85 as an ataxin-7 partner in a yeast two-hybrid screen
The yeast two-hybrid screen was performed with full-length mutated ataxin-7 (Q100) and a human retina cDNA library (29). Twenty-one independent library plasmids were isolated, one of which (R85) contained a 4 kb insert with an open reading frame (ORF) of 1218 bp encoding a protein fragment of 406 amino acids. In the absence of either R85 or ataxin-7, or the replacement of ataxin-7 by the unrelated protein lamin, no ß-galactosidase signal was generated by diploid cells (Fig. 1). The N-terminus of huntingtin (amino acids 1–163), another protein involved in a polyglutamine disease, also interacted with R85. The interactions between ataxin-7 and R85 and between huntingtin and R85 were not modulated by the size of the polyglutamine tract.
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R85 is a splice variant of the gene SH3P12 encoding multiple proteins
Human brain expressed sequenced tags (ESTs) overlapping with R85 were found in the NCBI database with the BLASTN program. One EST contained an initiation codon downstream of several stop codons. PCR primers derived from this EST and from R85 were used to isolate R85FL cDNA from an adult human brain cDNA library. A 2436 bp cDNA encoding a protein of 811 amino acids was obtained (Figs 2 and 3). The sequence of R85FL was found to be identical to several known cDNAs encoding the following proteins: CAP (27); SH3P12, a mouse protein identified during systematic isolation of SH3 domain-containing proteins (30,31); KIAA1296, a human fetal brain protein (32); a protein hypothetically expressed in human uterus; a mouse brain protein SOS-IP 14 (C. Leprince, unpublished data); mouse ponsin-1 and ponsin-2 (28). All these proteins seemed to be generated by alternative splicing of 10 exons (A–J) present in a single gene (Figs 2 and 3), designated SH3P12, which we have localized by fluorescence in situ hybridization (FISH) analysis on human chromosome 10q23–q24 (data not shown).
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R85FL, and all the SH3P12GPs, harbor two sorbin-like domains and three SH3 domains in their C-terminus (27). Unlike the other splice variants, R85FL also contains a bipartite NLS.
Expression pattern of SH3P12
Several splice variants of SH3P12GP have been shown to be ubiquitously expressed by northern blot analyses with cDNA probes including the SH3 domains common to all splice variants (27,28). To determine the expression pattern of splice variants targeted to the nucleus, we hybridized human multi-tissue and brain tissue RNA blots with exon I which encodes the nuclear localization sequence (Fig. 4). In heart and skeletal muscle, 7.5–8 kb transcripts containing exon I were strongly expressed, and in all brain structures examined a 7 kb transcript was detected. Interestingly, only brain and skeletal muscle expressed transcripts containing exon I with an NLS (Fig. 2). In brain, unlike previous studies, only a single variant was detected (Fig. 4). The regional distribution of SH3P12GPs in brain was examined by in situ hybridization and immunohistochemistry on mouse brain sections using a probe or an antibody that detects all splice variants. SH3P12 mRNAs were ubiquitously expressed throughout the brain (data not shown). Staining was exclusively neuronal. No labeling was observed in white matter. In cerebellum, SH3P12GPs are expressed in Purkinje cells (Fig. 5). Staining seems to be uniform throughout the cell. It is found in the cytoplasm of cell bodies and neurites (axon and dendrites).
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Ataxin-7 interacts with R85 through a polyproline-rich region in the yeast two-hybrid system
To delimitate the region of ataxin-7 essential for interaction with R85, truncated forms of ataxin-7 Q10 (T1–T7) were expressed as fusion proteins with LexA and tested for interaction with R85. R85 interacted with full-length ataxin-7 Q10 and with the N-terminal truncated forms (T1–T3). Interaction was stronger with subclones T2 (amino acids 1–230) and T3 (amino acids 1–636) than with T1 (amino acids 1–135) (Fig. 6). The minimal interacting domain T2 in ataxin-7 contained two polyproline-rich class II SH3-binding domains which were previously reported to interact with SH3 domains (33).
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Ataxin-7 interacts with an SH3P12GP in a glutathione S-transferase (GST) pull-down assay
In order to confirm that R85FL interacted with the polyproline-rich domains of ataxin-7, we used previously described GST fusion proteins with full-length or truncated forms of the mouse SH3P12GP ponsin-2 in a GST pull-down assay (28).
Cos-7 cells were transfected with Myc-tagged full-length ataxin-7 (Q10 or Q100). Cell lysates were incubated with immobilized GST fusion proteins containing different regions of the ponsin-2 protein (Fig. 7A and B). Interactions were observed between ataxin-7 and full-length ponsin-2, C-terminus ponsin-2, combinations of SH3 domains (first + second) or (second + third), and the first or the third SH3 domain. The strongest interaction was observed with the third SH3 domain. Both the normal (Q10) and the pathological form (Q100) of ataxin-7 produced in transfected Cos-7 cells interacted with ponsin-2. In contrast, no interactions were observed with GST, with N-terminus ponsin-2 or the second SH3 domain.
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In vitro translated 35S-labeled truncated (T2) ataxin-7 (amino acids 1–230) containing two polyproline SH3-binding domains bound to all GST fusion proteins which included the third SH3 domain (data not shown). Thus, the third SH3 domain of ponsin-2 appeared to be necessary and sufficient for SH3P12GP ponsin-2 binding to the first 230 amino acids of ataxin-7. Given their sequence identity and the fact that the SH3 domains are sufficient for interaction with ataxin-7, all SH3P12GPs might interact because all contain the SH3 domains.
Ataxin-7 co-immunoprecipitates with an SH3P12GP in HEK293 cells
Expression of Xpress-tagged R85FL in transfected Cos-7 cells was detected by western blot as a band of apparent molecular weight around 90 kDa (Fig. 8A, lanes 1 and 2). Similarly, the anti-CAP antibody which recognizes an epitope present in all human SH3P12GPs (Fig. 3) detected an endogenous protein of 90 kDa in total human lymphoblast extracts (Fig. 8A, lane 3) and in extracts from HEK293 cells (Fig. 8A, lane 5). Interaction between Cbl and SH3P12GPs in mammalian cells, shown by Ribon et al. (27), was confirmed here by co-immunoprecipitation (Fig. 8B, lane 3).
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To confirm the interaction between ataxin-7 and SH3P12GPs in mammalian cells, a HA-tagged truncated form (T2) of ataxin-7-Q100 (HA-T2-Q100) was expressed in HEK293 cells. Immunoprecipitation of endogenous SH3P12GP with anti-CAP antibody resulted in co-immunoprecipitation of ataxin-7-T2-Q100, as revealed on western blot with the anti-HA mAb (Fig. 8C, lane 5). Under the same lysis conditions, co-immunoprecipitation was not observed with TBP, another protein with a polyglutamine tract (Fig. 8D, lanes 4 and 5).
Ataxin-7 and R85FL colocalize in transfected Cos-7 cells
Cos-7 cells were co-transfected with plasmids expressing Myc-tagged ataxin-7 (Q10 or Q100) and Xpress-tagged R85FL (Fig. 9A–F). R85FL was found exclusively in the nucleus (Fig. 9A and D). Ataxin-7 (Q10) was also located exclusively in the nucleus of Cos-7 cells, as well as in Cos-1 cells as described previously (15), and colocalized with R85FL (Fig. 9B and C). Mutated ataxin-7 (Q100) was also found in the nucleus and formed aggregates, as expected (Fig. 9E) (15). R85FL did not seem to be sequestered in the nuclear inclusions or in cells with aggregates (Fig. 9F).
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Localization in SCA7 brain: brain SH3P12GP(s) is/are colocalized with ataxin-7 in NIIs
The subcellular localization of SH3P12GP was examined by immunohistochemistry with the anti-ponsin-2 antibody in the brain of a patient with SCA7 (Fig. 9G–L). SH3P12GP immunoreactivity was observed in both cytoplasm and nucleoplasm in neurons (Fig. 9G). In some cells, SH3P12GP immunoreactivity was present exclusively in the cytoplasm (Fig. 9H). When in the nucleus, ponsin-2-like immunoreactivity was present in NIIs (Fig. 9I) where it colocalized with ataxin-7 immunoreactivity (Fig. 9J–L). This was in contrast to what we had observed in Cos-7 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In order to approach the normal function of ataxin-7 and the molecular mechanism underlying the pathogenic effect of the mutated protein, we used the yeast two-hybrid method to identify proteins that interact with normal or mutated ataxin-7. We have identified R85 which harbors three SH3 domains. A database search revealed that R85FL and eight other proteins are splice variants of a gene designated SH3P12 which we have localized on chromosome 10q23–q24.
Northern blot analyses, performed with mouse SH3P12GP probes (full-length ponsin-2 and partial CAP cDNAs) containing the region encoding the three SH3 domains, have detected several splice variants (27,28). SH3P12 transcripts are ubiquitously expressed in brain and other tissues, although the size and number of variants differed among tissues. NLS-containing SH3P12GPs, including R85FL, are more selectively expressed in heart, skeletal muscle and brain, according to information in sequence databases (Fig. 2) and northern blot analysis with an exon I specific probe (Fig. 4). In our study on human brain, however, only one transcript was detected by northern blot analysis. We cannot explain this discrepancy. Since several transcripts containing NLS appear to be present in human brain according to the databases (like R85FL, KIAA1296, mouse SOS-IP 14) and the differences among them concern very small exons (Fig. 3), it is possible that they all migrated at the same apparent molecular weight in our study. Alternatively, several variants might be expressed at a very low level and escape detection by northern blot.
Yeast two-hybrid experiments, pull-down and in vitro binding studies demonstrated that the ataxin-7/SH3P12GP interaction requires the first 230 amino acids of ataxin-7 and the first and third SH3 domains of all SH3P12GPs. Specificity and affinity of ataxin-7 binding differs from one SH3 domain to another, because sequence differences among the three SH3 domains could modify their structure and conformation. Each SH3 domain can be involved in interaction with a specific protein: I-afadin binds to the region of ponsin containing the second and third SH3 domains and vinculin to the first and second SH3 domains (28). The strength of the interaction is not modulated by the number of glutamines in the polyglutamine stretch. The presence of SH3 domains in all proteins encoded by the SH3P12 gene is consistent with our hypothesis that all of these SH3P12GPs are potential molecular partners of ataxin-7. In yeast, SH3P12GP also interacts with the N-terminal region of huntingtin (amino acids 1–163) containing a large polyproline repeat but not a typical consensus SH3-binding domain. An SH3P12GP/huntingtin interaction has been mentioned previously (20). If the interaction is confirmed, SH3P12GP would be a common partner for two proteins involved in polyglutamine diseases. Ataxin-2 and atrophin also contain typical consensus SH3-binding domains and their interactions with SH3P12GP should also be tested. Other proteins without polyproline tracts but involved in polyglutamine diseases probably don’t interact with SH3P12GP, as we confirmed with TBP.
Co-immunoprecipitation of ataxin-7 and an endogenous SH3P12GP from transfected HEK293 cells with an antibody against the SH3P12GP CAP confirmed the interaction. The antibodies used in these experiments detect epitopes that are present in all known splice variants of SH3P12GP (Fig. 6) (28). Thus, in our experiments, we did not differentiate between the splice variants, but all of them have the required SH3 domains sufficient to bind ataxin-7. Importantly, immunohistochemical studies show strong SH3P12GP-like immunoreactivity in Purkinje cells in the cerebellum, which are extremely vulnerable to SCA7. SH3P12GP-like immunoreactivity was also localized in NIIs in brain from an SCA7 patient. However, with the same antibody, we were not able to detect SH3P12GPs in NIIs in a brain from a patient with Huntington’s disease (data not shown), suggesting that the protein partners or the pathological processes differ.
The cellular localization of the SH3P12GP varies, in part due to the presence or absence of an NLS. They also have different functions. CAP enhances actin stress fiber formation and focal adhesions, and interacts with signaling molecules such as the insulin receptor, focal adhesion kinase (FAK) and SOS (34). Ponsin, on the other hand, interacts with two actin filament (F-actin)-binding proteins, I-afadin and vinculin, at zonula adherens in epithelial cells, at cell–cell adherens junction (AJ) in non-epithelial cells, and at cell–matrix AJ in both types of cells (28). No function has been described in the CNS. R85FL, which we have isolated as an NLS-containing molecule, may have a novel function in the nucleus of neurons.
CAP was isolated as a result of its interaction with Cbl (27) which has a RING domain and E3 ubiquitin ligase activity. Cbl recognizes tyrosine-phosphorylated substrates, such as the EGF, CSF-1 and PDGF receptors, through its SH2 domain, and activates allosterically an E2 ubiquitin enzyme through its RING domain (35,36) to ubiquitinylate these receptors. This is a novel mechanism for targeting substrates to the ubiquitin system. Ubiquitinylated aggregates are a hallmark of various neurodegenerative disorders, including polyglutamine diseases (37,38). NIIs in SCA7 brains are known to be ubiquitinylated (4). Covalent binding of ubiquitin to proteins marks them to subsequent degradation by the 26S proteasome. The ubiquitin/proteasome pathway has been implicated in the pathophysiology of several polyglutamine diseases. First, NIIs have been shown to contain proteasome subunits (20S, 19S, 11S) (39–42) and molecular chaperones (Hsp70, HDJ2/HSDJ) (39,41–44). It has been suggested that molecular chaperones might be targeted to the NIIs to help maintain proteins with polyglutamine expansions in a conformation that can be recognized by the ubiquitin complex for hydrolysis by the proteasome. Furthermore, specific proteasome inhibitors, such as lactacystin, induce aggregation in cellular models of polyglutamine diseases (42,45). Finally, Cummings et al. (39) showed that, in vitro, mutated ataxin-1 is three times more resistant to proteasome-mediated degradation than normal ataxin-1 (45). These results directly implicate the ubiquitin/proteasome pathway in the pathophysiological process in polyglutamine diseases.
CAP was shown to interact with Cbl via its third SH3 domain (27). Since this SH3 domain is found in all SH3P12GPs, they are all potential Cbl partners. Thus, the binding of ataxin-7 to SH3P12GP might be a way for ataxin-7 to be targeted to Cbl, and directed to the ubiquitin/proteasome pathway.
The interaction between normal ataxin-7 and SH3P12GP suggests that it is involved in the physiological regulation, and perhaps the ubiquitinylation and degradation of ataxin-7. Mutated ataxin-7 also interacted with SH3P12GP, but ubiquitinylated proteins accumulate in NIIs in SCA7 brains, suggesting that the normal process thought to be mediated by this interaction may have been disrupted. The present results, including the recruitment of SH3P12GPs in NIIs in brain from a SCA7 patient, suggest that SH3P12GPs play an important role in the pathophysiology of SCA7.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid constructions
The plasmid for expression of the SCA7-Q10 LexA fusion protein (pLex-SCA7-Q10) was generated by PCR from previously cloned full-length ataxin-7 (6) and inserted in pLex10 (46). Oligonucleotide primers 1024 and 716, described previously (6), were used to isolate by PCR amplification an expansion of 100 CAG from a SCA7 patient lymphoblasts mRNA; pLex–SCA7–Q100 was generated by insertion of this PCR product with the Q100 expansion in pLex-SCA7-Q10. Various truncated forms of ataxin-7-Q10 were obtained by digestion of pLex-SCA7-Q10 with restriction enzymes, purification and ligation in pLex10 (Fig. 6).
Vectors used for construction of plasmids pCanMycSCA7-Q10 and SCA7-Q100 are from Onyx. The constructions were generated with inserts from pLex-SCA7-Q10 and pLex-SCA7-Q100. The plasmid HA-T2-Q100 was generated by insertion of a 5' HA and a 3' GFP tag by PCR in-frame with the truncated form T2 of ataxin-7 containing Q100 (amino acids 1–230) followed by sub-cloning in pEGFP-N1 (Clontech).
Cloning, plasmid amplification and other molecular biology procedures were performed according to standard procedures (47) using restriction enzymes from New England Biolabs and bacterial strains XL1-Blue or Sure from Stratagene. All the constructions generated by PCR were sequenced in ABI prism 377 (Perkin Elmer).
Cloning of full-length human R85FL cDNA
Oligonucleotide primers for PCR amplification were derived from a brain EST (AI452468) and the R85 insert cloned in the pGAD vector from an adult human brain cDNA library (Clontech) [primers sequences : R85FL, 5'-gcacgaattcATGAGTTCTGAATGTGATGGT-3' (forward) and 5'-gcacgtcgacTTATAGATACAGAGGTTTTAC-3' (reverse)]. The PCR product was subcloned in pcDNA3HisC (Invitrogen).
A bipartite NLS was identified with Profile Scan software (http://www.isrec.isb-sib.ch/software/PFSCAN_form.html). Amino acids were aligned with ClustalW 1.8 software (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html).
Yeast strains, transformations and two-hybrid screening for ataxin-7 interacting proteins
The yeast two-hybrid screen was performed as described previously (48). Yeast host strains were L40 (MATa) and AMR70 (MAT
). Yeasts were transformed according to a modified lithium acetate protocol (49) and grown at 30°C in appropriate complete dropout media (DO).
L40 yeast cells were sequentially transformed with pLex-SCA7-Q100 and with a human retina cDNA library containing 7 x 105 independent clones (oligodT and random primed cDNAs in the pGAD-GE vector) (29). A total of 1.3 x 106 independent yeast transformants were screened, representing about twice the complexity of the cDNA fusion library. After incubation for 3–5 days at 30°C, 158 colonies were orderly patched on a grid on DO minus tryptophan and leucine (DO–WL) and replicated on DO minus tryptophan, leucine and histidine (DO–WLH) plates with a stamp to assay for His+ growth and the ability to turn blue in a ß-galactosidase filter assay (see above). Primary His+/ß-galactosidase+ clones were picked, transferred into liquid DO–WL and grown overnight with shaking, at 30°C, to an OD600 of 1–2. Cells were then transferred into microtiter plates and spotted onto DO–WL, DO–WLH. ß-galactosidase+ clones were identified by filter assay.
To isolate the plasmids, the His+/ß-galactosidase+ colonies were grown overnight in DO minus leucine (DO–L) medium, then plated on dishes containing DO–L and grown at 30°C for 2 days. They were then patched on a grid on DO–L plates and replica plated on DO–WL. Trp–Leu+ colonies were lysed with acid-washed glass beads and the Escherichia coli DH5 strain was electroporated with yeast lysate for further manipulation. Transformants were selected on Luria-Bertani medium plates containing ampicillin for amplification of the plasmids.
For confirmation, pGAD plasmids isolated from positive colonies were transformed into AMR70 and mated with L40 harboring pLex-SCA7-Q10, pLex-SCA7-Q100, pBMT-Htt-Q15, pBMT-Htt-Q104, pLex-Lamin or pLex10 (empty vector). The diploid colonies were tested for activation of the lacZ and HIS3 reporter genes as before. After isolation of the library plasmids by segregation and restriction analysis with Sau3A, the plasmids of positive clones were classed by pattern of digestion and one insert of each class was then sequenced.
ß-galactosidase filter assay
ß-galactosidase expression was detected by a chromogenic filter assay. Yeast colonies were transferred onto Whatman 3M filters and lysed in liquid nitrogen for 15–20 s. Filters were allowed to dry at room temperature, placed on filter paper presoaked in Z-buffer (100 mM sodium phosphate pH 7.0, 10 mM KCl, 1 mM MgSO4) supplemented with 50 mM 2-mercaptoethanol and 2% Xgal (5-bromo-4-chloro-3-indoyl ß-D-galactoside) (Roche Molecular Biochemicals), then incubated at 30°C for up to 8 h.
Antibodies
Polyclonal anti-CAP antibody was directed against a peptide corresponding to R85FL (amino acids 200–215—CEKRAKDDSRRVVKST) and was purchased from Upstate Biotechnology. Polyclonal anti-ponsin-2 antibody was kindly provided by Prof. Takai (Osaka University Medical School, Japan) and was directed against a GST fusion protein encoding amino acids 460–558 of murine ponsin-2 containing the first SH3 domain (28). These two antibodies detect epitopes that are present in all known splice variants of SH3P12GPs. Anti-Myc mAb (9E10), polyclonal and monoclonal anti-TBP (N-12 and 58C9) were purchased from Santa Cruz Biotechnology, anti-Xpress mAb from Invitrogen, anti-HA mAb (HA.11) from Babco and anti-Cbl mAb from Transduction Laboratories. Anti-ataxin-7 mAb 1C1 was kindly provided by G. Yvert and Prof. J.L. Mandel (IGBMC, Illkirch, France). Polyclonal anti-ataxin-7 SCA7-5' was described previously (5). TRITC-conjugated anti-mouse Ig and Alexa 488 anti-rabbit IgG were from Dako and Molecular probes, respectively. Anti-mouse Ig and anti-rabbit Ig antibodies conjugated to horseradish peroxidase were from Jackson Immuno Research.
Tissue preparation for in situ hybridization (ISH)
Adult mice were perfused through the left ventricule with 4% paraformaldehyde (PFA) and postfixed overnight in the same fixative. Brains were rinsed in PBS after fixation, and cryoprotected in PBS containing 15% sucrose for 12 h at 4°C. After embedding in 15% sucrose and 7.5% gelatin in PBS, samples were frozen in melting isopentane and sections were cut on a Microm cryostat (20 µm thick) and collected on Superfrost plus glass slides (Menzel-Glaser).
Patterns of gene transcription were determined by ISH using digoxigenin (DIG)-labeled antisense cRNA probes (Roche Molecular Biochemicals) transcribed from a fragment of a mouse SOS-IP 14 cDNA including the coding region cloned into the polylinker of pBluescript SK. ISH was performed on cryostat sections according to the protocol of Strähle et al. (50) modified by Myat et al. (51). The alkaline phosphatase substrate was 5-bromo-4-indoyl phosphate/nitroblue tetrazolium chloride (BCIP/NBT) (Roche Molecular Biochemicals).
Northern blot analysis
Northern blot analysis was performed on commercially available multiple human adult tissue and brain tissue mRNA blots (Clontech). The 32P-labeled probe was prepared by random priming from a PCR product corresponding to R85FL exon I (bp 1543–1710, amino acids 515–570). Hybridization was performed in Expresshyb hybridization solution (Clontech) at 68°C for 1 h. Following hybridization, blots were washed according to the manufacturer’s recommendations and exposed to X-ray film (Kodak) for 5 days at –80°C.
FISH detection system and image analysis
The R85FL cDNA was mapped by FISH on normal human lymphocyte chromosomes counterstained with DAPI. A biotinylated probe was obtained by PCR, and was detected with avidin-fluorescein isothiocyanate (FITC). Metaphase preparations were viewed with an Axioplan microscope (Zeiss) coupled to a video camera and image analysis system. The DAPI (blue) and FITC (yellow) pseudocolored images were acquired separately, then merged.
Cell lines and cell transfection
Dulbecco’s modified Eagle’s medium (DMEM), modified Eagle’s medium (MEM), fetal calf serum, glutamine and penicillin/streptomycin were purchased from Life Technologies.
Transiently transfected Cos-7 cells (monkey kidney) were used for GST pull-down and colocalization experiments. They were grown in DMEM supplemented with fetal calf serum (10%), glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml). Transiently transfected HEK293 cells (human embryonic kidney) were used for co-immunoprecipitation experiments. They were grown in MEM supplemented with fetal calf serum (10%) and the antibiotics indicated above. Transfection was performed with DMRIE-C (Gibco BRL). Cells were plated to 70% confluence and transfected with 10 µg of DNA (10 cm dish). Transient expression was allowed to proceed for 48 h.
Immunofluorescence microscopy
Transfected Cos-7 cells were grown on coverslips and fixed 48 h post-transfection with 4% PFA for 15 min at room temperature. After washing cells in PBS, they were incubated for 15 min in 50 mM NH4Cl (in PBS), washed with PBS and permeabilized with 0.1% Triton X-100 (in PBS) for 10 min. The cells were then incubated with appropriate primary and secondary antibodies diluted in 3% BSA in PBS for 1 h. For double labeling, ataxin-7 was detected with polyclonal SCA7-5' antibody (1:100) and anti-rabbit Alexa 488-secondary antibody (1:500), whereas Xpress-tagged R85FL protein was detected with monoclonal anti-Xpress antibody (1:200) and anti-mouse TRITC secondary antibody (1:200). After extensive washing, the nuclei were counterstained with Hoechst 33258 (Sigma), rinsed in PBS, mounted in Mowiol and observed with a Leica TCD laser confocal microscope.
Western blot analysis
Cells were harvested at 70% confluence 48 h after transfection as described above. The cells were washed twice with PBS, then lysed for 30 min in 50 mM Tris–HCl pH 8, 150 mM NaCl, 1 mM EDTA with 1% NP40 for immunoprecipitation of ataxin-7 and TBP and 0.5% Triton X-100 for immunoprecipitation of Cbl. A cocktail of protease inhibitors, COMPLETE (Roche Molecular Biochemicals), Pefabloc (AEBSF) 1 mM (Uptima) and pepstatin A (1 µg/ml) was included in all extractions. Cells were scraped from the culture plates and debris was sedimented by centrifugation at 15 680 g for 20 min at 4°C.
Proteins present in the supernatant were denatured, separated by SDS–PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell). Non-specific binding to membranes was blocked with 5% non-fat dry milk in PBS containing 0.2% Tween-20, before incubation with the appropriate primary and secondary antibodies. Proteins were detected with anti-Xpress mAb (1:5000), anti-Myc mAb (9E10) (1:200), polyclonal anti-CAP antibody (1:1000), anti-Cbl mAb (1:5000), anti-HA mAb (1:1000), polyclonal anti-TBP antibody (1:500) or with anti-TBP mAb (1:200). After incubation with secondary antibodies (1:50 000) (Jackson Laboratories), immunoreactive proteins were detected with an enhanced chemiluminescence reagent (Super Signal Kit, Pierce).
Fusion protein expresssion and pull-down assay
GST fusion proteins were expressed in E.coli strain AD202 by induction with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 5 h at 30°C. The cells were then incubated in binding buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) with Triton-X100 (1%) and 100 µg/ml lysozyme (Sigma), and the lysate centrifuged at 15 680 g for 30 min at 4°C. The supernatant was used for the pull-down experiments. The GST fusion proteins were affinity purified using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). After incubation in the binding buffer for 30 min at 4°C, they were separated by SDS–PAGE and detected using Coomassie brilliant blue staining.
35S-labeled wild-type and mutated ataxin-7 proteins were generated using a transcription/translation kit (Promega), according to the supplier’s recommendations. The 35S-labeled products and Myc-Ataxin-7-Q10/Q100 proteins produced in Cos-7 cells were incubated in NP40 lysis buffer with equal amounts of washed GST fusion protein immobilized on Sepharose beads for 2 h at 4°C. The beads were washed extensively then boiled in SDS–PAGE sample buffer, separated on 8% SDS–PAGE and blotted.
Co-immunoprecipitation experiments
Cells were scraped from the plates and lysed under mild conditions (1% NP-40 or 0.5% Triton X-100). Cell debris was eliminated by centrifugation at 15 680 g for 20 min at 4°C. The supernatant (total soluble extract) was incubated with antibody overnight at 4°C, then, according to standard procedures, with a mix of protein A– and protein G–Sepharose (Roche Molecular Biochemicals). The Sepharose beads were collected by centrifugation and washed three times with lysis buffer. Bound protein was eluted from the beads with SDS sample buffer and analyzed by western blotting.
Immunohistochemical analyses of mouse and human brain
Immunostaining of mouse brain was performed as described previously (52). Sections were treated with 0.3% H2O2 (in PBS) for 30 min, preincubated in 0.2% Triton X-100 and 0.2% gelatine in PBS at 37°C for 30 min, then incubated with polyclonal anti-CAP antibody (1:100) for 48 h at 4°C. Seven micron sections were cut from formaline-fixed paraffin-embedded blocks of the cerebral cortex from a patient with 85 CAG repeats in the SCA7 gene (4), autoclaved in 10 mM citric buffer (pH 6.0) for 10 min, and incubated with the anti-ponsin-2 antibody (1:300) (28) or anti-ataxin-7 1C1 mAb (1:12 000) (18,53) for 48 h at 4°C. Immunoreactivity was revealed with biotinylated secondary antibodies (Vector Laboratories or ChemMate system, DAKO). Human brain sections were counterstained with Harris’ hematoxylin. For immunofluorescence, FITC-coupled anti-rabbit (AMRAD Biotech) and CY3-coupled anti-mouse antibodies (Jackson Immuno Research Laboratories) were used. Images were obtained with a Leica TCD laser confocal microscope.
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ACKNOWLEDGEMENTS |
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We thank Drs M. Ruberg, G. Stevanin and O. Corti for critical reading of the manuscript, Prof. Y. Takai (Osaka University Medical School, Japan) for the GST-fusion constructs of ponsin-2 and the anti-ponsin-2 antibody, Drs G. Yvert and D. Devys and Prof. J.L. Mandel (INSERM U 184, Illkirsch, France) for the gift of the 1C1 mAb and plasmids pBMT-Htt-Q15 and Q104, and Dr V. Ribon (University of Michigan School of Medicine, Ann Arbor, MI) for his help. This study was supported by the Association Française contre les Myopathies (AFM) and the VERUM foundation. A.S.L. was supported by INSERM. L.J. and C.Z. were supported by fellowships from the VERUM foundation and Association Française de recherche contre le Cancer, respectively.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 42162182; Fax: +33 1 44243658; Email : brice@ccr.jussieu.fr
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Stevanin, G., Durr, A. and Brice, A. (2000) Clinical and molecular advances in autosomal dominant cerebellar ataxias: from genotype to phenotype and physiopathology. Eur. J. Hum. Genet., 8, 4–18.
2 Gouw, L.G., Digre, K.B., Harris, C.P., Haines, J.H. and Ptacek, L.J. (1994) Autosomal dominant cerebellar ataxia with retinal degeneration: clinical, neuropathologic, and genetic analysis of a large kindred. Neurology, 44, 1441–1447.
3 Martin, J.J., Van Regemorter, N., Krols, L., Brucher, J.M., de Barsy, T., Szliwowski, H., Evrard, P., Ceuterick, C., Tassignon, M.J., Smet-Dieleman, H. et al. (1994) On an autosomal dominant form of retinal-cerebellar degeneration: an autopsy study of five patients in one family. Acta Neuropathol. (Berl.), 88, 277–286.[Medline]
4 Holmberg, M., Duyckaerts, C., Durr, A., Cancel, G., Gourfinkel-An, I., Damier, P., Faucheux, B., Trottier, Y., Hirsch, E.C., Agid, Y. and Brice, A. (1998) Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions. Hum. Mol. Genet., 7, 913–918.
5 Cancel, G., Duyckaerts, C., Holmberg, M., Zander, C., Yvert, G., Lebre, A.S., Ruberg, M., Faucheux, B., Agid, Y., Hirsch, E. and Brice, A. (2000) Distribution of ataxin-7 in normal human brain and retina. Brain, 123, 2519–2530.
6 David, G., Abbas, N., Stevanin, G., Dürr, A., Yvert, G., Cancel, G., Weber, C., Imbert, G., Saudou, F., Antoniou, E. et al. (1997) Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat. Genet., 17, 65–70.[Web of Science][Medline]
7 Zoghbi, H.Y. and Orr, H.T. (2000) Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci., 23, 217–247.[Web of Science][Medline]
8 Koide, R., Kobayashi, S., Shimohata, T., Ikeuchi, T., Maruyama, M., Saito, M., Yamada, M., Takahashi, H. and Tsuji, S. (1999) A neurological disease caused by an expanded CAG trinucleotide repeat in the TATA-binding protein gene: a new polyglutamine disease? Hum. Mol. Genet., 8, 2047–2053.
9 Nakamura, K., Jeong, S.Y., Ichikawa, Y., Nagashima, K., Nagashima, T., Goto, J., Ikeda, S. and Kanazawa, I. (2000) SCA15, a novel autosomal dominant cerebellar ataxia caused by the expanded polyglutamine in TATA-binding protein identified with 1C2 antibody immunoscreening. Am. J. Hum. Genet., 67 (Suppl. 2), 2185.
10 Mauger, C., Del-Favero, J., Ceuterick, C., Lubke, U., Van Broeckhoven, C. and Martin, J. (1999) Identification and localization of ataxin-7 in brain and retina of a patient with cerebellar ataxia type II using anti-peptide antibody. Brain Res. Mol. Brain Res., 74, 35–43.[Medline]
11 Ishikawa, K., Fujigasaki, H., Saegusa, H., Ohwada, K., Fujita, T., Iwamoto, H., Komatsuzaki, Y., Toru, S., Toriyama, H., Watanabe, M. et al. (1999) Abundant expression and cytoplasmic aggregations of [alpha]1A voltage-dependent calcium channel protein associated with neurodegeneration in spinocerebellar ataxia type 6. Hum. Mol. Genet., 8, 1185–1193.
12 Shimohata, T., Nakajima, T., Yamada, M., Uchida, C., Onodera, O., Naruse, S., Kimura, T., Koide, R., Nozaki, K., Sano, Y. et al. (2000) Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription. Nat. Genet., 26, 29–36.[Web of Science][Medline]
13 Evert, B.O., Wullner, U. and Klockgether, T. (2000) Cell death in polyglutamine diseases. Cell Tissue Res., 301, 189–204.[Web of Science][Medline]
14 Lin, X., Cummings, C.J. and Zoghbi, H.Y. (1999) Expanding our understanding of polyglutamine diseases through mouse models. Neuron, 24, 499–502.[Web of Science][Medline]
15 Kaytor, M.D., Duvick, L.A., Skinner, P.J., Koob, M.D., Ranum, L.P. and Orr, H.T. (1999) Nuclear localization of the spinocerebellar ataxia type 7 protein, ataxin-7. Hum. Mol. Genet., 8, 1657–1664.
16 Mushegian, A.R., Vishnivetskiy, S.A. and Gurevich, V.V. (2000) Conserved phosphoprotein interaction motif is functionally interchangeable between ataxin-7 and arrestins. Biochemistry, 39, 6809–6813.[Medline]
17 Gurevich, V.V., Dion, S.B., Onorato, J.J., Ptasienski, J., Kim, C.M., Sterne-Marr, R., Hosey, M.M. and Benovic, J.L. (1995) Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors. J. Biol. Chem., 270, 720–731.
18 Lindenberg, K.S., Yvert, G., Muller, K. and Landwehrmeyer, G.B. (2000) Expression analysis of ataxin-7 mRNA and protein in human brain: evidence for a widespread distribution and focal protein accumulation. Brain Pathol., 10, 385–394.[Web of Science][Medline]
19 Li, X.J., Li, S.H., Sharp, A.H., Nucifora, F.C.,Jr, Schilling, G., Lanahan, A., Worley, P., Snyder, S.H. and Ross, C.A. (1995) A huntingtin-associated protein enriched in brain with implications for pathology. Nature, 378, 398–402.[Medline]
20 Faber, P.W., Barnes, G.T., Srinidhi, J., Chen, J., Gusella, J.F. and MacDonald, M.E. (1998) Huntingtin interacts with a family of WW domain proteins. Hum. Mol. Genet., 7, 1463–1474.
21 Sittler, A., Walter, S., Wedemeyer, N., Hasenbank, R., Scherzinger, E., Eickhoff, H., Bates, G.P., Lehrach, H. and Wanker, E.E. (1998) SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polygln-containing protein aggregates. Mol. Cell, 2, 427–436.[Web of Science][Medline]
22 Bao, J., Sharp, A.H., Wagster, M.V., Becher, M., Schilling, G., Ross, C.A., Dawson, V.L. and Dawson, T.M. (1996) Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin. Proc. Natl Acad. Sci. USA, 93, 5037–5042.
23 Matilla, A., Koshy, B.T., Cummings, C.J., Isobe, T., Orr, H.T. and Zoghbi, H.Y. (1997) The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1. Nature, 389, 974–978.[Medline]
24 Yanagisawa, H., Bundo, M., Miyashita, T., Okamura-Oho, Y., Tadokoro, K., Tokunaga, K. and Yamada, M. (2000) Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine. Hum. Mol. Genet., 9, 1433–1442.
25 Kalchman, M.A., Koide, H.B., McCutcheon, K., Graham, R.K., Nichol, K., Nishiyama, K., Kazemi-Esfarjani, P., Lynn, F.C., Wellington, C., Metzler, M. et al. (1997) HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane- associated huntingtin in the brain. Nat. Genet. 16, 44–53.[Web of Science][Medline]
26 Wanker, E.E., Rovira, C., Scherzinger, E., Hasenbank, R., Walter, S., Tait, D., Colicelli, J. and Lehrach, H. (1997) HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system. Hum. Mol. Genet., 6, 487–495.
27 Ribon, V., Printen, J.A., Hoffman, N.G., Kay, B.K. and Saltiel, A.R. (1998) A novel, multifuntional c-Cbl binding protein in insulin receptor signaling in 3T3-L1 adipocytes. Mol. Cell Biol., 18, 872–879.
28 Mandai, K., Nakanishi, H., Satoh, A., Takahashi, K., Satoh, K., Nishioka, H., Mizoguchi, A. and Takai, Y. (1999) Ponsin/SH3P12: an l-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherens junctions. J. Cell. Biol., 144, 1001–1017.
29 Kussel-Andermann, P., El-Amraoui, A., Safieddine, S., Hardelin, J.P., Nouaille, S., Camonis, J. and Petit, C. (2000) Unconventional myosin VIIA is a novel A-kinase-anchoring protein. J. Biol. Chem., 275, 29654–29659.
30 Sparks, A.B., Hoffman, N.G., McConnell, S.J., Fowlkes, D.M. and Kay, B.K. (1996) Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins. Nat. Biotechnol., 14, 741–744.[Web of Science][Medline]
31 Hoffman, N.G., Sparks, A.B., Carter, J.M. and Kay, B.K. (1996) Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries. Mol. Divers., 2, 5–12.[Web of Science][Medline]
32 Nagase, T., Kikuno, R., Ishikawa, K.I., Hirosawa, M. and Ohara, O. (2000) Prediction of the coding sequences of unidentified human genes. XVI. The complete sequences of 150 new cDNA clones from brain which code for large proteins in vitro. DNA Res., 7, 65–73.[Abstract]
33 Ren, R., Mayer, B.J., Cicchetti, P. and Baltimore, D. (1993) Identification of a ten-amino acid proline-rich SH3 binding site. Science, 259, 1157–1161.
34 Ribon, V., Herrera, R., Kay, B.K. and Saltiel, A.R. (1998) A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions. J. Biol. Chem., 273, 4073–4080.
35 Joazeiro, C.A., Wing, S.S., Huang, H., Leverson, J.D., Hunter, T. and Liu, Y.C. (1999) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science, 286, 309–312.
36 Freemont, P.S. (2000) RING for destruction? Curr. Biol., 10, R84–R87.[Web of Science][Medline]
37 Hardy, J. and Gwinn-Hardy, K. (1998) Genetic classification of primary neurodegenerative disease. Science, 282, 1075–1079.
38 Alves-Rodrigues, A., Gregori, L. and Figueiredo-Pereira, M.E. (1998) Ubiquitin, cellular inclusions and their role in neurodegeneration. Trends Neurosci., 21, 516–520.[Web of Science][Medline]
39 Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T. and Zoghbi, H.Y. (1998) Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet., 19, 148–154.[Web of Science][Medline]
40 Chai, Y., Koppenhafer, S.L., Shoesmith, S.J., Perez, M.K. and Paulson, H.L. (1999) Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro. Hum. Mol. Genet., 8, 673–682.
41 Stenoien, D.L., Cummings, C.J., Adams, H.P., Mancini, M.G., Patel, K., DeMartino, G.N., Marcelli, M., Weigel, N.L. and Mancini, M.A. (1999) Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Hum. Mol. Genet., 8, 731–741.
42 Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J. and Rubinsztein, D.C. (2000) Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease. Proc. Natl Acad. Sci. USA, 97, 2898–2903.
43 Chai, Y., Koppenhafer, S.L., Bonini, N.M. and Paulson, H.L. (1999) Analysis of the role of heat shock protein (Hsp) molecular chaperones in polyglutamine disease. J. Neurosci., 19, 10338–10347.
44 Kobayashi, Y., Kume, A., Li, M., Doyu, M., Hata, M., Ohtsuka, K. and Sobue, G. (2000) Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract. J. Biol. Chem., 275, 8772–8778.
45 Cummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y., Ciechanover, A., Orr, H.T., Beaudet, A.L. and Zoghbi, H.Y. (1999) Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron, 24, 879–892.[Web of Science][Medline]
46 Romero, F., Dargemont, C., Pozo, F., Reeves, W.H., Camonis, J., Gisselbrecht, S. and Fischer, S. (1996) p95vav associates with the nuclear protein Ku-70. Mol. Cell Biol., 16, 37–44.
47 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1989) Plasmid vectors. In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
48 Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature, 340, 245–246.[Medline]
49 Schiestl, R.H. and Gietz, R.D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet., 16, 339–346.[Web of Science][Medline]
50 Strahle, U., Blader, P., Adam, J. and Ingham, P.W. (1994) A simple and efficient procedure for non-isotopic in situ hybridization to sectioned material. Trends Genet., 10, 75–76.[Web of Science][Medline]
51 Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J. (1996) A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis. Dev. Biol., 174, 233–247.[Web of Science][Medline]
52 Côté, S.L., Ribeiro-da-Silva, A. and Cuello, A.C. (1993) Current protocols for Light Microscopy Immunocytochemistry. In Cuello, A.C. (ed.), Immunohistochemistry II, Wiley, West Sussex, pp. 147–168.
53 Yvert, G., Lindenberg, K.S., Picaud, S., Landwehrmeyer, G.B., Sahel, J.A. and Mandel, J.L. (2000) Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice. Hum. Mol. Genet., 9, 2491–2506.
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