Human Molecular Genetics, 2000, Vol. 9, No. 15 2281-2289
© 2000 Oxford University Press
Alzheimer’s disease-associated presenilin 2 interacts with DRAL, an LIM-domain protein
Division of Demyelinating Disease and Aging, National Institute of Neuroscience, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
Received 29 May 2000; Revised and Accepted 7 August 2000.
DDBJ/EMBL/GenBank accession nos. AB008571, AB038991S1–AB038991S5.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Using the yeast two-hybrid system, we screened for proteins interacting with presenilin 2 (PS2) and cloned DRAL. DRAL is an LIM-only protein containing four LIM domains and an N-terminal half LIM domain. Previously DRAL has been cloned as a co-activator of the androgen receptor and as a protein interacting with a DNA replication regulatory protein, hCDC47. Our yeast two-hybrid assay showed that DRAL interacted with a hydrophilic loop region (amino acids 269–298) in the endoproteolytic N-terminal fragment of PS2, but not that of PS1, although the region 269–298 of PS2 and the corresponding PS1 sequence differ by only three amino acids. Each point mutation within this region, R275A, T280A, Q282A, R284A, N285A, P287T, I288L, F289A and S296A, in PS2 abolished the binding. This suggests that DRAL recognizes the PS2 structure specifically. The in vitro interaction was confirmed by affinity column assay and the physiological interactions between endogenous PS2 and DRAL by co-immunoprecipitation from human lung fibroblast MRC5 cells. Furthermore, in PS2-overexpressing HEK293 cells, we found an increase in the amount of DRAL in the membrane fraction and an increase in the amount of DRAL that was co-immunoprecipitated with PS2. The potential role of DRAL in the cellular signaling suggests that DRAL functions as an adaptor protein that links PS2 to an intracellular signaling.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Alzheimer’s disease (AD) is characterized clinically by progressive dementia and neuropathologically by neuronal loss, formation of senile plaques and neurofibrillary tangles. Senile plaques have deposits primarily composed of a 40–42 amino acid peptide called ß-amyloid peptide (Aß), which is derived from the proteolytic processing of amyloid precursor protein (APP). Three genes [APP, presenilin 1 (PS1), presenilin 2 (PS2)] have been identified as harboring pathogenic mutations that affect APP processing (1). We confirmed the mutation of the PS1 gene in Japanese patients and showed that mutation also occurs in a sporadic manner (2). PS1 and PS2 are structurally similar membrane proteins (PS2 is 63% homologous in amino acid sequence to PS1) with eight likely transmembrane domains. Presenilins are primarily located in the endoplasmic reticulum and the Golgi apparatus (3), but are also found on nuclear and plasma membranes (4,5) and cytosolic vehicles (6,7). Both their N- and C-terminal hydrophilic regions as well as a large hydrophilic loop region between the 6th and 7th transmembrane regions are proposed to face towards the cytosolic compartment (8). To date, 16 independent mutations including two splice site mutations have been identified in the large hydrophilic loop of PS1 (Human Gene Mutation Database, http://www.uwcm.ac.uk/uwcm/mg/hgmd0.html ). PS1 and PS2 holoproteins are mainly processed by endoproteolysis between amino acids 298 and 299, and 306 and 307, respectively, in this loop region (9–11), but are also cleaved by caspase-3 within the loop region (12–14). These findings suggest that this region is an important functional domain. The three genes (APP, PS1 and PS2) implicated in the etiology of autosomal dominant AD account for <20% of cases (2,15). The only well-replicated genetic risk factor for non-autosomal dominant factors of AD is the
4 allele of the gene encoding apolipoprotein E (APOE), estimated to account for <50% of the genetic variance (16). These findings suggest that other pathogenic genetic or risk factors exist. Therefore, we tried to identify proteins interacting within this region to obtain information on the role of presenilin-associated protein in the pathogenesis of AD. Biochemical interactions between the presenilins and a variety of proteins imply potential functions of presenilins in Wg/Wnt signaling, apoptosis, stabilization of the cytoskeleton and intracellular Ca2+ homeostasis (17–22). Here we report that DRAL, a member of the four and a half LIM protein family, interacted specifically with amino acids 269–296 within the loop region of PS2, but not in PS1. |
RESULTS |
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DRAL interacts with a large hydrophilic loop region in PS2
Using the two-hybrid system we screened a human fetal brain cDNA library as described in Materials and Methods, with amino acids 269–388 containing the PS2 large hydrophilic loop as bait, and 16 putative interacting clones were isolated. These clones were tested for the specific interaction with the PS2 large hydrophilic loop region. Six clones showed the specific interaction with the PS2. These clones were the same and the cDNA contained the full coding sequence of DRAL which consists of 279 amino acids with a predicted molecular weight of 32 kDa. Our cloned DRAL cDNA insert lacked the reported nucleotide sequence 64–114 (GenBank accession no. L42176) and had the additional 5' sequence CGGAGC. Previously, DRAL has been cloned as a protein expressed in primary myoblasts and down-regulated in rhabdomyosarcoma cells by subtractive cloning (23). DRAL is an LIM-only protein containing four LIM domains and an N-terminal half LIM domain. The LIM domain is defined by two adjacent zinc fingers with the consensus sequence C-X2-C-X17–19-H-X2-C-X2-C-X2-C-X16–20-C-X2-C/H/D, which is able to coordinate two metal ions and mediate protein–protein interaction. The LIM domain is found in various proteins, including homeodomain-containing transcription factors, cytoskeletal proteins and other signaling proteins that are known to be involved in cell fate determination, growth regulation and oncogenesis (24). Our yeast two-hybrid assay showed that DRAL interacted with PS2L (codons 269–388) and PS2Ln (codons 269–306) but not with PS2Lc (codons 307–388), PS2C (codons 410–448), PS1L (codons 263–407) and PS1C (codons 429–467). DRAL did not interact with irrelevant proteins such as human lamin C (codons 66–230), murine p53 (codons 72–390) and GAL4 DNA-binding domain (codons 1–147). To further verify DRAL as a PS2-binding protein, cloning vectors were switched by changing the DRAL insert from pACT2 to pAS2-1 and PS1 or PS2 inserts as specified (Fig. 1) from pAS2-1 to pACT2 and then the two-hybrid assay was repeated. The results demonstrated that DRAL specifically interacted with a hydrophilic loop region (amino acids 269–306) in the endoproteolytic N-terminal fragment, but not with that of PS-1 in yeast two-hybrid assay.
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Mutational analysis of amino acids 269–306 of PS2 for DRAL-binding activity
To identify the DRAL-binding region of PS2, we generated a set of PS2 deletion mutants fused to the GAL4 activating domain. As shown in Figure 2A, we found that the domain essential for the interaction was located between residues 269–298, in which 27 of the 30 amino acids are identical to the corresponding PS1 sequence (residues 263–292). In the region 263–292 of PS1, 12 pathogenic mutations have been found. This suggests that this region is important in presenilin function. Amino acids 269–298 of PS2 and the corresponding PS1 sequence differ by only three amino acids. To examine how these three residues influence the interaction with DRAL in yeast two-hybrid assays, the PS2 bait plasmid pACT2/PS2Lnd1 was mutated singly by introducing the corresponding PS1 sequence into the PS2 sequence, P287T, I288L and A297T. Furthermore, other PS2 amino acids in pACT2/PS2Lnd1 were mutated singly by substitution with alanine in order to identify the amino acid residues necessary for DRAL binding and these bait plasmids were transformed into Y190 harboring the plasmid pAS2-1/DRAL. Colony-lift ß-galactosidase assay using X-gal showed that no staining was observed when bait plasmids carrying R275A, T280A, Q282A, R284A, N285A, P287T, I288L, F289A and S296A substitutions were used. The P287T and I288L substitutions are the introduction of the corresponding PS1 sequence into the PS2 sequence. Liquid ß-galactosidase assay using O-nitrophenyl-ß-D-galactopyranoside (ONPG) as a substrate showed that E283A, E286A, P290A and L292A substitutions reduced the activity to 31, 23, 54 and 60%, respectively, of that obtained with the original bait plasmid (Fig. 2B). These results show that amino acids 280–292 and distal arginine at 275 and serine at 296 are important for the association with DRAL, suggesting that DRAL recognizes the PS2 structure specifically.
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DRAL needs four LIM domains to bind PS2, and other DRAL family members do not interact with PS2
To identify the PS2-binding region of DRAL, a set of DRAL deletion mutants fused to the GAL4-binding domain was generated and these bait plasmids were transformed into Y190 harboring the plasmid pACT2/PS2Lnd1, and a colony-lift ß-galactosidase assay was performed. As shown in Figure 3, DRAL needs four LIM domains to bind PS2. DRAL is a member of a family of proteins that have four closely connected LIM domains and an N-terminal single zinc finger domain that meets the consensus of the C-terminal half of the LIM domain motif. Other human members, FHL1/Slim1 (25,26) and FHL3/Slim2 (25), have 48 and 52% sequence identity to DRAL, respectively. We tested whether FHL1/Slim1 and FHL3/Slim2 interact with PS2 by the yeast two-hybrid assay. Figure 4 shows that only DRAL interacted with PS2. This suggests that the PS2–DRAL interaction is not due to the non-specific interaction caused by the zinc finger motif.
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Tissue distribution and subcellular localization of DRAL
Recent work reported that DRAL expression was restricted to the heart (27). We analyzed the tissue distribution and subcellular localization of DRAL. Northern analysis of DRAL expression in different human adult and fetal tissues was performed. DRAL mRNA was detectable in all tissues examined, mostly in heart but also in skeletal muscle and placenta, and all regions of the brain sampled (Fig. 5). Compared with PS1 mRNA, PS2 mRNA is expressed at higher levels in the heart, skeletal muscle and in the pancreas (28,29). To study further the PS2 and DRAL protein interactions, we generated rabbit polyclonal anti-PS2 antibody (
-PS2N) and anti-DRAL antibodies (-CRN and -CTN). -PS2N was raised against the GST fusion protein, GST–PS2N, which contained amino acids 1–87 of N-terminal region of human PS2. As shown in Figure 6A, -PS2N reacted with the 55 kDa full-length PS2 and the 35 kDa N-terminal fragment. -CRN and -CTN were raised against the synthetic peptides CRNSLVDKPFAAKED (amino acids 71–85 of human DRAL) within the first LIM domain and CTNPISGLGGTKYIS (amino acids 224–238) within the fourth LIM domain, respectively. Western blot analysis of COS cells expressing DRAL exogenously with -CRN and -CTN showed that these antibodies recognized a 32 kDa DRAL band, consistent with the respective predicted molecular weight. The DRAL protein was highly expressed in normal human lung fibroblast MRC5 cells (Fig. 6B). To check whether our antibodies recognize other DRAL family members, FHL1/Slim1 or FHL3/Slim2, we performed western blot analysis of yeast extracts expressing GAL4–FHL1 or GAL4–FHL3 fusion protein. The antibodies did not recognize these fusion proteins (data not shown). We also cloned rat DRAL cDNA (GenBank accession no. AB008471). The predicted open reading frame from the cDNA encoded 279 residues and the predicted rat DRAL protein has 92% identity compared with that of the human DRAL protein. Amino acids 224–238 of human and rat DRAL protein, against which the -CTN was raised, were the same. Western blot analysis of DRAL expression in different human and rat tissues was performed using -CTN. The 32 kDa DRAL protein was highly expressed in rat heart and detectable in other tissues including the brain (Fig. 6C). Western blot analysis of fractionated lysates from MRC5 cells showed that DRAL was mainly localized in the cytosolic and nuclei fraction, although a portion of the protein also distributed in the membrane-containing fraction after extensive washing of the membrane-containing pellets (Fig. 6D). This membrane-containing fraction was fractionated in the presence of 1% Triton X-100 to dissolve the membrane. DRAL was present in the 1% Triton X-100 soluble fraction containing the membrane fraction but not in the insoluble fraction containing the cytoskeleton (Fig. 6D). As DRAL has no transmembrane region, our result implies that a portion of DRAL may attach to proteins that are intrinsic to the membrane.
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Affinity chromatography and immunoprecipitation
The MRC5 cytosolic fraction was incubated with GST–PS2L, GST–PS1L or GST and glutathione beads. The beads were then poured into a column and washed and eluted with reduced glutathione. Co-elution of DRAL with GST fusion protein was determined by western blot analysis with
-CTN. Figure 7 shows that DRAL was co-eluted with GST–PS2L but not with GST–PS1L and GST. The results from the yeast two-hybrid assay and affinity chromatography with GST fusion proteins showed that DRAL interacts with the large hydrophilic loop region of PS2 (PS2L) but not with PS1L. The results using the MRC5 cytosolic fraction containing 10 mM EDTA showed negative interactions (data not shown). To investigate the physiological interaction between the endogenous N-terminal fragment of PS2 and DRAL, the extract of MRC5 cells was immunoprecipitated with -PS2N. DRAL was co-immunoprecipitated with -PS2N. In the negative control experiment, an irrelevant antibody, anti-GST antibody, did not immunoprecipitate DRAL (Fig. 8). Furthermore, comparing the PS2-transfected HEK293 cells with the empty vector transfected cells, we found an increase in the amount of DRAL in the membrane fraction of PS2-overexpressing cells (Fig. 9A). The efficiency of the subcellular fraction was confirmed by analysis with an antibody against a typical endoplasmic reticulum marker, GRP78. The amount of GRP78 in the membrane fraction of the PS2-transfected cells was similar to that of the empty vector transfected cells. We also found an increase in the amount of DRAL that was co-immunoprecipitated with PS2 in PS2-overexpressing HEK293 cells (Fig. 9B). These results suggest that PS2 works as a membrane receptor for DRAL. We concluded that DRAL forms stable complexes in vivo.
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Genome structure of human DRAL
We determined the genomic structure of the human DRAL gene by long and accurate PCR (LA-PCR). The gene was composed of five coding exons distributed over 26 kb (Fig. 10) and the genomic organization was quite similar to that of the FHL1/Slim1 gene (30). DRAL and FHL1/Slim1 are located on chromosomes 2q12–q13 and Xq27, respectively (26,30). It is likely that DRAL and FHL1/Slim1 are derived from a common ancestral gene and split onto different chromosomes through evolution. This determination of the DRAL genome structure will facilitate linkage analysis and the search for mutations in the DRAL gene in patients with AD.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study we showed that DRAL interacted with a large hydrophilic loop region (amino acids 269–298) in the endoproteolytic N-terminal fragment of PS2, but not that of PS1, by yeast two-hybrid and GST-binding assays. In an immunoprecipitation study using MRC5 cell extracts, we also demonstrated that endogenous DRAL interacted with PS2. Furthermore, in PS2-overexpressing HEK293 cells we found an increase in the amount of DRAL in the membrane fraction and an increase in the amount of DRAL that was co-immunoprecipitated with PS2. Thus, it is clear that DRAL interacts with PS2 physiologically. Each of nine single amino acid substitutions within the region 269–298 abolished the binding with DRAL. These results suggest that DRAL recognizes the PS2 structure specifically. It is interesting that DRAL interacts with the loop region of PS2 but not that of PS1, although amino acids 269–298 of PS2 and the corresponding PS1 sequence differ by only three amino acids. On the other hand, we showed previously that
-catenin interacted with a large hydrophilic loop region in the endoproteolytic C-terminal fragment of PS1, but not of PS2 (31). In addition, PSAP containing a PDZ-like domain interacts with the C-terminus of PS1 but not of PS2 (32), sorcin interacts with PS2 but not PS1 (22) and calmyrin preferentially interacts with amino acids 270–319 of PS2 in yeast two-hybrid assays (18). Furthermore, whereas PS1-null mice show developmental defects that culminate in a perinatal lethality, PS2-null mice exhibit no obvious defects (33). These results suggest that PS1 and PS2 partly differ in function. In the study by Stabler et al. (18), the double mutants of the PS1 loop C region (amino acids 264–294 of PS1), PS1(L282I, T291A), PS1(T281P, L282I) and PS1(T281P, T291A), are equivalent to our single mutants of PS2, P287T, A297T and I288L, respectively. Although the DRAL binding activities were abolished by single mutations of P287T and I288L in PS2, calmyrin binding activities were retained at 32 and 219% of wild-type PS2 (18). This suggests that DRAL and calmyrin recognize different structures in PS2. The LIM domains have been reported to bind various motifs and sequences that are structurally quite different. LIM domains can associate with other LIM domains to form homo- or heterodimers (34,35). In addition, LIM domains also bind tyrosine-containing tight-turn structures, PDZ domains, ankyrin repeats, the SH3 domain and helix–loop–helix domains in proteins lacking LIM domains including tyrosine and serine/threonine kinase, cytoskeletal proteins and transcription factors (36–42). The interaction between the LIM domains of DRAL and PS2 does not belong to these categories. The DRAL binding domain within amino acids 269–298 of PS2 has two predicted polyproline type II helix structures, 284RNEPIFP290 and 270PKGPLR275, which have consensus sequences for SH3 binding sites (class 1, RXXPXXP; and class 2, PXXPXR; where P and R are crucial for the binding) (43). Although the single amino acid substitution in the 284RNEPIFP290 sequence abolished or reduced the binding activity, the P290A substitution did not abolish the binding. Moreover, other residues affected the binding, so the binding site cannot be simply adapted to the SH3 binding motif. However, PS2 may interact with proteins containing the SH3 domain. Since DRAL may shuttle between the cytoplasm and nucleus and has multiple protein–protein interactive domains, there is a possibility that it interacts with other protein partners and participates in an intracellular signal transduction pathway. Recently, Chan et al. (44) reported that DRAL interacted with the DNA replication regulatory protein, hCDC47 (44). hCDC47 is a human member of the minichromosome maintenance family that has been implicated in the regulatory machinery causing DNA to replicate once in the S phase. On the other hand, DRAL has been cloned as a co-activator of the androgen receptor: the transcription of the probasin gene, one of the targets for the prostate-specific androgen receptor, is co-activated by DRAL (27). These findings suggest that DRAL acts as a modulator for DNA replication and gene transcription. Furthermore, a typical androgen, testosterone, increases the secretion of the non-amyloidgenic APP fragment, sAPP, and decreases the secretion of Aß peptides from N2a and rat primary cerebrocortical neurons (45). So it is possible that DRAL affects APP metabolism. Although further studies are required to elucidate the relevance to the PS2–DRAL interaction and the role in AD, it appears that DRAL may function as an adaptor protein that links PS2 to an intracellular signalling. |
MATERIALS AND METHODS |
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Strains and plasmid construction
Yeast expression vectors, pAS2-1 containing the GAL4 DNA-binding domain (BD), pACT2 containing the GAL4 activation domain (AD), and false-positive detection control plasmids, pVA3-1 (BD/murine p53(codons 72–390) fusion), pLAM5'-1 [BD/human lamin C(codons 66–230) fusion] and pTD1-1 [AD/SV40 large T antigen(codons 84–708) fusion] were purchased from Clontech (Palo Alto, CA). To construct a series of BD/presenilin fusion plasmids (pAS2-1/PS2L, pAS2-1/PS2Ln, pAS2-1/PS2Lc, pAS2-1/PS2C, pAS2-1/PS1L, pAS2-1/PS1Ln, pAS2-1/PS1Lc, pAS2-1/PS1C), codons 269–388, 269–306, 307–388 and 410–448 in PS2 and codons 263–407, 263–298, 299–407 and 429–467 in PS1 were PCR-amplified and fused in-frame at the EcoRI–BamHI site in pAS2-1. To construct a series of AD/presenilin fusion plasmids (pACT2/PS2L, pACT2/PS2Ln, pACT2/PS1L, pACT2/PS1Ln), each SfiI–SalI fragment of the above-mentioned BD/presenilin fusion plasmids was subcloned into the SfiI–XhoI site of pACT2. A pACT2/PS2Lnd1 plasmid was constructed by subcloning the NcoI fragment of pACT2/PS2L plasmid including codons 269–298 of PS2 into the NcoI site of pACT2 in frame. To construct pACT2/PS2Lnd2 and pACT2/PS2Lnd3, NcoI anchored-PCR products including codons 269–296 and 269–291 of PS2 were subcloned into the NcoI site of pACT2 in frame. To construct a series of pACT2/point-mutated PS2 fusion plasmids, a P270A, K271A, G272A, P273A, L274A, R275A, M276A, L277A, V278A, E279A, T280A, Q282A, E283A, R284A, N285A, E286A, P287T, I288L, F289A, P290A, L292A, I293A, Y294A, S295A, S296A or A297T mutation was introduced into pACT2/PS2Lnd1 plasmid by PCR. To change the cloned DRAL insert from pACT2 to pAS2-1, the SfiI–XhoI fragment including the DRAL cDNA was subcloned into the SfiI–SalI site of pAS2-1. Several BD/DRAL fusion plasmids having truncated C-terminal LIM domains of DRAL, pAS2-1/DRAL(-L4), pAS2-1/DRAL(-L3, 4), pAS2-1/DRAL(-L2, 3, 4), pAS2-1/DRAL(-L1, 2, 3, 4) and pAS2-1/DRAL(-L1/2, 1, 2, 3, 4) were constructed as follows. PCR was performed using a primer (5'-CAGATTACGCTAGCTTGGGTGGTC-3') derived from the sequence immediately upstream of the cloning site of pACT2 and DRAL-specific anchored primers (GenBank accession no. L42176; antisense of nucleotides 798–779, 621–602, 438–419, 258–241 and 138–119 with a stop codon and BamHI site, respectively) and the pACT2/DRAL as a template. Then the PCR products were digested with NcoI and BamHI and subcloned into the NcoI–BamHI site of pAS2-1. Two BD/DRAL fusion plasmids having a truncated N-terminus, pAS2-1/DRAL(-U, -L1/2, 1) and pAS2-1/DRAL(-U, -L1/2, 1, 2), were constructed as follows. EcoRI-anchored PCR products (nucleotides 415–984, 598–984 of DRAL, respectively) were subcloned into the EcoRI site of pAS2-1 in frame. To construct pAS2-1/FHL1 and pAS2-1/FHL3, FHL1 (nucleotides 31–939, GenBank accession no. U60115) and FHL3 (nucleotides 20–911, GenBank accession no. U60116), cDNAs were cloned by RT–PCR from human brain RNA and FHL1 cDNAs were cloned into the EcoRI–Sal I site of pAS2-1 and FHL3 cDNA was cloned into the NcoI–EcoRI site of pAS2-1 in frame. The plasmid constructs that express GST–presenilin fusion proteins GST–PS2N, GST–PS2L and GST–PS1L were described previously (31). To express DRAL or PS2 exogenously in mammalian cells, a full-length DRAL or PS2 cDNA was cloned into a pSG5 or pCEP4 expression vector (Invitrogen, Leek, The Netherlands).
Yeast two-hybrid screening
Yeast cell culture, transformation, ß-galactosidase assay, plasmid isolation from yeast and selection of pACT2/library plasmid by transformation in Escherichia coli strain HB101 were carried out with the Matchmaker two-hybrid system 2 (Clontech). Yeast host cells Y190 (Clontech) were first transformed with pAS2-1/PS2L and subsequently with plasmid DNA prepared from a human fetal brain Matchmaker cDNA library fused with the AD (Clontech). The transformants were grown on selection plates without trytophan, leucine and histidine supplemented with 25 mM 3-amino-1,2,4-triazole (Sigma, St Louis, MO) for 7 days. After transferring yeast colonies onto Hybond-N nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, UK), a colony-lift ß-galactosidase filter assay was carried out. Master plates were incubated for a further 4 days and candidates were then cultured. The pACT2/library plasmid DNA was propagated in HB101 cells and used in subsequent experiments. The cDNA inserts of the library plasmids were analyzed by nucleotide sequencing. ß-galactosidase activities were also measured by liquid culture assay using ONPG as the substrate and the enzyme activity was calculated by the equation of Miller.
Cloning of cDNA for rat DRAL and genomic DNA for human DRAL
Rat DRAL cDNA was cloned by screening a rat brain cDNA library (Clontech) using human DRAL cDNA as a probe. The genomic DNA for human DRAL was cloned by LA–PCR using several oligonucleotide primer pairs derived from the cDNA sequence and human genomic DNA as a template. PCR products were subjected to direct sequencing.
Northern analysis
Northern analysis was carried out using human DRAL (GenBank accession no. L42176, nucleotides 139–978) cDNA as a probe and the probe was hybridized to multiple tissue northern blots (Clontech). Human ß-actin or GAPDH cDNA was used for an internal control.
Antibodies
A rabbit anti-PS2 polyclonal antibody, -PS2N, was raised against the GST fusion protein, GST–PS2N, which contained amino acids 1–87 of the N-terminal region of the human PS2. The antisera were pre-cleared on an FMP-activated cellulose affinity column coupled with GST and purified on an FMP-activated cellulose affinity column coupled with GST–PS2N. Rabbit anti-DRAL polyclonal antibodies, -CRN and -CTN, were raised against synthetic peptides CRNSLVDKPFAAKED (amino acids 71–85 of human DRAL) and CTNPISGLGGTKYIS (amino acids 224–238), respectively. The antisera were purified on an FMP-activated cellulose affinity column coupled with each peptide. Rabbit anti-GST antibody, Z-5, and goat anti-GRP78 antibody, N-20, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell fraction
For subcellular fractionation, normal human lung fibroblast MRC cells or human embryo kidney HEK293 cells were homogenized with a glass Dounce homogenizer in lysis buffer (20 mM HEPES–KOH pH7.5, 10 mM KCl, 1.5 mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin and 1 mM p-APMAF). Nuclei were removed by centrifuging the homogenates at 1000 g for 10 min and the supernatant was centrifuged again. Nuclei were washed with lysis buffer once. To obtain the cytosolic and the membrane-containing fraction, the supernatant was centrifuged at 100 000 g for 60 min. The membrane-containing pellets were washed with lysis buffer twice. The pellets were lysed in lysis buffer containing 1% Triton X-100. After 30 min incubation, the lysates were centrifuged at 100 000 g for 60 min to obtain a Triton X-100 soluble membrane fraction and insoluble fraction containing the cytoskeletal fraction.
Affinity chromatography
Five hundred micrograms of the cytosolic fraction of MRC5 cells (as determined by BCA protein assay; Pierce, Rockford, IL) was incubated overnight at 4°C with 100 µl of 50% (v/v) glutathione–Sepharose beads (Amersham Pharmacia Biotech) and an equivalent amount (100 µg) of purified GST–PS2L, GST–PS1L or GST with or without 10 µM EDTA. The beads were then poured into the column and washed three times with 300 µl of phosphate-buffered saline (PBS) containing 0.5 mg/ml bovine serum albumin (BSA) and 50 µM ZnSO4 or 10 mM EDTA and eluted with PBS containing 0.5 mg/ml BSA, 50 µM ZnSO4 and 10 mM reduced glutathione. Each 50 µl fraction was collected and 10 µl of the fraction was subjected to western analysis using -CTN or Z-5.
Transfection, immunoprecipitation and western blot analysis
For transient expression, the pSG5/DRAL or pSG5/PS2 plasmid (4 µg) was transfected using lipofectamine (Life Technologies, Rockville, MD), according to the manufacturer’s instructions, into COS cells in 10 cm plates. The pCEP4 (Amersham Pharmacia Biotech) or pCEP4/PS2 plasmid (10 µg) was transfected using lipofectamine 2000 (Life Technologies) into HEK293 cells. Two days after transfection, transfected cells were homogenated in 1 ml of SDS sample buffer without dithiothreinine, briefly sonicated and centrifuged at 100 000 g for 1 h at 4°C. Human adult brain extracts and brain, kidney and heart extracts from adult rats were also prepared as described above. Their supernatants were collected and 20 µg of each extract was separated by 13% SDS–PAGE. For immunoprecipitation, MRC5 cells or transfected HEK293 cells were collected and washed three times in PBS. Cell pellets were then resuspended in 4x PBS containing 1% (w/v) Triton X-100 and a mixture of protease inhibitors, homogenized by 20 strokes in a glass Dounce homogenizer and stirred slowly for 1 h at 4°C. Insoluble material was removed by centrifugation at 100 000 g for 1 h at 4°C. The supernatants were incubated at 4°C with -PS2N or an irrelevant antibody, anti-GST Z5, for 1 h and the immunocomplexes were collected with Protein A–agarose (Roche, Basel, Switzerland). The beads were washed three times in PBS containing 0.5 mg/ml BSA, 50 µM ZnSO4, 1% (w/v) Triton X-100 and a mixture of protease inhibitors, subjected to SDS–PAGE and blotted. The blots were immunostained with -CTN.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 42 341 2711; Fax: +81 42 346 1747; Email: tanahash@ncnp.go.jp
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REFERENCES |
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