From the Cancer Research Program, Garvan Institute of
Medical Research, St. Vincent's Hospital, Sydney, New South Wales
2010, Australia, the § Department of Cell Biology and
Genetics, Rockefeller University, New York, New York 10021, the
¶ Department of Medicine, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021, and the
Centre for Medical
Genetics, Department of Cytogenetics and Molecular Genetics, Women's
and Children's Hospital, Adelaide,
South Australia 5006, Australia
Received for publication, October 25, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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Tankyrase is an ankyrin repeat-containing
poly(ADP-ribose) polymerase originally isolated as a binding partner
for the telomeric protein TRF1, but recently identified as a
mitogen-activated protein kinase substrate implicated in regulation of
Golgi vesicle trafficking. In this study, a novel human tankyrase,
designated tankyrase 2, was isolated in a yeast two-hybrid screen as a
binding partner for the Src homology 2 domain-containing adaptor
protein Grb14. Tankyrase 2 is a 130-kDa protein, which lacks the
N-terminal histidine/proline/serine-rich region of tankyrase, but
contains a corresponding ankyrin repeat region, sterile It is now evident that protein-protein interactions play a
critical role in signal transduction, not only mediating recruitment of
signaling proteins to receptors and assembly of multiprotein signaling
complexes, but also directing the correct subcellular compartmentalization of such complexes and hence providing signal fidelity (1). A variety of protein modules have been identified that
mediate these interactions including
SH21 domains, which bind
specific phosphotyrosine-containing peptide sequences; SH3 domains,
which target specific proline-rich motifs with a PXXP core;
and PDZ domains, which interact with the C-terminal consensus
(S/T/Y)X(V/I) (2, 3). Another module, the PH domain, also
mediates intermolecular interactions, but here the targets are
predominantly specific polyphosphoinositides and inositol polyphosphates (4). These modules may be found in signaling proteins
that possess a catalytic activity (e.g. c-Src and
phospholipase C- The Grb7 family is a group of related SH2 domain-containing adaptors,
comprising Grb7, -10, and -14 (7). These proteins share significant
sequence homology and a conserved molecular architecture, consisting of
a N-terminal region containing the motif P(S/A)IPNPFPEL, a central PH
domain-containing region (designated the GM domain), which bears
homology to the Caenorhabditis elegans protein Mig10 and a
C-terminal SH2 domain. The family members differ in their specificity
and modes of receptor recruitment. Grb7 binds via its SH2 domain to a
variety of receptor tyrosine kinases and tyrosine-phosphorylated
proteins, including erbB2, erbB3, and Shc (7-10). In the case of
Grb10, most attention has focused on its recruitment by the IR and
IGF-1R (7, 11). Grb14 is also bound by the IR (12) and has recently
been identified as a fibroblast growth factor receptor 1 target (13). A
50-amino acid region between the PH and SH2 domains (BPS domain)
contributes to binding of Grb10 and Grb14 to the IR (11, 12).
The signaling function of the Grb7 family is poorly understood. One
role for Grb10 and -14 may be as negative regulators of IR signaling.
For example, overexpression of Grb14 reduces insulin-induced DNA and
glycogen synthesis (12) and inhibition of insulin-induced insulin-like
receptor substrate-1 phosphorylation occurs upon overexpression of
hGrb10 In this paper we describe the identification of a novel tankyrase,
tankyrase 2, as a binding partner for the Grb14 N terminus. Tankyrase
was originally identified by virtue of an interaction with the
telomeric protein TRF1, and consists of a N-terminal HPS region, 24 consecutive ankyrin-type repeats, a SAM module, and a C-terminal region
with homology to the PARP catalytic domain (19). A small fraction of
tankyrase co-localizes with TRF1 at telomeres, and tankyrase can
ADP-ribosylate TRF1 in vitro, leading to a reduction in
binding of TRF1 to telomeric DNA. Consequently, one function of
tankyrase may be in regulation of telomere function via
ADP-ribosylation. However, the majority of tankyrase is extranuclear, and a recent study identified it as a peripheral membrane protein associated with the Golgi, where it localizes to Glut4 vesicles via the
IRAP cytosolic domain and acts as a substrate for insulin and growth
factor-induced MAP kinase activity (20). Interestingly, tankyrase 2 is
also predominantly cytoplasmic and associates with the LDM fraction.
The association of tankyrase 2 with Grb14 supports the hypothesis that
tankyrases may provide a link between signal transduction pathways and
vesicle trafficking.
Yeast Two-hybrid Library Screening--
A plasmid construct
encoding a Gal4 DNA-BD-Grb14 fusion was generated as follows. The
plasmid GRB14/pRcCMVF containing full-length GRB14 cDNA (21) was digested with HindIII and
Klenow-treated to create blunt ends, and then digested with
BclI to release three fragments of ~1.1, 4.2, and 1.7 kb.
The 1.7-kb fragment was isolated and cloned into the NdeI
(Klenow-treated) and BamHI sites of the yeast expression
vector pAS2-1 (CLONTECH, Palo Alto, CA) to
generate GRB14/pAS2-1 containing an in-frame fusion of
full-length Grb14 with the Gal4 DNA-BD. This construct was introduced
by electroporation into the yeast strain CG1945 selecting for
tryptophan prototrophy. Following preparation of yeast cell extracts by
trichloroacetic acid protein extraction, the expression of the fusion
protein was verified by Western blot analysis with antibodies directed against the Flag epitope or the Gal4 DNA-BD. The recipient strain was
then grown to mid-log phase and a human liver cDNA library in the
vector pACT2 (CLONTECH) introduced using the LiAc
procedure (22). Transformants were selected for tryptophan, leucine,
and histidine prototrophy in the presence of 5 mM
3-aminotriazole and then tested for
Clones scoring positive in the
The DNA sequences of the cDNA inserts were then obtained by cycle
sequencing (Promega, Annandale, New South Wales, Australia) using
pACT2-specific and/or clone-specific primers.
Analysis of Protein-Protein Interactions Using the Yeast
Two-hybrid System--
In order to identify the region of Grb14 that
interacts with tankyrase 2, a series of Grb14 deletion mutants were
generated by cloning polymerase chain reaction fragments synthesized
using the appropriate flanking primers into the vector pAS2-1. These fragments spanned the following regions: N terminus (amino acids 1-110), the central region encompassing the Mig10 homology and the BPS
domain (amino acids 110-437), and the N-terminal and central regions
(amino acids 1-437). These plasmids were individually transformed into
the yeast strain Y190. Following transformation of the resulting yeast
strains with the TANKYRASE 2 cDNA clone L1 in pACT-2,
the strength of the interaction was determined by either liquid- or
filter-based
In order to investigate the interaction of tankyrase 2 with
TRF1, a fragment of tankyrase 2 corresponding to the 10-ankyrin repeat
region of tankyrase responsible for TRF1 binding (TR1L12) (19) was
expressed as a Gal4 AD fusion in pGAD10 (CLONTECH). Binding of this to LexA fusions of full-length TRF1 and TRF1 lacking the tankyrase binding site (amino acids 1-67) was then performed as
described previously (19).
Library Screening and Clone Characterization--
Following the
isolation of the original TANKYRASE 2 cDNA, further
clones were isolated by standard cDNA library screening methodology
(24). DNA probes were labeled by random primer extension (Promega)
using [ Assembly and Transcription/Translation of the TANKYRASE 2 cDNA--
The cDNA was first assembled in the vector
Bluescript SK+ (Stratagene) containing alterations to the
multiple cloning site (MCS). The MCS was changed by insertion of
annealed oligonucleotides 5'-GGCCGCGGATCCCGGCTCGAGCGGGAATTCCATGCCATGGCATGCCAAGCTTTCTAGAG-3' and
5'-TCGACTCTAGAAAGCTTGGCAT- GCCATGGCATGGAATTCCCGCTCGAGCCGGGATCCGC-3' into the NotI/XhoI sites to provide the
modified cloning site NotI, BamHI,
XhoI, EcoRI, NcoI, HindIII,
XbaI and to destroy the original XhoI site,
creating the vector BSK (
Coupled transcription/translation reactions were performed according to
the manufacturer's instructions (Promega).
Genomic Localization of TANKYRASE 2--
The original
TANKYRASE 2 cDNA clone (L1) subcloned into pGEX-4T-2
(Amersham Pharmacia Biotech) was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 15 ng/µl to metaphases from two normal males. The FISH method was
modified from that described previously (25) in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a cooled charged coupled device camera
using the ChromoScan image collection and enhancement system (Applied
Imaging Corporation, Newcastle, United Kingdom). FISH signals and the
DAPI banding pattern were merged for figure preparation.
Northern Blot Analysis--
Human multiple tissue Northern blots
(CLONTECH) were hybridized under conditions
recommended by the manufacturer. The radiolabeled probe utilized was
the TANKYRASE 2 cDNA clone L1 labeled by random primer
extension (Promega) using [ Generation of GST Fusion Proteins--
The following regions of
tankyrase 2 were expressed as GST fusion proteins; amino acids 324-980
(corresponding to clone L1 and construct 1 in Fig. 8), amino acids
324-870 (construct 2), amino acids 324-630 (construct 3), amino acids
631-980 (construct 4, also used to generate Ab-1), amino acids
486-630 (used to generate Ab-5), and amino acids 871-935 (construct
5). Construct 1 was generated by subcloning a
SalI-XhoI fragment from pACT2 into the NdeI site of pGEX-4T-2 (Amersham Pharmacia Biotech). DNA
fragments encoding the other regions were synthesized by polymerase
chain reaction using flanking primers containing restriction enzyme sites for in-frame directional insertion into this vector. Following cloning and transformation of the resulting plasmids into
Escherichia coli DH5 Cell Culture--
DU145 human prostate cancer cells, HEK293
cells, and HEK293 cells stably transfected with the
GRB14/pRcCMVF expression vector were maintained
as described previously (21). Where indicated, the cells were starved
overnight in medium containing 0.5% fetal calf serum.
Cell Lysis, Immunoprecipitation, and Western Blotting--
These
techniques were as described previously (27), except that the lysis and
wash buffers used for detection of Grb14-tankyrase 2 co-immunoprecipitation contained 0.1% Triton X-100.
Cell Fractionation--
DU145 cells were serum-starved overnight
in RPMI/0.5% fetal calf serum and then harvested (1 ml/150-mm dish) in
subcellular fractionation buffer (250 mM sucrose, 10 mM Tris, pH 7.5, 0.5 mM EDTA, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). The cell suspensions were subjected to three freeze-thaw cycles and then Dounce homogenization until, by microscopic inspection, the majority of the nuclei were released. The samples were then centrifuged at 800 × g for 10 min (to isolate the low
speed pellet), 50,000 × g for 20 min (to isolate the
HDM), and 160,000 × g for 70 min (to isolate the LDM).
The pellets from each centrifugation step were resuspended in
subcellular fractionation buffer at 10% of the original volume, and
the remaining supernatant was then concentrated to the same volume
using a Microcon YM-10 centrifugal filter device (Millipore Corp.,
Bedford, MA). Equivalent amounts of each fraction (i.e.
normalized for cell number) were then analyzed by Western blotting.
Affinity Purification of Tankyrase 2 Antisera--
GST or the
appropriate GST fusion protein were purified on glutathione-agarose
beads (Sigma, Castle Hill, New South Wales, Australia) (26) and then
cross-linked to the beads using dimethylpimelimidate (Sigma) (28) to
generate affinity columns. The rabbit antiserum was diluted 1:10 with
10 mM Tris-HCl, pH 7.5, and applied to the GST column. The
flow-through was then applied to the GST fusion protein column.
Following washing with 10 mM Tris-HCl, pH 7.5, 500 mM NaCl, the bound antibodies were eluted with 100 mM glycine, pH 2.5, and immediately neutralized with 1 M Tris-HCl, pH 8.0. The antibodies were finally subjected
to buffer exchange with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl using a Centricon 30 microconcentrator (Amicon,
Beverly, MA) and stored in aliquots at Commercial Antibodies--
Commercially available antibodies
used were as follows: M2 monoclonal anti-FLAG antibody (Sigma),
monoclonal anti-golgi 58 kDa protein FTCD (29) (Sigma), monoclonal
anti-GAL4 AD antibody (CLONTECH), monoclonal
anti-GAL4 DNA-BD antibody (CLONTECH), goat polyclonal anti-Grb14 antibody (Santa Cruz Biotechnology, Santa Cruz,
CA), and D8 polyclonal anti-Flag antibody (Santa Cruz Biotechnology).
Binding Assays Using GST Fusion Proteins--
Five
µg of GST or GST fusion protein immobilized on glutathione-agarose
beads were incubated with 400 µl of lysate (~5 mg/ml total protein)
from serum-starved HEK/Grb14 cells (21) for 2 h at 4 C. The beads
were then washed three times with cell lysis buffer and subjected to
SDS-PAGE. Following transfer to a polyvinylidene difluoride membrane,
bound Grb14 was detected by Western blotting with antibody D8 against
the Flag epitope tag.
Indirect Immunofluorescence--
Cells grown on culture slides
in RPMI/10% fetal calf serum were rinsed twice in PBS, fixed at room
temperature in 3.7% paraformaldehyde in PBS for 20 min, and then
permeabilized with 0.2% Triton X-100 in PBS for 10 min. After
extensive washing, fixed cells were blocked in 10% normal goat serum
or 2% bovine serum albumin in PBS containing 0.05% Tween 20 at room
temperature for 45 min and subsequently incubated with antibodies
against tankyrase 2 (Ab-5, 1:50) and Golgi 58-kDa protein (1:50) for
1 h at room temperature or overnight at 4 C. After extensive
washes in PBS containing 0.05% Tween 20, cells were incubated with
Alexa FluorTM 594-conjugated goat anti-rabbit IgG antibody (1:50,
Molecular Probes Inc, Eugene, OR) and Alexa FluorTM 488-conjugated
goat anti-mouse IgG antibody (1:50, Molecular Probes) for 45 min at
room temperature. To detect Grb14, cells were stained as above with
anti-Grb14 antibody (1:100), followed by Texas Red-conjugated donkey
anti-goat antibody (1:50, Jackson Immunoresearch Laboratories Inc, West
Grove, PA). Following washing, samples were mounted in Vectashield plus
DAPI (Vector Laboratories Inc., Burlingame, CA). Images were acquired
on a Leica DMR microscope (Leica Microsystems Pty Ltd, Gladesville, New
South Wales, Australia) using the TCS SP software.
Identification of Grb14-interacting Proteins by the Yeast
Two-hybrid Technique--
In order to identify binding partners for
the Grb14 adaptor protein, a human liver cDNA library in the Gal4
AD vector pACT2 was screened using a full-length Grb14 bait expressed
as a Gal4 DNA-BD fusion. From a screen of ~1 × 106
clones, 31 colonies were initially selected on synthetic complete Cloning and Characterization of TANKYRASE 2--
Screening of
cDNA libraries using the original TANKYRASE 2 clone L1
as probe led to the isolation of a series of overlapping cDNA
clones, which provided the full-length TANKYRASE 2 cDNA
sequence.3 This revealed an
open reading frame for tankyrase 2 spanning 1166 amino acids, encoding
a polypeptide with a predicted molecular mass of 130 kDa. The protein
sequence for tankyrase 2 aligned with that of tankyrase (19) is shown
in Fig. 1. The original cDNA clone
isolated by the two hybrid screen, clone L1, spans amino acids 324-980
of the full-length sequence.
The major difference between the two proteins is the absence of a HPS
domain in tankyrase 2. The molecular architecture of tankyrase 2, starting at the ankyrin repeat region, is then similar to tankyrase.
Both proteins possess 24 ankyrin-type repeats, aligned in Smith
et al. (19), with an overall sequence identity of 83% and
sequence similarity of 90%. The major differences between the two
proteins in this region occur at the C termini of ankyrin repeats 1, 14, and 24 and the N terminus of repeat 24, where there are five or
more non-conservative changes, and the C terminus of repeat 23, where
there is a non-conservative change and then an insertion of 7 amino
acids in tankyrase 2 relative to tankyrase (Fig. 1). The ankyrin repeat
region is then followed by a SAM domain, exhibiting 77% sequence
identity and 89% similarity. The most closely related region is the
C-terminal PARP homology domain, with 93% sequence identity and 96%
similarity. Critical residues required for NAD+ binding and
catalysis are entirely conserved.
Genomic Localization of TANKYRASE 2--
The TANKYRASE
2 gene was localized by FISH. Twenty metaphases from a normal male
were examined for fluorescent signal. All of these metaphases showed
signal on one or both chromatids of chromosome 10 in the region 10q22
to 10q24; 92% of this signal was at 10q23.2 (Fig.
2). There was a total of 13 nonspecific
background dots observed in these 20 metaphases. A similar result was
obtained from hybridization of the probe to 15 metaphases from a second normal male (data not shown). To increase the mapping resolution, 15 metaphases expressing the folate-sensitive fragile site FRA10A, and
showing signal, were then examined. All of these metaphases showed
signal proximal to the fragile site. The precise localization of FRA10A
was described by Sutherland and Hecht (30) as toward the distal margin
of band 10q23.3. Since the TANKYRASE 2 probe hybridized to
band 10q23 and proximal to FRA10A, its likely location is 10q23.2.
Northern Blot Analysis of TANKYRASE 2 Gene Expression--
The
tissue specificity of TANKYRASE 2 expression was
investigated by hybridizing Northern blots of poly(A)+ RNA
isolated from a variety of human tissues to a TANKYRASE
2-specific cDNA probe. This resulted in the detection of a
widely expressed mRNA transcript of ~7 kb (Fig.
3). Expression of TANKYRASE 2 mRNA was particularly high in skeletal muscle and placenta and
moderate in leukocytes, small intestine, ovary, testis, prostate,
thymus, spleen, and pancreas. Prolonged exposure of the autoradiograph indicated that expression was low in colon, liver, lung, brain, and
heart and undetectable in kidney.
Detection of Tankyrase 2 Protein--
In order to further
characterize tankyrase 2, rabbit polyclonal antisera were raised
against GST fusion proteins of two non-overlapping regions of the
protein. Following affinity purification, these antisera, designated
Ab-1 and Ab-5, were used to Western blot cell lysates from DU145
prostate carcinoma cells. This cell line was initially chosen as a
model because it expresses high levels of Grb14 (21). Both antisera,
and not their respective pre-immune controls, detected a protein of
~130 kDa (Fig. 4A), which
agrees with the size predicted from the open reading frame of the
TANKYRASE 2 cDNA. The weaker band of ~170 kDa detected
on these blots is likely to represent tankyrase, which exhibits close
similarity to tankyrase 2 in the regions used for antibody production
but, due to the presence of the HPS domain, migrates more slowly upon SDS-PAGE.
In order to compare the size of tankyrase 2 in cells with that
translated from the cDNA, a coupled transcription-translation reaction was performed using the full-length TANKYRASE 2 cDNA in pcDNA 3.1 as template. This reaction produced a
specific [35S]methionine-labeled product of ~130 kDa,
which was immunoprecipitated with Ab-1 (Fig. 4B).
Furthermore, Western blotting of these samples run adjacent to DU145
cell lysate indicated that this translation product exhibited the same
mobility as DU145-derived tankyrase 2, confirming that the open reading
frame encoded the full-length protein. The lower molecular mass bands
in lanes 1 and 2 of both panels are probably due to the use of downstream
translational start sites as the sequence surrounding the initiation
methionine is very GC-rich.
Subcellular Localization of Tankyrase 2--
Although a small
fraction of the cellular tankyrase pool localizes to telomeres (19),
the majority appears to reside in the cytoplasm in a perinuclear
location (20, 31). In 3T3-L1 fibroblasts, tankyrase co-localizes with
the Golgi marker FTCD by immunofluorescence and co-fractionates with
this marker upon subcellular fractionation (20). We were therefore
interested in determining the subcellular localization of tankyrase 2. DU145 cells were separated by centrifugation into low speed pellet
(residual intact cells, nuclei), HDM (mitochondria, Golgi, endoplasmic
reticulum), LDM (vesicles endosomes), and supernatant (cytosol)
fractions, which were then analyzed by Western blotting (Fig.
5). Blotting with Ab-1 revealed that both
tankyrase (170 kDa) and tankyrase 2 (130 kDa) were predominantly
recovered in the LDM fraction, with lower amounts in the HDM fraction
and the supernatant. FTCD was mainly recovered in the LDM fraction,
with lower amounts in the cytosol, as would be expected for a
peripheral membrane protein in the Golgi. Interestingly, Grb14
exhibited a similar distribution to tankyrase 2, preferentially
associating with the LDM fraction. These results indicate that
tankyrase, tankyrase 2, and Grb14 co-reside in a subcellular fraction
enriched in Golgi vesicles and endosomes.
In order to characterize the subcellular location of the tankyrases
further, DU145 cells were stained with Ab-5, an antibody against the
Golgi marker FTCD, or the two in combination, and then analyzed by
confocal microscopy (Fig. 6). The Golgi
marker exhibited a perinuclear location, as would be expected for this organelle (Fig. 6A) whereas staining with Ab-5 revealed both
diffuse and punctate cytoplasmic staining (Fig. 6B). This
staining was more widespread than the FTCD staining, often extended to
more peripheral regions of the cell, and generally did not co-localize with FTCD upon overlay (Fig. 6C). Similar results were
obtained using Ab-1. These results therefore differ from those
presented by Chi and Lodish (20), where tankyrase strongly co-localized with the Golgi marker FTCD. This may reflect a cell type difference (Chi and Lodish used 3T3-L1 fibroblasts) or preferential detection of
tankyrase 2 by our antibody upon immunostaining, with tankyrase 2 exhibiting a different distribution to tankyrase. Since, in DU145
cells, both tankyrases are enriched in the LDM fraction (Fig. 5), the
punctate staining obtained in Fig. 6B may represent early
endosomes.
Unfortunately, we were unable to establish conditions for co-staining
of DU145 cells for both tankyrase 2 and Grb14. However, indirect
immunofluorescence using an anti-Grb14 antibody revealed largely
diffuse, but also punctate, cytoplasmic staining that was more
concentrated around the nucleus (Fig. 6D). A pool of Grb14
also localized to the plasma membrane. Although plasma membrane localization was not observed for the tankyrases, the staining pattern
for the tankyrases and Grb14 is consistent with the interaction of
these proteins in the cytoplasm.
Comparison of the Binding Selectivity of the Ankyrin Repeat Regions
of Tankyrase 2 and Tankyrase--
The interaction of tankyrase 2 with
Grb14, coupled with its predominantly cytoplasmic localization, led us
to investigate whether the properties of this protein might differ from
tankyrase, which binds the telomeric protein TRF1 (19). A region of
tankyrase containing 10 consecutive ankyrin repeats (denoted TR1L12) is sufficient for binding the N-terminal acidic domain of TRF1 (19). In
order to investigate whether the corresponding region of tankyrase 2 binds TRF1, this fragment (denoted Tank2-L12 and encompassing amino
acids 278-644; Fig. 7A) was
expressed as a Gal4 AD fusion in yeast together with a LexA-TRF1 bait.
Interestingly, this region of tankyrase 2 did not bind TRF1, whereas a
strong induction of A Subset of Tankyrase 2 Ankyrin Repeats Is Sufficient for Binding
Grb14--
The original tankyrase 2 cDNA clone isolated by the two
hybrid screen (clone L1, corresponding to construct 1 in Fig.
8A) stretched from midway
through ankyrin repeat 10 to the region between the SAM and PARP
domains. This region also bound Grb14 in a GST fusion protein pull-down
assay (Fig. 8B). In order to further delineate a Grb14
binding region, a series of deletion constructs were generated and used
in this assay (Fig. 8). We accept that regions of tankyrase 2 outside
of construct 1 may also contribute to binding of full-length tankyrase
2 to Grb14. Deletion of the SAM domain (construct 2) did not affect
Grb14 binding, and the SAM domain alone (construct 5) did not bind
Grb14, indicating that the SAM domain is dispensable for Grb14
interaction. However, construct 4, encoding the C terminus of repeat 19 through to, and including, the SAM domain did not bind Grb14,
implicating the N-terminal set of repeats in Grb14 binding. This was
confirmed using a GST fusion protein corresponding to a region between
mid-repeat 10 and mid-repeat 19 (construct 3), which bound Grb14 almost
as strongly as construct 1.
The N Terminus of Grb14 Is Sufficient for Tankyrase 2 Binding--
In order to map the regions of Grb14 involved in binding
to tankyrase 2, a series of Grb14 deletion mutants fused to the Gal4 DNA-BD (Fig. 9) were co-expressed in the
yeast strain Y190 with the Gal4 AD fusion of tankyrase 2 isolated by
the two hybrid screen. Deletion of the SH2 domain did not markedly
affect binding of tankyrase 2 (Fig. 9). In contrast, removal of the
N-terminal region prevented tankyrase 2 interaction. Furthermore,
expression of a fusion protein containing only the Grb14 N terminus
resulted in significant Association of Grb14 with Tankyrase 2 in Vivo--
In order to
investigate whether Grb14 associates with tankyrase 2 in living cells,
HEK-293 cells stably transfected with Flag epitope-tagged Grb14 (21),
were utilized. These cells express tankyrase 2 but at ~5-fold lower
levels than DU145 cells, and subcellular fractionation of these cells
revealed that, as in DU145 cells, both tankyrase 2 and Grb14
preferentially associate with the LDM
fraction.4 Anti-Flag
immunoprecipitations were performed on lysates from cells transfected
with Grb14 or control HEK293 cells, and the immunoprecipitates then
Western-blotted for the presence of tankyrase 2 (Fig.
10). This led to the detection of
tankyrase 2 in the immunoprecipitate containing Grb14, but not in the
control immunoprecipitate. On longer exposure of the autoradiograph,
tankyrase was also detected in the Grb14
immunoprecipitate.4 However, at this stage, we do not know
whether the Grb14-tankyrase association is direct. This association of
Grb14 with tankyrase 2 in vivo suggests that the interaction
of these two proteins detected in GST pull-down assays (Fig. 8) and in
the yeast two-hybrid system (Fig. 9) is physiologically relevant.
Both the signaling mechanism and function of the Grb7 family of
adaptor proteins are currently poorly understood, but the identification of non-receptor tyrosine kinase binding partners for
their conserved protein modules may shed light on both these properties. Such interacting molecules may perform effector roles or
regulate such processes, for example, by modulating a catalytic activity or regulating subcellular localization. To date, two studies
have identified candidate proteins for such roles. The serine/threonine
kinases Raf1 and MEK1 (32) and the E3 ubiquitin ligase Nedd4 (33)
associate with Grb10, these interactions being mediated primarily by
the Grb10 SH2 domain in a phosphotyrosine-independent manner. However,
tankyrase 2 represents the first protein identified that binds the N
terminus of a Grb7 family protein. Deletion analysis identified the
N-terminal 110 amino acids of Grb14 as being sufficient for this
interaction (Fig. 9). The N-terminal region contains the conserved
proline-rich motif of the Grb7 family (PSIPNPFPEL in Grb14) flanked by
two short stretches of charged amino acids (21). We have yet to
determine the relative contribution of these two types of sequence
motif to tankyrase 2 binding, although it is interesting that the
binding partner identified for this region of Grb14 does not contain an
SH3 or WW domain, protein-protein interaction modules that target
specific proline-rich sequences (34). In GST pull-down experiments, a
region of tankyrase 2 encompassing amino acids 324-630 (ankyrin
repeats 10-19) was sufficient for binding Grb14 (Fig. 8). These
experiments also demonstrated that the SAM domain of tankyrase 2 is not
involved in Grb14 interaction. The role of the SAM domain in the
tankyrases is not known, but these domains can mediate both homotypic
and heterotypic protein-protein interactions (35).
Our identification of a second ankyrin repeat-containing PARP is
interesting in the context of the possible evolutionary relationship between the genes encoding the tankyrases and ankyrins. The
localization of both the TANKYRASE (TNKS) and
ANK1 genes to chromosome 8 and the significant structural
and sequence homology of the repeat regions of the two encoded proteins
led Zhu et al. (36) to suggest a common ancestral origin for
these two genes. The localization of the TANKYRASE 2 gene to
chromosome 10q23.2, close to the ANK3 gene at 10q21 (37),
provides further support for this hypothesis and indicates that these
two gene families have co-segregated during evolution. Both
tankyrase-encoding genes are widely expressed, but TANKYRASE
expression, unlike that of TANKYRASE 2 (Fig. 3) is very high
in testis compared with other tissues (19), arguing for some functional
specificity. The major structural difference between tankyrase and
tankyrase 2 is the absence of a HPS domain in the latter (Fig. 1). The
remainder of the proteins possess the same domain structure and are
highly related in amino acid sequence (91% amino acid similarity). The
role of the HPS domain is not known at present, although the
identification of tankyrase as a substrate for growth factor-induced
MAP kinase activity (20) and the presence of four PXSP
consensus sites for phosphorylation by MAP kinases (38) in the HPS
domain, suggest that this region may be targeted by these enzymes. It
is interesting that the ankyrin repeat regions are highly related but
exhibit a different binding selectivity, in that a 10-ankyrin repeat
segment of tankyrase, but not tankyrase 2, binds TRF1 in a two hybrid
assay (Fig. 7). The region involved spans repeats 9-18, and sequence
comparisons suggest that a divergent stretch of amino acids at the
C-terminal end of repeat 14 may be responsible for this difference in
binding activity (Fig. 1). We have yet to determine whether the ankyrin repeat region of tankyrase binds Grb14. However, it is noteworthy that
the TRF1-binding region of tankyrase (ankyrin repeats 9-18) is very
similar to a Grb14-binding region of tankyrase 2 (ankyrin repeats
10-19, Fig. 8), suggesting that this region of the tankyrases presents
a binding surface for protein-protein interaction.
Smith and de Lange (31) localized tankyrase in HeLa cells to telomeres,
nuclear pore complexes, or the pericentriolar matrix, depending on the
stage of the cell cycle. However, under certain staining conditions, a
strong punctate juxtanuclear staining was also evident. The recent work
of Chi and Lodish (20) indicates that this is probably due to a pool of
tankyrase in the Golgi, since tankyrase in 3T3-L1 fibroblasts
associated with the LDM fraction upon subcellular fractionation and
co-localized with the Golgi marker FTCD by immunofluorescence.
Furthermore, tankyrase bound the cytoplasmic domain of IRAP in
vitro, and in 3T3-L1 adipocytes co-localized with perinuclear, but
not cytoplasmic, Glut4. Unfortunately, we have not been able to raise a
tankyrase 2-specific antibody to allow definitive determination of the
subcellular location of this protein by immunofluorescence, as an
anti-peptide antibody against amino acids 811-830, which are divergent
between tankyrase and tankyrase 2 (Fig. 1), exhibited very low affinity
binding.4 However, our subcellular fractionation studies
indicate that, in DU145 cells, both tankyrase and tankyrase 2 associate
with the LDM fraction (Fig. 5), and indirect immunofluorescence using an antibody that recognizes both tankyrase species detects punctate cytoplasmic staining, which generally does not co-localize with the
Golgi marker FTCD (Fig. 6). The difference between our results and
those of Chi and Lodish may reflect cell type specificity in
localization and/or differences in the localization of tankyrase and
tankyrase 2. However, it is now clear that the function of the
tankyrases is not restricted to the nucleus, and that they must also
perform a function in the cytoplasm where they associate with the Golgi
or endosome fractions. The lack of association of the ankyrin repeat
region of tankyrase 2 with the telomeric protein TRF1 (Fig. 7) may
indicate that tankyrase 2 does not function at telomeres, but further
experimentation will be required to resolve this issue.
What may be the function of the tankyrases in the cytoplasm? Tankyrase
exhibits PARP activity in vitro toward both itself and the
exogenous substrates TRF1 and the IRAP cytoplasmic domain (19, 20),
although endogenous substrates in cells have yet to be identified. The
high conservation of tankyrase 2 in the PARP homology domain (Fig. 1),
including the essential residues for catalytic activity (19), strongly
suggests that tankyrase 2 will also possess PARP activity.
Interestingly, the activity of C-terminal-binding protein 1/brefeldin
A-ADP-ribosylated substrate, which acylates lysophosphatidic acid to
promote the fission of Golgi membranes, is inhibited by
ADP-ribosylation (39), and this protein becomes ADP-ribosylated by an
unknown activity upon treatment of cells by the Golgi-disrupting agent
brefeldin A (40). Consequently, ADP-ribosylation may regulate vesicle
formation in this and other cytoplasmic compartments. As indicated by
the mode of tankyrase 2 binding to Grb14, the repeat region of the tankyrases is likely to participate in protein-protein interactions in
a manner similar to that of the ankyrins themselves, which link
specific integral membrane proteins to the underlying cytoskeleton (41). A precedent for the interaction of ankyrins with signaling proteins associated with the plasma membrane is the binding of the
ankyrin-repeat regions of Ank1 and Ank3 to the Rho family GDP-GTP
exchange factor Tiam 1 (42). However, the ankyrins are not restricted
to a plasma membrane location. Particular ankyrin isoforms, some
retaining the ankyrin-repeat region, are associated with the Golgi and
endolysosomes, where they form part of a Golgi-associated spectrin
skeleton, which may function in structural organization, cargo
selection, and/or linkage to motor proteins (43). Finally, an
intriguing observation in relation to the potential role of tankyrase 2 in regulating vesicle dynamics is that a region of mouse chromosome 19 syntenic with human chromosome 10q23 (which harbors TANKYRASE
2, Fig. 2) contains two loci, pale ear (ep) and ruby-eye (ru), responsible for phenotypes
similar to human Hermansky-Pudlak syndrome, a condition associated with
defects in multiple cytoplasmic organelles, including lysosomes (44, 45). The ep locus corresponds to the human gene
HPS, which is mutated in a subset of Hermansky-Pudlak
syndrome patients (45). However, the gene responsible for the defect in
ru mice remains to be identified, and our studies highlight
Tankyrase 2 as a candidate.
A surprising result was that Grb14 preferentially
associates with the LDM fraction (Fig. 5). Since Grb14 associates with
tankyrase 2 in GST pull-down assays (Fig. 8), the yeast two hybrid
system (Fig. 9) and in vivo (Fig. 10), tankyrase 2 may
tether Grb14 to vesicles in this fraction, but the PH domain of Grb14
may also participate. What could be the significance of this
localization? Conceivably, vesicle trafficking may deliver Grb14 to a
specific subcellular location. A precedent for this is that movement of paxillin from the Golgi apparatus to focal adhesions is dependent on
the activity of the small GTP-binding protein ARF1, which regulates coatamer assembly on Golgi transport vesicles (46). Alternatively, given the association of Grb14 with specific activated growth factor
receptors (12, 13), the tankyrase 2-Grb14 interaction may play a role
in sorting of internalized receptors. In this context it is interesting
that we also isolated Nedd4, which regulates the ubiquitination and
endocytosis of cell surface proteins (47), as a Grb14 binding partner
in our two hybrid screen. Similar to the Grb10-Nedd4 interaction
reported by Morrione et al. (33), the SH2 domain of Grb14
binds in vitro to a region of Nedd4 encompassing the
calcium-lipid binding/C2 domain in a phosphotyrosine independent manner, although to date we have been unable to co-immunoprecipitate these two proteins.4 These hypotheses concerning the
signaling mechanism of Grb14 are currently under investigation.
motif
module, and poly(ADP-ribose) polymerase homology domain. The
TANKYRASE 2 gene localizes to chromosome 10q23.2 and is
widely expressed, with mRNA transcripts particularly abundant in
skeletal muscle and placenta. Upon subcellular fractionation, both
Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be
detected by co-immunoprecipitation analysis. Deletion analyses
implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats
10-19 of tankyrase 2 in mediating this interaction. This study
supports a role for the tankyrases in cytoplasmic signal transduction
pathways and suggests that vesicle trafficking may be involved in the
subcellular localization or signaling function of Grb14.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
); the adaptor class (e.g. Grb2), which
provide a molecular link to separate effector molecules; and proteins
that provide an anchoring or scaffolding function, e.g.
PSD-95 (1). As well as initiating signaling events,
protein-protein interactions are also important in regulating the
internalization of cell surface receptors and their subsequent sorting
to lysosomal or recycling compartments (5, 6).
2 (Grb-IR) (14) or
Grb14 (12). However, data supporting a positive role for mGrb10
in
insulin-, IGF-1-, and platelet-derived growth factor BB-stimulated
mitogenesis have recently been presented (15). It is also likely that
the functional role of the Grb7 family extends beyond signal
modulation. For example, inhibition of Grb7 expression suppresses the
invasive potential of esophageal cancer cells (16), and overexpression
of Grb7 and its targeting to focal contacts correlates with increased
cell motility (17). Definition of the molecular interactions mediated
by Grb7 family proteins, particularly those involving the N-terminal
and GM domains, may provide a valuable insight into their signaling
mechanism and how it is regulated. With regard to the N-terminal
region, the SH3 domain of c-Abl binds the conserved proline-rich motif of Grb10 in vitro (18), but an in vivo binding
partner has yet to be identified.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-galactosidase activity by
either a liquid culture-based method (Galacto-Light, Tropix, Bedford,
MA) or colony lift filter assay (CLONTECH).
-galactosidase assays were subjected
to CHX curing to remove the bait plasmid by streaking out on synthetic
complete-leu media containing 10 µg/ml CHX (pAS2-1 contains the
CYH2 gene which restores CHX sensitivity to CG1945 cells).
This enabled confirmation of the bait dependence of LacZ activation and subsequent isolation of the pACT2 plasmids encoding interacting proteins by standard methodology (23). Back transformations were then performed in which these pACT2 plasmids were introduced into
CG1945 strains containing the bait plasmid (GRB14/pAS2-1) or constructs encoding non-related Gal4 DNA-BD fusions in order to
confirm the specificity of the interactions.
-galactosidase assays. Expression of the constructs was
confirmed by Western blotting of yeast extracts with Gal4 DNA-BD- and
Gal4 AD-specific antibodies.
-32P]dCTP (110 TBq/mmol, Amersham Pharmacia
Biotech Pty Ltd, Castle Hill, New South Wales, Australia). Following
isolation of phage or phagemid DNA (Promega Wizard kits), sequencing of
the cDNA inserts was performed by cycle sequencing. The cDNA
cloning strategy was as follows, and further cDNA clone details can
be provided upon request. The original TANKYRASE 2 cDNA
isolated from the two hybrid screen (L1) was used as a probe to screen
a
gt10 human placental cDNA library (5' Stretch Plus,
CLONTECH). This isolated two clones, designated P8
and P12. P8 was ~2.0 kb and provided the C-terminal end of the
tankyrase 2 protein sequence. P12 was ~3.5 kb and extended the
cDNA sequence 0.9 kb in the 5' direction. Screening of the human
placental cDNA library and a
gt11 human small intestine cDNA
library (5' Stretch, CLONTECH) with 5'-located probes led to the isolation of two clones, designated P5 and SI4, respectively, which both extended the sequence further 5' and provided
a putative translation initiation codon. Screening of a
ZAP II human
fetal brain cDNA library (Stratagene, La Jolla, CA) with a 414-bp
probe including the extended sequence isolated two further clones, FB3
and FB11, which confirmed this sequence. Sequence alignments were
performed using the program ClustalW.
MCS). The first 495 bp of TANKYRASE
2 were obtained as a BamHI/XhoI fragment
from FB11, and inserted into the BamHI/XhoI sites
of BSK (
MCS) creating BSK(I). The next 840 bp were obtained as a
XhoI/EcoRI fragment from SI4 and cloned into the
XhoI/EcoRI sites of BSK(I) creating BSK(II). The
following 1104 bp were obtained as a EcoRI/NcoI
fragment from L1 and inserted into the EcoRI/NcoI
site in BSK(II), creating BSK(III). The final 1361 bp were obtained as
a NcoI/HindIII fragment from P8, and cloned into
the NcoI/HindIII site in BSK(III), creating BSK(IV). The assembled TANKYRASE 2 cDNA was subcloned
into the NotI/XbaI sites of pcDNA 3.1(+)
(Invitrogen, Groningen, The Netherlands).
-32P]dCTP (Amersham
Pharmacia Biotech).
, GST fusion proteins were purified
from isopropyl-
-D-thiogalactopyranoside-induced bacterial cultures as described previously (26).
70 °C.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu
His
Trp medium and were then tested for
-galactosidase
activity. Nine clones gave significant activity in the latter assay and were characterized by DNA sequencing. One of these pACT2 clones harbored a novel cDNA of 1971 bp. This clone encoded a polypeptide of 657 amino acids in frame with the Gal4 DNA-BD and exhibited homology
to tankyrase (19), but the absence of translation start and stop codons
revealed that the cDNA clone was incomplete.
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Fig. 1.
Sequence of tankyrase 2 and alignment with
tankyrase. An alignment of the tankyrase and tankyrase 2 protein
sequences was generated using the program ClustalW. Identical amino
acids (bold letters, dark
shading) or conservative changes (light
shading) are boxed. The ankyrin repeat region is
indicated by a single underline, the SAM module
by a broken underline, and the PARP-related
domain by a double underline. Note the absence of
the N-terminal HPS domain in tankyrase 2. Numbers
above the ankyrin repeat region indicate the start of
individual repeats. Amino acid residues for each protein are
numbered (from the initiation methionine) on the
left and right of the figure.
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Fig. 2.
Localization of the human TANKYRASE
2 gene by FISH. Metaphase showing FISH with the
TANKYRASE 2 probe. The chromosomes have also been stained
with DAPI for identification. Hybridization sites on chromosome 10 are
indicated by an arrow.
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Fig. 3.
Northern blot analysis of TANKYRASE
2 gene expression. Northern blots of
poly(A)+ RNA isolated from a variety of human tissues were
hybridized to a TANKYRASE 2 cDNA probe labeled with
32P by random primer extension. The exposure time for the
autoradiograph was 16 h with two intensifying screens.
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Fig. 4.
Detection of tankyrase 2 protein.
A, detection of tankyrase 2 by Western blot analysis.
Lysates from DU145 prostate carcinoma cells were separated by SDS-PAGE
(6% gel), transferred to nitrocellulose, and Western blotted with
either preimmune serum (P) or affinity-purified antisera
(I) Ab-1 or Ab-5. Detection of bound antibody was by ECL.
The positions of tankyrase and tankyrase 2 are indicated by
open and closed arrowheads,
respectively. B, comparison of endogenous tankyrase 2 with
that obtained from translation of the TANKYRASE 2 cDNA.
Left panel, the following samples were subjected
to SDS-PAGE and transferred to nitrocellulose. Lane
1, 35S-labeled transcription-translation
reaction programmed with TANKYRASE 2 cDNA;
lane 2, Ab-1 immunoprecipitate of sample in
lane 1; lane 3, DU145 cell
lysate. The filter was then Western-blotted (WB) with Ab-1.
Detection of bound antibody was by ECL. Open and
closed arrowheads, as for A. The
filter was then subjected to autoradiography (right
panel, 3-day exposure).
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Fig. 5.
Analysis of tankyrase 2 and Grb14
localization by subcellular fractionation. Low speed pellet
(lane 1), HDM (lane 2), LDM
(lane 3), and supernatant (lane
4) fractions were prepared from DU145 cells as described
under "Experimental Procedures." Following separation by SDS-PAGE
and transfer to a polyvinylidene difluoride membrane, replicate filters
were blotted for tankyrase 2 (using Ab-1) (top
panel), Grb14 (middle panel), and the
Golgi 58-kDa marker FTCD (bottom panel).
Detection of bound antibodies was by ECL.
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Fig. 6.
Immunolocalization of tankyrases and Grb14 in
DU145 prostate carcinoma cells. Cells were fixed in 3.7%
paraformaldehyde and stained with antibodies raised against the Golgi
58-kDa protein FTCD (A; green Alexa Fluor 488) and tankyrase
2 (B; red Alexa Fluor 594) as described under
"Experimental Procedures." The overlay is shown in C,
and any co-localization between tankyrases and FTCD appears
yellow. In panel D, cells were stained
with an anti-Grb14 antibody followed by a Texas Red-conjugated
secondary antibody.
-galactosidase activity occurred with positive
controls involving either TR1L12-TRF1 interaction or TRF1 dimerization
(Fig. 7B). Western blotting of yeast extracts confirmed
expression of the appropriate fusion proteins. Consequently, although
tankyrase and tankyrase 2 are closely related in their ankyrin repeat
regions (90% amino acid similarity), the small number of amino acid
substitutions present are sufficient to markedly alter the binding
selectivity of these protein-protein interaction domains.
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Fig. 7.
Comparison of the binding selectivity of the
tankyrase and tankyrase 2 ankyrin-repeat regions. A,
schematic representation of the TRF1, tankyrase, and tankyrase 2 fusion
partners utilized in yeast two hybrid analysis. In TRF1, A
denotes the acidic region, D the dimerization domain, and
M the Myb-related region. Full-length TRF1 (FL-TRF1) or the
N-terminal truncation N-TRF1 were fused to LexA. In tankyrase
(T) and tankyrase 2 (T2), ANK denotes
the ankyrin repeat region, S the SAM module, and
P the PARP homology domain. The solid
bars represent the 10-ankyrin repeat region of tankyrase
responsible for TRF1 binding (TR1L12) and the corresponding region of
tankyrase 2 (Tank2-L12) with the amino acid boundaries indicated. These
two regions were fused to the Gal4 AD. B, analysis of the
interaction between the ankyrin repeat regions of tankyrase or
tankyrase 2 with TRF1. TR1L12 and Tank2-L12 were expressed as Gal4 AD
fusions in yeast strain L40 with the TRF1 or
N-TRF1 bait constructs.
The AD vector pGAD10 (V) was used as a negative control.
Interaction of LexA and the Gal4 AD via TRF1 dimerization was used as
an additional positive control.
-Galactosidase activity assays were
performed by the liquid culture-derived method and represent the mean
of triplicate determinations. Results are expressed as mean
-galactosidase units (× 10
3). ND, not
determined.
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Fig. 8.
Delineation of a Grb14 binding region on
tankyrase 2. A, schematic representation of the
different regions of tankyrase 2 expressed as GST fusion proteins. The
different constructs are numbered on the left of
the figure. The individual ankyrin repeats are drawn to scale, and key
repeats are labeled. The region labeled S is the SAM domain.
B, mapping of a Grb14-binding region on tankyrase 2 by a GST
fusion protein pull-down assay. The fusion proteins illustrated in
A were coupled to glutathione-agarose beads and incubated
with lysates from cells expressing Flag epitope-tagged Grb14. Following
washing of the beads, bound Grb14 was detected by Western blotting with
an antibody against the epitope tag.
-galactosidase activity, demonstrating that
this region is not only required, but also sufficient, for tankyrase 2 binding. However, since we did not utilize a construct lacking the N
terminus but containing the SH2 domain, we cannot completely rule out a
contribution of the SH2 region to Grb14-tankyrase 2 interaction.
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Fig. 9.
Mapping of the tankyrase 2 binding site on
Grb14. Constructs encoding fusions of full-length Grb14
(FL), the N-terminal region (N), central region
(C), and N-terminal + central regions
(N+C) were generated in the vector pAS2-1. The
table shows results of -galactosidase activity assays following
transformation of plasmids encoding the above Grb14-BD fusions or
pAS2-1 vector alone (V) into yeast strain Y190 together
with a tankyrase 2 cDNA clone in pACT2 encoding amino acids
324-980 (clone L1). Assays were performed in triplicate by the liquid
culture-derived method and expressed as a percentage relative to the
activity obtained with the full-length Grb14 construct. Additionally,
the results of a colony lift filter assay are shown in
parentheses, with the intensity of blue color development
scored from
(absent) to +++ (strong).
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Fig. 10.
Tankyrase 2 and Grb14 associate in
vivo. Lysates from serum-starved control HEK293 cells
(HEK) or HEK293 cells stably transfected with Flag
epitope-tagged Grb14 (HEK/Grb14) were subjected to
immunoprecipitation with the anti-Flag monoclonal antibody M2. The
immunoprecipitates (IP) were then separated by SDS-PAGE,
transferred to nitrocellulose, and Western-blotted (WB) with
anti-Flag antibodies (M2) or anti-tankyrase 2 antibodies (Ab-1).
Lysates from these cell lines were analyzed in parallel.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Anne Cunningham (Confocal Facility, Garvan Institute of Medical Research, Sydney, New South Wales, Australia) for assistance with immunofluorescence techniques and confocal microscopy and Dr. Keith Stanley (Center for Immunology, St. Vincent's Hospital, Sydney, New South Wales, Australia) for helpful discussion.
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FOOTNOTES |
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* This work was funded by research grants from the National Health and Medical Research Council of Australia, the New South Wales Cancer Council, and the Kathleen Cuningham Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF329696.
** To whom correspondence should be addressed. Tel.: 61-2-9295-8333; Fax: 61-2-9295-8321; E-mail: r.daly@garvan.org.au.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M009756200
2 The nomenclature used for particular Grb10 isoforms is that proposed by André Nantel following consultation with workers in the field. This system allows for the possibility that the same variant will be identified in different species, and should therefore be given the same isoform designation (indicated by a Greek letter).
3 The nucleotide sequence for the human TANKYRASE 2 cDNA has been deposited in the GenBankTM database under GenBank accession no. AF329696. We note close matches with sequences deposited under the following accession numbers: AF264912, AX029397, AF305081, and AK023746.
4 R. J. Lyons, R. Deane, G. M. Sanderson, and R. J. Daly, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
SH, Src homology;
Ab, antibody;
AD, activation domain;
BD, binding domain;
bp, base pair(s);
BPS, between pleckstrin homology and Src homology 2;
CHX, cycloheximide;
DAPI, 4', 6-diamidine-2-phenylindole;
FISH, fluorescence
in situ hybridization;
FTCD, formiminotransferase
cyclodeaminase;
GM, Grb-Mig;
Grb, growth factor receptor-bound;
GST, glutathione S-transferase;
HDM, high density microsome;
HPS, histidine/proline/serine-rich;
IGF-1(R), insulin-like growth factor 1 (receptor);
IR, insulin receptor;
IRAP, insulin-responsive
aminopeptidase;
kb, kilobase(s);
LDM, low density microsome;
MAP, mitogen-activated protein;
PAGE, polyacrylamide gel electrophoresis;
PARP, poly(ADP-ribose) polymerase;
PBS, phosphate-buffered saline;
PDZ, PSD-95/Dlg/ZO1;
PH, pleckstrin homology;
SAM, sterile motif;
TRF, telomere repeat binding factor.
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