From the Department of Biological Chemistry, Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, the § Graduate Program, Department of Biochemistry and
Molecular Genetics, University of Alabama at Birmingham, Birmingham,
Alabama 35294, and the ¶ Department of Anatomy, Kyorin University
School of Medicine, Mitaka, Tokyo 181-8611, Japan
Received for publication, September 12, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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The abundant and dynamic post-translational
modification of nuclear and cytosolic proteins by
Dynamic modification of Ser/Thr residues of nucleocytoplasmic
proteins by single Enzymes that cycle the O-GlcNAc modification are analogous
to those that catalyze phosphorylation (i.e. kinases and
phosphatases). The enzyme that attaches the saccharide to proteins is
uridine diphospho-N-acetylglucosamine:polypeptide
OGT localizes to both the nucleus and cytoplasm of cells, but it is
present at higher levels in the nucleus (25). Aside from being
tyrosine-phosphorylated and O-GlcNAc-modified, the rat
110-kDa OGT enzyme contains 111/2 tetratricopeptide repeats (TPRs), a protein-protein interaction domain found in many proteins (25, 29, 30). TPRs have been shown to mediate protein-protein interactions in a variety of proteins (37). Recently, the crystal structure of the TPR domain of two of these proteins, protein phosphatase 5 (31) and Pex5p (32), were solved. OGT exists as a
homotrimer, and the TPR domain was shown to mediate the trimerization (33). Studies performed on recombinant OGT overexpressed and purified
in baculovirus (33) and Escherichia coli (13) have shown
that the TPR domain plays a key role in intrasubunit interaction and in
substrate recognition.
Unlike the large numbers of genes encoding kinases, thus far, there is
evidence for only a single OGT catalytic subunit despite the myriad of
different O-GlcNAc-modified proteins (1). Virtually nothing
is known about the regulation of OGT's subcellular localization or
substrate specificity. The presence of multiple TPR repeats implies
that proteins might specifically interact with the enzyme, targeting
the catalytic domain to specific protein substrates within the nucleus
and cytoplasm. To examine this hypothesis, we screened a rat brain
library with OGT using the yeast two-hybrid approach, in order to begin
to identify potential OGT-interacting proteins (OIPs). Initially, 250 positively interacting clones were identified. Here, we report the
cloning and characterization of GRIF-1, a recently cloned
GABAA receptor-associated protein, and its novel homolog
KIAA1042/OIP106 as novel coiled-coil domain proteins that interact
strongly with the TPR domain of OGT. Our findings suggest that these
proteins possibly function to target OGT to RNA polymerase II and
GABAA receptor complexes to mediate transcriptional and
signaling events.
Plasmid Constructs--
The OGT-Gal4 BD fusion bait plasmid
pJL59-OGT was generated by inserting the rat OGT cDNA into pJL59.
The negative control pJL59-C-termOGT plasmid was generated
by inserting the C terminus (residues 476-1037) of OGT. The plasmids
encoding the full-length OGT, C-termOGT, and TPR domain for
E. coli expression were generated by subcloning the
respective cDNAs into pET32 (Novagen) as thioredoxin fusion proteins. Constructs encoding for an N-terminal hexa-His-tagged TPR and
C-termOGT fusion proteins (pRSET-TPR and pRSET-C,
respectively) were generated by subcloning the TPR and
C-termOGT fragments into the pRSET vector (Invitrogen). The
pACT2 plasmid encoding the partial rat OIP98 (AD-OIP98) clone (residues
105-878) fused to the activation domain of Gal4 was obtained from the
initial yeast two-hybrid screen as a result of plasmid rescue.
Full-length rat OIP98 was generated by performing 5'-rapid
amplification of cDNA ends using the rat brain Marathon-Ready
cDNA library (Clontech) and by assembling the
5'-rapid amplification of cDNA ends product with the preexisting
insert derived from the yeast two-hybrid screen using overlapping
restriction sites. OIP98 is a partial clone of GRIF-1, lacking exon 15. The missing exon was obtained by using a QuickClone rat brain cDNA
library (Clontech) as template and GRIF-1
gene-specific primers flanking exon 15 in a PCR and inserted into the
OIP98 cDNA using overlapping restriction sites. An E. coli expression plasmid of rat GRIF-1 (OIP98) was generated by
subcloning the cDNA insert into pET32. Plasmids for in
vitro transcription/translation reactions and mammalian
overexpression were generated by subcloning the GRIF-1 cDNA into
pCITE4c (Novagen) and pcDNA3.1 His A (Invitrogen), respectively.
The insert encoding KIAA1042/OIP106 cDNA was obtained from Dr.
Takahiro Nagase from the Kazusa DNA Research Institute (Chiba, Japan)
in pBluescript II SK+. The insert was then used as template
to generate a cassette via PCR. This cassette was then subcloned into
pCITE5b and pcDNA3.1 His B (Invitrogen) for use in in
vitro transcription/translation reactions and mammalian
expression. Finally, pCITE-p62 was constructed by subcloning the
cDNA encoding rat nucleoporin p62 (48) into pCITE4c. The fidelity
of all DNA constructs was verified by nucleotide sequencing.
Yeast Two-hybrid Screen and Interaction Testing--
The
Matchmaker rat brain library in pACT2 (prey vector with Gal4 activation
domain) (Clontech) was used to isolate interacting clones, according to the manufacturer's instructions. Screening was
performed on SD/ Protein Expression and Purification--
The E. coli
strain BL21 (DE3) codon plus RIL strain (Stratagene) was used for the
overexpression of OGT and GRIF-1 (OIP98) protein constructs in either
LB or double yeast tryptone-ampicillin. Unless indicated otherwise, all
purified proteins were desalted in 20 mM Tris, pH 7.8, 20-40% (v/v) glycerol (v/v), 0.02% (w/v) sodium azide and stored at
Blot Overlay Assays--
Equal amounts of E. coli
expressed recombinant OGT, TPR, and C-termOGT and BSA
proteins were separated on SDS-PAGE and blotted on polyvinylidene difluoride membranes. Proteins were renatured en blot in
renaturation buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 100 mM potassium acetate, 1 mM dithiothreitol, 5 mM MgCl2, 1 mM EDTA, 0.1% (v/v) Tween 20, 0.1 mM
ZnCl2, 5% milk (w/v), and 0.1 M Met) at
4 °C. [35S]Met-labeled GRIF-1 and OIP106 probes were
synthesized in vitro using the TnT coupled reticulocyte
lysate system (Promega) according to the manufacturer's instructions.
Reactions were desalted and added to 15 ml of cold renaturation buffer
and used to probe the blots containing the immobilized proteins at
4 °C. Following probing, the blots were washed extensively with
renaturation buffer, dried, and subjected to autoradiography by
exposure to Biomax MR film (Eastman Kodak Co.) at Preparation and Purification of Rabbit and Chicken Polyclonal
Antibodies--
Rabbit and chicken antiserum to GRIF-1 and OIP106 were
generated by immunizing rabbits and chickens with recombinant
His-tagged GRIF-1 (OIP98) purified as described above.
Gel-purified protein was used as an immunogen by Covance Research
Products (Denver, PA) to produce polyclonal antisera in one
rabbit designated as JH 3286. In a similar manner, chickens were
immunized by Aves Laboratories (Tigard, OR) to produce chicken IgY
antibodies. Affinity-purified antibodies were generated by purifying JH
3286 antiserum or chicken IgY over a GRIF-1 (OIP98) affinity
column as previously described (36). KIAA1042/OIP106-specific
polyclonal antibodies were generated by immunizing one rabbit
designated as SAI1 with a peptide containing the first 20 amino acids
of the N terminus (N20) of KIAA1042. This region is specific and unique
to KIAA1042. Affinity-purified antibodies were generated by purifying
SAI1 antiserum over an N20 peptide affinity column as previously
described (36). The anti-OGT antibody AL28 was generated in a similar
manner by using His-tagged C-termOGT protein to immunize
two rabbits designated AL28 and AL29. AL28 antiserum was
affinity-purified over a thioredoxin-C-termOGT (pET32-C)
column as previously described (36).
Antibodies and Western Blot Analysis--
AL28 was used at a
final concentration of 25-50 ng/ml in 5% (w/v) milk in Tris-buffered
saline containing 0.05% (v/v) Tween 20 for 16 h at 4 °C.
Similarly, JH 3286 was used at a final concentration of 100 ng/ml.
Chicken anti-OIP106/GRIF-1 was used at a final concentration of 0.5-1
µg/ml. S-protein HRP conjugate (Novagen) was used at 1:5000 according
to the manufacturer's instructions. Anti-actin (Sigma) and anti-Rb
p107 (Santa Cruz Biotechnology, Inc.) antibodies were used at 1:5000.
8WG16 mouse monoclonal antibodies to hypophosphorylated RNA polymerase
II (Neoclone) and anti-tubulin (Sigma) were used at 1:10,000. Mouse
Omniprobe anti-Xpress antibody (D-8; Santa Cruz
Biotechnology) was used at 1:2000, and anti-O-GlcNAc CTD 110.6 mouse monoclonal antibody was used at 1:2500 as described before,
either in the absence or presence of 50 mM GlcNAc (51). All
blots were developed with the enhanced chemiluminescence (ECL) reagent
(Amersham Biosciences).
Cell Culture--
HeLa and HEK 293 cells were grown in
Dulbecco's modified Eagle's medium, 10% (v/v) fetal bovine serum
containing 0.1 mM nonessential amino acids, penicillin, and streptomycin.
Preparation of Tissue Extracts--
For preparation of whole
tissue extracts, frozen tissue from male Sprague-Dawley rats were
homogenized in ice-cold radioimmune precipitation assay lysis buffer
(PBS, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate)
containing protease inhibitors, and soluble crude extracts were
generated by spinning the homogenate at 100,000 × g
for 90 min at 4 °C. Extracts were assayed for protein concentration
via the Bio-Rad protein assay reagent, and equal amounts of each tissue
extract were separated on SDS-PAGE for JH 3286 Western blot analysis.
HeLa whole cell lysate was prepared in a similar manner. For analysis
of human cell line extracts, Cruz-Blot B was purchased (Santa Cruz
Biotechnology) and analyzed with SAI1 and
For immunoprecipitation experiments, frozen rat brains were homogenized
in Hepes-buffered saline (HBS) containing 20 mM Hepes pH
7.4, 0.3 M NaCl, 5 mM MgCl2, 1 mM NaF, 100 mM GlcNAc, and protease inhibitors
using a Polytron homogenizer. The homogenate was then centrifuged at
100 000 × g for 90 min at 4 °C to generate soluble crude brain extract and stored at OIP106 Confocal and Electron Microscopy--
HeLa cells were
fixed in 4% (v/v) formaldehyde in PBS (pH 7.3) for 1 h at
4 °C, permeabilized with 0.5%(v/v) Triton X-100 in PBS for 5 min
and treated with 5% (w/v) BSA in PBS for 10 min. The specimens were
then incubated with the anti-OIP106 antibody (JH 3286) (dilution,
1:200) or with pre-immune rabbit IgG for 1 h at room temperature,
washed with PBS, and subsequently incubated for 1 h with
Cy3-conjugated donkey anti-rabbit IgG antibody (dilution, 1:500)
(Jackson Immunoresearch). Specimens were then incubated with the mouse
monoclonal anti-hypophosphorylated RNA polymerase II (dilution, 1:1000)
antibody 8WG16 (Neoclone) for 1 h at room temperature, washed with
PBS, and incubated for 1 h with fluorescein isothiocyanate-conjugated donkey anti mouse IgG (Jackson
Immunoresearch). After a wash with PBS, the specimens were mounted in
90% (v/v) glycerol, 0.1 M Tris buffer (pH 8.5) containing
0.5 mM p-phenylene diamine and observed under
laser-scanning confocal microscope (MRC-1024; Bio-Rad). Electron
microscopy was performed as previously described (47), using a JEM-1010
electron microscope (JEOL, Tokyo, Japan), using the above dilutions for
JH 3286 and 8WG16.
Immunoprecipitation--
Crude rat brain extract was filtered
through a 0.22-µm syringe filter and was precleared by
anti-IgY-agarose (Promega). Precleared extract was incubated with
either 2 µg of preimmune chicken IgY or anti-OIP106/GRIF-1 IgY to
immunoprecipitate native OIP106 and GRIF-1 16 h at 4 °C.
OIP106/GRIF-1 immune complexes were collected by incubation with
anti-IgY-agarose for 1 h at 4 °C. The beads were extensively
washed in cold HBS extract buffer, and bound proteins were eluted with
SDS-PAGE sample buffer. Samples were boiled, separated by SDS-PAGE, and
analyzed by Western blotting with either rabbit anti-OIP106/GRIF-1
antibody JH 3286 or rabbit anti-OGT antibody AL28.
Similarly, for OGT immunoprecipitations, crude rat brain extract was
incubated with AL28 in HBS (0.3 M NaCl) or radioimmune precipitation assay lysis buffer for 16 h at 4 °C. Immune
complexes were collected by incubation with protein A-Sepharose
(Amersham Biosciences), and bound proteins were eluted with SDS-PAGE
sample buffer, followed by analysis by Western blotting.
HEK 293 cell immunoprecipitations were performed by lysing transfected
cells in HBS (0.3 M NaCl) buffer containing 0.5% (v/v) Triton X-100 and 0.5 mM phenylmethylsulfonyl fluoride for
30 min at 4 °C. Whole cell extracts were generated by centrifuging
the lysates at 16,000 × g at 4 °C for 10 min.
Precleared extracts were incubated with anti-Xpress Omniprobe
D-8 antibody (Santa Cruz Biotechnology) and protein A/G
plus agarose (Santa Cruz Biotechnology) to immunoprecipitate
recombinant Xpress-tagged OIP106 and GRIF-1 for 3 h at 4 °C.
Immunoprecipitates were washed extensively in lysis buffer and eluted
with SDS-PAGE sample buffer, followed by Western blotting with
Omniprobe D-8, anti-OGT AL28, and CTD 110.6 antibodies.
Immunoprecipitations from TnT rabbit reticulocyte lysates expressing
Xpress-tagged OIP106 and GRIF-1 were performed with Omniprobe
D-8 antibody in a similar manner as described above.
For HeLa nuclear extract immunoprecipitations, 5 µg of either
preimmune chicken IgY or anti-OIP106/GRIF-1 IgY bound to
anti-IgY-agarose were incubated with 330 µg of precleared HeLa
nuclear extract in 20 mM Hepes, pH 7.9, 300 mM
NaCl, 1 mM EDTA, 10 mM MgCl2, 20 µM
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate, 0.1% (v/v) Triton X-100 overnight at 4 °C to immunoprecipitate native OIP106. OIP106 immune complexes were washed extensively with
binding buffer containing 0.2% (v/v) Triton X-100, and bound proteins
were eluted by boiling in SDS-PAGE sample buffer, followed by Western
blot analysis with JH 3286, 8WG16, AL28, and anti-Rb p107 antibodies.
Protein-Protein Interactions--
For OIP106/GRIF-1 pull-down
experiments, S-tagged OIP106 and GRIF-1 were synthesized in TnT
rabbit reticulocyte lysates (Promega). Following synthesis, lysates
were incubated with S-protein-agarose to purify S-tagged OIP106 and
GRIF-1 for 3 h at room temperature in HBS (0.3 M NaCl)
lysis buffer containing 0.5% (v/v) Triton X-100. Pull-downs were
washed extensively in lysis buffer, and bound proteins were eluted with
SDS-PAGE sample buffer and analyzed by silver staining.
For the p62 and OIP106-AL28 pull-down assays, S-tagged p62 and OIP106
were synthesized in TnT rabbit reticulocyte lysates. Following
synthesis, proteins were incubated with preimmune or AL28 in
Tris-buffered saline containing 0.5 M NaCl and 0.1% (v/v) Triton X-100 at 4 °C. Immune complexes were collected by incubating the reactions with protein A-Sepharose (Amersham Biosciences). Beads
containing the immune complexes were washed extensively with binding
buffer, and bound proteins were eluted by boiling in SDS-PAGE sample
buffer. Samples were then analyzed with S-protein HRP and AL28 Western blots.
Expression of OIP106 and GRIF-1 cDNAs--
OIP106 and GRIF-1
were expressed in vitro in rabbit reticulocyte lysates using
the TnT in vitro transcription/translation system (Promega)
from either pcDNA 3.1 His or pCITE vectors. For HEK 293 cell
transfections, 4 µg each of OIP106 and GRIF-1 in their respective
pcDNA 3.1 His vectors were transfected using LipofectAMINE
2000 and expressed as Xpress-tagged fusion proteins. HEK 293 cell
transfections were carried out for 48 h, and harvested and
transfected cells were subjected to anti-Xpress Omniprobe antibody
immunoprecipitation as described under
"Immunoprecipitation."
Yeast Two-hybrid Screen to Identify Proteins That Interact with
O-GlcNAc Transferase--
To identify OGT-interacting proteins, ~ 7 × 106 clones (6-7-fold redundancy) were screened
from a rat brain two-hybrid cDNA library, and 250 positive yeast
colonies were isolated. We chose a rat brain library because OGT
protein levels and its enzymatic activity are high in brain (25).
Eighty of these colonies were screened for the presence of cDNA
inserts that were larger than 2 kb, in hopes of isolating full-length
cDNAs. Nine of these 80 clones encoded for cDNAs ranging in
size from 2.3 to 3.5 kb and were sequenced and identified. Four of the
nine clones contained a cDNA that encoded for a 774-amino acid
polypeptide, which was lacking its 5'-end. We performed 5'-rapid
amplification of cDNA ends using a rat brain cDNA library to
isolate the 5'-end and isolated a 900-bp fragment that encoded for an
additional 104 amino acids. Assembly of both cDNA fragments yielded
a ~3.4-kb cDNA fragment (GenBankTM accession number
AF474163) that encoded for a 878-amino acid polypeptide, with a
predicted molecular mass of 98 kDa, which we named OIP98 (for
OGT-interacting protein of
98 kDa). To ensure that the original interaction observed
in yeast was not a false positive, we repeated the yeast two-hybrid
assay using the original isolated partial OIP98 target cDNA
(AD-OIP98) fused to the activation domain of Gal4 and the OGT bait
cDNA (BD-OGT) fused to the binding domain of Gal4. As seen in Fig.
1A, only the yeast colonies
containing both the interacting OGT bait and the OIP98 target plasmids
were able to grow on plates lacking the histidine-selective marker, by
activation of the HIS3 reporter gene. Furthermore, this was confirmed by additional liquid phase assays where the OIP98-OGT interaction was quantified by the activation of the
At the time of its isolation, OIP98 did not bear any resemblance to any
known protein; thus, we considered it a novel protein. However, during
revision of this manuscript, homology searches performed in
GenBankTM revealed that OIP98 displayed 93% identity to a
rat 914-amino acid protein named GRIF-1 (GABAA
receptor-associated protein) (GenBankTM accession number
AJ288898) (52). GRIF-1 was found to interact with the
Further GenBankTM searches showed considerable sequence
identity (~40%) between GRIF-1 and another novel human gene of
unknown function, KIAA1042 (41) (GenBankTM accession number
AB028965). KIAA1042 encodes for a 953-amino acid polypeptide, with a
predicted molecular mass of 106 kDa (Fig. 2A) and is mapped
to 3p22.1.2 We expressed the
KIAA1042 and GRIF-1 cDNAs in a rabbit reticulocyte lysate system
and analyzed the [35S]Met-labeled protein products on a
7.5% SDS-PAGE, followed by autoradiography. Surprisingly, as shown in
Fig. 3A (left
panel), both KIAA1042 and GRIF-1 migrated similarly on
SDS-PAGE, at an apparent molecular mass of about 115 kDa. Whereas the
migratory pattern of GRIF-1 is consistent with the reported migration
(52), our observation that KIAA1042 migrated similarly was unexpected, since KIAA1042 is 40 amino acids larger than GRIF-1. In a separate reaction using pCITE vectors, translated GRIF-1 and KIAA1042 proteins were purified via S-tag affinity chromatography. As seen in Fig. 3A (middle panel), equal amounts of
S-tagged GRIF-1 and KIAA1042 were purified, as detected by S-protein
HRP blot. Affinity-purified anti-GRIF-1/OIP98 polyclonal antibody (JH
3286) cross-reacted well with both purified GRIF-1 and KIAA1042 (Fig.
3A, right panel). This is not
surprising, since KIAA1042 shares significant sequence identity with
GRIF-1 (Fig. 2). Thus, our GRIF-1 antibody JH 3286 reacts with both
KIAA1042 and GRIF-1.
We performed further JH 3286 Western blot analysis on whole
rat tissue and human HeLa cell lysates. Beck et al. (52)
have reported that GRIF-1 is only expressed in excitable tissue such as
brain, heart, and skeletal muscle, based on Western blot analysis with
a GRIF-1-specific antibody. Our JH 3286 antibody, however, reacted with
a ~115-kDa band in all rat tissues that were examined, as well as
HeLa cell lysates (Fig. 3B). In addition, JH 3286 reacted with a ~106-kDa band in heart, brain, lung, and smooth muscle tissue
(Fig. 3B, top asterisk). An additional
reactivity at ~98 kDa was observed in lung tissue (Fig.
3B, bottom asterisk). Thus, JH 3286 reacted with GRIF-1 in excitable tissue. The additional immunoreactivity and aberrant migratory patterns of GRIF-1 are consistent with the patterns reported by Beck et al. (52).
Since JH 3286 reacts with both KIAA1042 and GRIF-1, the reactivity
noticed in nonexcitable tissue as well as in HeLa cell lysates is
probably due to the presence of KIAA1042. Interestingly, the protein
species in liver and kidney migrated slightly differently from the
protein reactivities in the other tissues. This could be a result of
differential post-translational modifications that might occur in these
tissues. It is curious that the anti-GRIF-1 antibody generated against residues 8-633 of GRIF-1 by Beck et al. (52) did not react
with KIAA1042 in nonexcitable tissue, since both KIAA1042 and GRIF-1 share a high degree of homology in that region (Fig.
2A).
Currently, however, since JH 3286 recognizes both GRIF-1
and KIAA1042, we cannot distinguish between the two proteins in brain and heart tissue. In order to confirm KIAA1042 expression in HeLa cell
lysates, whole cell lysates were probed with a KIAA1042 N terminus
(N20; first 20 amino acids of human KIAA1042) specific antibody SAI1.
As seen in Fig. 3C, SAI1 specifically reacted with a
~115-kDa KIAA1042 band, consistent with the JH 3286 reactivity in
Fig. 3B (right panel). This reactivity
was competed away in the presence of the KIAA1042 N20 peptide antigen
(Fig. 3C, right panel), thus
confirming the identity of the band as KIAA1042. Further specificity of
SAI1 toward KIAA1042 was examined by probing recombinant Xpress-tagged
KIAA1042 and GRIF-1 with SAI1. As seen in Fig. 3D, the
Biochemical Confirmation of GRIF-1 and KIAA1042 Interaction with
OGT--
In order to confirm that the interactions observed in the
original yeast two-hybrid screen were valid and not false positives, we
performed a variety of in vitro and in vivo
protein-protein interaction assays. To show the physiological existence
of native GRIF-1-OGT and KIAA1042-OGT complexes from rat brain, we
performed co-immunoprecipitation (IP) experiments. GRIF-1 and KIAA1042
were immunoprecipitated with anti-GRIF-1 antibody (raised in chicken) from rat brain extracts. This antibody reacts with both GRIF-1 and
KIAA1042, similar to JH 3286. Immunoprecipitates were separated on a
10% SDS-PAGE and analyzed with JH 3286, anti-OGT AL28 antibody, anti-actin, and anti-tubulin antibodies. As seen in Fig.
4A, the JH 3286 Western blot
(top panel) shows that chicken anti-GRIF-1 antibody specifically immunoprecipitated GRIF-1/KIAA1042 from rat brain
extracts. Western blot analysis by anti-OGT AL28 antibody (second panel) clearly shows the presence of OGT
in the anti-GRIF-1 IP, indicating that OGT co-immunoprecipitated with
GRIF-1/KIAA1042. The absence of tubulin and actin (bottom
two panels, respectively), which are abundant
proteins in brain, in the anti-GRIF-1 IP demonstrate that the
interactions between OGT and GRIF-1/KIAA1042 are specific and not a
result of nonspecific binding.
To show that GRIF-1/KIAA1042 reciprocally co-immunoprecipitated with
OGT, OGT was immunoprecipitated with AL28 from rat brain extract. IPs
were performed in HBS (0.3 M NaCl) and high stringency radioimmune precipitation assay lysis buffer (containing 0.1% SDS).
Immunoprecipitates were washed and analyzed with AL28 and chicken
anti-GRIF-1. As is seen in Fig. 4B, AL28 immunoprecipitated OGT in both native and high stringency conditions, as analyzed by the
AL28 Western blot (top panel). Western blot
analysis with chicken anti-GRIF-1 (bottom panel)
clearly shows the presence of GRIF-1/KIAA1042 in OGT IPs but not in the
control preimmune IP, under native and high stringency binding conditions.
Since our anti-GRIF-1 antibodies do not distinguish between GRIF-1 and
KIAA1042, it was not clear which protein interacted with OGT. We
addressed this issue by transiently transfecting KIAA1042 and GRIF-1
cDNAs or vector alone (mock control) separately in HEK 293 cells as
Xpress-tagged fusion proteins. Xpress-tagged GRIF-1 and KIAA1042 were
immunoprecipitated using anti-Xpress Omniprobe antibody, and IPs were
analyzed by Omniprobe and AL28 Western blots. As seen in Fig.
4C, the Omniprobe blot (left panel) clearly shows the presence of immunoprecipitated Xpress-tagged GRIF-1
and KIAA1042 in the Omniprobe IPs. The AL28 Western blot (bottom panel) shows that endogenous OGT in HEK
293 cells specifically co-immunoprecipitated with Xpress-tagged GRIF-1
and KIAA1042 but not from mock-transfected lysates (bottom
panel, right lane). Therefore, OGT
interacts with both KIAA1042 and GRIF-1 individually, confirming the
previous rat brain native IP results.
These data clearly indicate that KIAA1042 and GRIF-1 interact with OGT
quite strongly (even in the presence of 0.1% SDS) and probably exist
in a complex in vivo, thus validating the interactions observed from the yeast two-hybrid experiments. Since we identified KIAA1042 as an O-GlcNAc transferase-binding protein, we
named it OIP106 (for OGT-interacting
protein of 106 kDa), based on its predicted molecular mass.
The TPR Domain of OGT Interacts with OIP106--
Since the OGT
contains a TPR domain at its amino terminus, which is a protein-protein
interaction domain, we hypothesized that the TPR domain may be the
region of OGT that interacted with GRIF-1 and OIP106. In addition, the
carboxyl terminus of OGT failed to interact with GRIF-1 (OIP98) in the
yeast two-hybrid system (Fig. 1B). To examine this
hypothesis, OGT and its individual domains were tested for interactions
with GRIF-1 and OIP106 using an in vitro blot overlay
interaction assay. Recombinant OGT, TPR, and C-termOGT (C)
proteins synthesized in E. coli were separated on SDS-PAGE and blotted onto polyvinylidene difluoride membranes. BSA was similarly
blotted to use as a negative control. Immobilized proteins were then
renatured en blot and probed with radioactively labeled GRIF-1 and OIP106. Blots were washed and then subjected to
autoradiography. As can be seen in Fig.
5, A and B, the TPR
domain strongly bound radiolabeled GRIF-1 and OIP106 (middle
panels). Similar binding was exhibited by the full-length
OGT. In contrast, no signal was observed in the C protein lanes or the
BSA control lanes, indicating that the binding was specific. Therefore,
this indicates that GRIF-1 and OIP106 interact with OGT via the TPR
domain.
We wanted to examine whether GRIF-1 and OIP106 bound OGT
stoichiometrically. S-tagged GRIF-1 and OIP106 were synthesized in rabbit reticulocyte lysates and pulled down by S-protein-agarose. Beads
were washed extensively in high salt buffer and analyzed by silver
staining. As is seen in Fig. 5C, S-tagged GRIF-1 and OIP106
were purified by S-protein-agarose (middle and
right lanes, respectively). The endogenous OGT
band, which migrates at ~110 kDa, is clearly seen in both GRIF-1 and
OIP106 pull-downs. AL28 Western blot had identified this co-purifying
band as OGT in preliminary experiments (data not shown). This band is
not present in the control S-protein-agarose pull-down lane
(left lane). Therefore, whereas it appears that
the binding of GRIF-1 and OIP106 to OGT appears to be stoichiometric,
quantification of relative amounts of the protein species here is not
applicable, since the response of silver staining varies from one
protein to another. The above experiments, in addition to the co-IP
experiments, confirmed the initial yeast two-hybrid studies and clearly
show that OIP106 and GRIF-1 are a novel family of highly homologous
proteins that interact strongly with OGT via its TPR repeats.
GRIF-1 and OIP106 Are Modified by O-GlcNAc--
PROSITE analysis
of GRIF-1 and OIP106 sequences revealed that the carboxyl halves of
both proteins contained many potential sites of O-GlcNAc
modification. Since both proteins interact strongly with OGT, we
examined the potential existence of O-GlcNAc on
immunoprecipitated Xpress-tagged GRIF-1 and OIP106 by probing with CTD
110.6 (51), which is an anti-O-GlcNAc-specific mouse
monoclonal antibody. As is seen in Fig. 4C
(middle panel), when probed in the absence of 50 mM GlcNAc, both GRIF-1 and OIP106 reacted strongly with CTD
110.6. This reactivity was competed away when probed in the presence of
50 mM GlcNAc (Fig. 4C, right
panel), indicating that the reactivity observed was due to
the presence of the O-GlcNAc modification and not due to
nonspecific binding of the antibody. This indicates that both GRIF-1
and OIP106 are modified by O-GlcNAc and are substrates for
OGT.
We wanted to further examine whether other substrates of OGT also
interacted with OGT in a similar manner. Nucleoporin p62 is a well
studied, high affinity substrate for OGT (13). We performed in
vitro binding experiments with p62 and looked for its ability to
stably interact with OGT. An identical experiment was performed with
OIP106 to serve as a positive control for OGT binding. OIP106 and p62
were synthesized as S-tagged proteins in reticulocyte lysates.
Following synthesis, lysates were incubated with anti-OGT AL28 antibody
to immunoprecipitate endogenous OGT, and IPs were assayed for
the presence of either OIP106 or p62 by S-protein HRP blot. As seen in
the left bottom panel of Fig. 6 (long exposure), the AL28 blot on AL28
IPs shows that the antibody immunoprecipitated OGT from lysates, as
expected. No OGT was immunoprecipitated by the preimmune IgG
(lane 4, bottom panel). The
S-protein HRP blot in Fig. 6 (long exposure; left
top panel) shows that OIP106 clearly
co-immunoprecipitated with OGT (lane 5) but not
with the preimmune IgG (lane 4). In contrast, no
p62 was detected in the AL28 IP (lane 2). The
short exposure panels on the
right show that the concentration of OIP106 used in the
input for the IPs was less than that of the amount of p62 used
(right top panel; compare
lane 1 with lane 3),
indicating that the binding observed are specific and not effects of
mass action. This indicates that p62 did not stably interact with OGT
in this system, but OIP106 clearly did, as consistent with previous
data.
We performed a similar experiment on rat brain extracts, which would
reflect an in vivo native system, and obtained the same result (data not shown). These results indicate that p62 does not
interact with OGT in a stable complex, but OIP106 and GRIF-1 do,
although all three proteins are substrates for OGT.
OIP106 Localizes to the Nucleus in HeLa Cells--
Since the
characterization of GRIF-1 and its subcellular localization have
already been reported by Beck et al. (52), we decided to
focus on OIP106. In order to more closely examine subcellular distribution of OIP106, we performed biochemical fractionation on HeLa
cells. HeLa cells were biochemically fractionated into nuclear and
cytosolic extracts, and equal amounts of each extract were analyzed by
JH 3286. As seen in Fig. 7A,
OIP106 was only detected in the nuclear (N) fraction (Fig.
7A, top panel). OGT, which is a
nucleocytoplasmic enzyme, is present in higher levels in the nucleus
and is shown as a control in the bottom
panel of Fig. 7A by AL28 Western blotting (25).
Similar results were obtained using the OIP106-specific SAI1 antibody
(data not shown). Thus, in HeLa cells, OIP106 is a nuclear protein.
OIP106 Co-localizes with RNA Polymerase II in Vivo--
We decided
to take advantage of the observation that endogenous OIP106 was
adequately detected in HeLa cells by JH 3286 (Fig. 7A) and
performed native OIP106 immunofluorescence with JH 3286 via
laser-scanning confocal microscopy. As seen in Fig. 7A,
panel a, OIP106 localized to distinct punctate
regions in the nucleus. Thus, the nuclear staining of OIP106 correlated
well with the biochemical fractionation in Fig. 7A. In order
to further examine OIP106's nuclear punctate localization, we
performed laser-scanning confocal microscopy co-staining for proteins
that are known to be present in these punctate regions. Recently, von
Mikecz et al. (44) showed that the IIA form of RNA
polymerase II exhibited a broad staining of the nonnucleolar portion of
the nucleus in Hep-2 cells when stained with the 8WG16 mouse monoclonal
antibody. The 8WG16 antibody is specific for the hypophosphorylated
form of polymerase II (IIA) (45), which has been previously shown to be
extensively O-GlcNAc-modified (7). We felt that this was a
suitable marker for the observed punctate staining and wanted to
examine whether OIP106 possibly co-localized with RNA polymerase IIA
in vivo. We performed co-staining of OIP106 and RNA
polymerase IIA using JH 3286 (from rabbit) and 8WG16 (from mouse) on
HeLa cells to determine their co-localization. As is seen in Fig.
7B, panels b and c, subsets
of OIP106 co-localized with RNA polymerase IIA in the distinct dotlike
regions, as evident in the merged image (panel
c). Fig. 7B, panel d, is an
enlarged image of the inset in panel
c, clearly showing the co-localized subsets of OIP106 and
RNA polymerase IIA in yellow. To further confirm this observation, we performed immunogold electron microscopy. This is shown
in Fig. 7C, panels a and b.
The large 18-nm colloidal gold particles represent OIP106 molecules,
and the smaller 12-nm particles represent RNA polymerase IIA. As seen
in the inset in Fig. 7C, panel
b, subsets of OIP106 co-localize with RNA polymerase IIA,
further supporting the confocal microscopy data.
OIP106 Exists in a Complex with RNA Polymerase II and
OGT--
Since OIP106 and RNA polymerase II co-localize (Fig. 7,
B and C), we wanted to biochemically confirm
whether the two proteins are present in a complex in vivo.
We performed co-IP experiments with the anti-GRIF-1 IgY (which reacts
well with OIP106) and immunoprecipitated OIP106 from HeLa nuclear
extracts. IPs were washed extensively and analyzed by JH 3286, AL28,
8WG16, and anti-Rb p107 Western blotting. As is seen in Fig.
7D, a subset of RNA polymerase II (second
panel, lane 3) co-immunoprecipitated
with OIP106 (top panel, lane
3) but not by the preimmune antibody (lane
2). The presence of OGT in the OIP106 IP, but not in the
preimmune, serves as a positive control for the IP (Fig. 7D,
third panel, lanes 3 and
2, respectively). Furthermore, Rb p107, which is a
nonrelated abundant nuclear protein, did not co-IP with OIP106
(bottom panel), demonstrating that the polymerase
II-OIP106 interactions are specific. Similar results were obtained with
the OIP106-specific SAI1 antibody (data not shown). These data support
the hypothesis that OIP106 exists in a complex with a subset of RNA
polymerase II and OGT, providing evidence for the possible targeting of
OGT by OIP106 to transcriptional complexes.
OGT Interacts with Many Proteins--
A myriad of different
nucleocytoplasmic proteins are modified by O-GlcNAc, yet
there appears to be only a single OGT catalytic subunit (25). The OGT
cDNA was cloned from rat (25) and human (26) 4 years ago.
Subsequent knockout of the OGT gene in mouse resulted in embryonic
lethality, at the single cell level (27). Mapping of the OGT gene in
humans and mice revealed it to lie on the X chromosome as a single copy
gene (27, 28). This clearly demonstrated that the OGT enzyme was
absolutely essential for life. However, virtually nothing is known
about the mechanisms regulating OGT protein specificity. The N-terminal
half of the OGT contains a TPR domain, which in rat OGT, is composed of
11.5 TPR repeats. Recently, contrary to earlier reports (26), it has
been shown that the human OGT has the same number of TPR repeats as the
rat enzyme (28). TPR domains have been shown to be responsible for
intra- and intermolecular protein-protein interactions in a variety of
proteins, spanning a variety of cellular functions (29, 30). Thus, in
order to identify putative binding proteins that might potentially
regulate the OGT's specificity or its subcellular localization, we
performed an unbiased yeast two-hybrid screen of a rat brain library
using the entire OGT protein as a bait. Our final round of screening
resulted in about 250 clones, which, with a ~6-7-fold
redundancy, we estimated to represent at least 30 unique clones.
Whereas we selected for clones that were larger than 2 kb in hopes that
we would find complete cDNAs, our PCR screening revealed that a
large number of the clones were less than 2 kb, indicating that these
either encoded for smaller proteins or were partial clones. Thus, it is
reasonable to estimate, even conservatively, that the number of
potential OGT-interacting proteins is quite substantial. Thus, for such
a single, unique enzyme to glycosylate a myriad of proteins, we
hypothesize that it is regulated by a large number of TPR-interacting proteins.
GRIF-1, OIP106, and OGT: Implications for Targeting of
OGT--
GRIF-1 and OIP106 interact with OGT quite strongly and
stoichiometrically. The interactions are resistant to high salt,
nonionic, and ionic detergents such as SDS. As is seen with the blot
overlay assays, these interactions occur in the TPR domain.
Interestingly, in HEK 293 cell transfection experiments, binding to OGT
by a FLAG-tagged OIP106 construct was not competed away when
co-transfected with increasing amounts of Xpress-tagged GRIF-1 (data
not shown), indicating that possibly GRIF-1 and OIP106 may bind to
different TPR repeats of OGT. Thus, mutually exclusive GRIF-1-OGT and
OIP106-OGT complexes could exist in the cell. This could help explain
how each protein differentially might affect OGT's localization.
GRIF-1 was recently isolated as a novel GABAA
receptor-interacting protein that is expressed only in excitable tissue
(52). The function of GRIF-1 is unknown. However, by virtue of its
association with OGT, we propose that perhaps it functions to target
OGT to GABAA receptor complexes. This would implicate
O-GlcNAc, OGT, and GRIF-1 as being involved in GABA
signaling. GRIF-1 would function as an adaptor/scaffolding protein,
bridging OGT to GABAA receptor in this model. Signaling
through such scaffolding/anchoring protein networks has been well
documented, especially for protein kinases such as protein kinase A and
its various A kinase-anchoring proteins and in
N-methyl-D-aspartate receptor signaling via PDZ
domain proteins (53). Interestingly, GRIF-1 contains several
PXXP motifs in its carboxyl terminus (Fig. 2).
PXXP motifs are known to bind to Src homology 3 domains
(53). The function of Src homology 3 domains in proteins that contain
it is to form functional oligomeric complexes at defined subcellular
sites, usually in concert with other modular domains. A canonical
example of such proteins is PSD-95, which contains both an Src homology
3 and a PDZ domain and has been shown to mediate
N-methyl-D-aspartate receptor signaling via
these domains (53). Thus, GRIF-1 may interact with Src homology 3 domain proteins via its PXXP motifs, recruiting OGT to these complexes, in the context of GABAA receptor signaling.
Furthermore, regulation of GABAA receptor via
phosphorylation has been well documented, and it is the large
intracellular cytoplasmic loops of the various receptor subunits
(
OIP106-OGT complexes occur in punctate regions within the nucleus.
These nuclear punctae have been shown to contain proteins involved in
splicing and transcription, including RNA polymerase II and CBP/p300
histone acetyltransferases (44). Our laboratory and others have shown
that most transcription factors that have been examined as well as
various proteins involved in splicing are heavily modified by
O-GlcNAc (46). The CTD of RNA polymerase II has been shown
to be extensively modified by O-GlcNAc (7), and it is the
IIA form of polymerase II (found in preinitiation complexes) that has
been shown to be glycosylated. Very recently, our laboratory has shown
that basal transcription factors, including the TFIID complex are also
extensively modified by O-GlcNAc (50). Thus, in our model of
OIP106 function with respect to OGT, the enzyme would be targeted to
sites of transcription by OIP106, to glycosylate proteins in the
preinitiation complex such as the IIA form of RNA polymerase II. Our
co-localization of OIP106 with RNA polymerase IIA, using both confocal
and electron microscopy and its interaction with polymerase II via
co-IP experiments, strongly supports to this model. Thus, based on our
data, we hypothesize that OIP106 and OGT may represent novel components
of preinitiation complexes. Our model in which preinitiation complexes
are glycosylated has been in existence for a number of years (1, 7,
46), and the discovery of OIP106's associations with OGT and RNA
polymerase IIA further supports this notion.
OIP106 and GRIF-1--
Both GRIF-1 and OIP106 contain very highly
identical coiled-coil domains. Coiled-coil domains are known to
be involved in homo- or heterodimerization. GRIF-1 has been shown to be
able to homodimerize via its coiled-coil domain (52). We therefore reasoned that GRIF-1 and OIP106 may be able to interact with each other, presumably via their respective coiled-coil domains. In preliminary in vitro pull-down experiments performed with
rabbit reticulocyte lysate synthesized proteins, GRIF-1 strongly bound to OIP106 (data not shown). Whether this interaction occurs
physiologically is unknown. However, this does raise the potential of
cross-talk between GRIF-1 and OIP106 to occur, possibly coupling GABA
receptor signaling to transcriptional events. GRIF-1 has been reported to be found in the nuclear fractions in excitable tissue (52), and this
notion would be consistent with that finding. Thus, the identification
of these novel proteins and their species-specific orthologs comprise a
novel family of OGT-interacting proteins that potentially regulate OGT
by influencing its targeting and subcellular localization.
-O-linked N-acetylglucosamine (O-GlcNAc) is catalyzed by O-GlcNAc transferase
(OGT). Here we used the yeast two-hybrid approach to identify and
isolate GABAA receptor-associated protein, GRIF-1 (Beck,
M., Brickley, K., Wilkinson, H. L., Sharma, S., Smith, M., Chazot,
P. L., Pollard, S., and Stephenson, F. A. (2002)
J. Biol. Chem. 277, 30079-30090), and its novel
homolog, OIP106 (KIAA1042), as novel OGT-interacting proteins. The
proteins are highly similar to each other but are encoded by two
separate genes. Both GRIF-1 and OIP106 contain coiled-coil domains and
interact with the tetratricopeptide repeats of OGT. GRIF-1 and OIP106
are modified by O-GlcNAc and therefore are substrates for
OGT. However, unlike another high affinity protein substrate, such as
nucleoporin p62, OIP106 and GRIF-1 co-immunoprecipitate with OGT,
exhibiting stable in vitro and in vivo
associations. Whereas GRIF-1 has been reported to be expressed only in
excitable tissue, OIP106 is expressed in all human cell lines that were
examined. Confocal and electron microscopy show that OIP106 localizes
to nuclear punctae in HeLa cells and co-localizes with RNA polymerase
II. Co-immunoprecipitation experiments confirm the presence
of an in vivo RNA polymerase II-OIP106-OGT complex, suggesting that OIP106 may target OGT to transcriptional complexes for
glycosylation of transcriptional proteins, such as RNA polymerase II, and transcription factors. Similarly, GRIF-1 may serve to target OGT to GABAA receptor complexes for mediating GABA
signaling cascades.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-O-linked
N-acetylglucosamine
(O-GlcNAc)1
monosaccharides is ubiquitous in multicellular eukaryotes (1-3). The
proteins modified by O-GlcNAc are myriad and diverse in form and function, ranging from transcription factors (4-6) to RNA polymerase II (7), oncoproteins (8), tumor suppressors (9), viral
proteins (10), cytoskeletal proteins (11, 12), kinases (13) and
phosphatases (14). Furthermore, O-GlcNAc-modified proteins
are phosphoproteins as well, often belonging to large multimeric and
reversible protein complexes. Indeed, in many cases, the sites of
O-GlcNAc modification are either the same or adjacent to
those modified by O-phosphate (15). Thus, there is mounting evidence supporting the hypothesis that O-GlcNAc is a
regulatory modification analogous to phosphorylation. For example, the
transcription factor Sp1 is extensively modified by
O-GlcNAc, and it has been shown that the presence of the
sugar in the transactivation domain inhibits its homomultimerization
and transcriptional capability (4, 5, 16). O-GlcNAc
modification of p67 regulates protein synthesis by controlling the
phosphorylation status of eukaryotic initiation factor-2
(17, 18).
In cases where the sites of modification of O-GlcNAc and
O-phosphate are the same, a reciprocal relationship between
the two modifications has been suggested (19, 20).
-N-acetylglucosaminyltransferase, or O-GlcNAc
transferase (OGT), and its counterpart is a
O-GlcNAc-specific
-N-acetylglucosaminidase
known as the O-GlcNAcase. Both of these enzymes have been
purified and characterized (21-26). Recently, the cDNAs that
encode for OGT (25, 26) and O-GlcNAcase (23), have been
cloned from rat, Caenorhabditis elegans, and human. The OGT
is a highly unique and ubiquitous glycosyltransferase, encoded by a
single gene. OGT is highly conserved throughout evolution from C. elegans to humans. The gene for OGT has been mapped to the X
chromosome (Xq13 in humans) (27, 28). Targeted deletion of the OGT gene
results in ES cell lethality in mice (27). Thus, OGT is essential for
life at the single cell level.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu/
Trp/
His/
Ade plates in the presence of 10 mM 3-aminotriazole, in yeast strain PJ69-4A. Three rounds of screening were performed, resulting in about 250 final positive clones. 80 of the resulting positives were screened for the presence of
the AD fusion inserts using pACT2-specific primers via a yeast colony
PCR approach as described (34). Nine inserts greater than 2 kb were
selected for DNA sequencing, and selected plasmid DNAs were recovered
by performing yeast plasmid preparations as described previously (34).
Finally, confirmation of the originally observed interactions was
performed by retransforming yeast with pJL59-OGT bait or empty pJL59
and pACT2-prey or empty pACT2 vectors and plated on
SD/
Leu/
Trp/
His/
Ade plates containing 30 mM
3-aminotriazole. The carboxyl terminus of OGT fused to the BD of Gal4
was used as the negative control bait vector. Liquid
-galactosidase
assays were performed as described (34).
20 °C. Recombinant hexa-His-tagged thioredoxin-OGT,
C-termOGT, and GRIF-1 (OIP98) fusion protein expression was
induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside for 4 h at
37 °C. Cells were harvested, and His-tagged proteins were purified under denaturing conditions via nickel affinity chromatography using
HiTrap chelating columns (Amersham Biosciences) according to the
manufacturer's instructions. His-tagged C-termOGT
(pRSET-C) was expressed and purified in a similar manner for use as an
antigen to raise anti-OGT polyclonal antibodies generated in rabbits.
Soluble thioredoxin-tagged TPR (pET32-TPR) was overexpressed and
purified under native, nondenaturing conditions via nickel affinity chromatography.
80 °C. Typical
exposure times were 8-24 h.
-tubulin antibodies.
80 °C. Fractionation of nuclear and cytoplasmic extracts from HeLa cells was performed as described (38).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase reporter gene (Fig. 1B). We performed an additional control
by testing the interaction of the putative catalytic domain of OGT (BD-C termOGT) with OIP98. As can be seen in Fig.
1B, the C terminus of OGT did not interact with OIP98,
indicating that the amino-terminal half of OGT containing the TPR
domain may be the region of interaction with OIP98.
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Fig. 1.
Isolation and identification of GRIF-1
(OIP98) as a novel OGT-interacting protein using the yeast interaction
trap approach. A, OGT was fused to the BD of GAL4
(pJL59-OGT) and used as bait to screen a rat brain GAL4 AD cDNA
fusion library for interacting proteins. Yeast strain PJ69 was
co-transformed with pJL59-OGT and the Matchmaker rat brain AD fusion
library (Clontech) and plated onto
Leu/
Trp plates.
Leu/
Trp transformants were plated onto
Leu/
Trp/
His/
Ade 30 mM 3-aminotriazole plates. Only
colonies containing both interacting bait and target
proteins survive and grow on
Leu/
Trp/
His/
Ade 30 mM
3-aminotriazole plates. B,
-galactosidase reporter assays
show the interaction of BD-OGT and AD-OIP98.
2
subunit of the GABAA receptor and was isolated from a yeast
two-hybrid screen. Rat GRIF-1 is the rat ortholog of human ALS2CR3
(40). Interestingly, sequence comparison between OIP98 and GRIF-1
revealed that the region of difference between the two proteins was
from residue 620 (in GRIF-1 and OIP98) to 688 (in GRIF-1; 653 in OIP98)
(positions 644-712 in Fig.
2A). This region corresponds
to exon 15 in human ALS2CR3/GRIF-1. Thus, OIP98 is the GRIF-1 cDNA
product but lacking exon 15; therefore, it is a partial clone of
GRIF-1. We cloned the missing exon by PCR, using GRIF-1 gene-specific
primers that flanked exon 15, from a rat brain cDNA library
template (see "Experimental Procedures"). Interestingly, sequence
comparison of our assembled GRIF-1/OIP98 to the published GRIF-1
sequence (52) revealed three major differences in the predicted
sequence (52). These are Leu579 (our GRIF-1) to
Trp579 (52), Thr595 (ours) to
Ser595 (52), and Gln596 (ours) to
Glu596 (52) (Fig. 2B). These differences
were not found when we compared our cloned GRIF-1 sequence to the human
GRIF-1 (ALS2CR3) (Fig. 2B). Similar results were found when
exon 15 was cloned from a Matchmaker (Clontech) rat
brain cDNA library (data not shown).
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Fig. 2.
A, ClustalW alignment of human
OIP106/KIAA1042, rat GRIF-1 (QuickClone), rat GRIF-1 (52),
human ALS2CR3 and rat OIP98. Exon 15 (and part of exon 16) in GRIF-1,
the region missing in OIP98, is bracketed. B,
differences between cloned GRIF-1 (QuickClone), published
GRIF-1 (52), human ALS2CR3, and OIP98. Differences in the sequences are
indicated by the arrows. These residues are conserved in our
cloned GRIF-1 (and OIP98) as well as the published human ALS2CR3 but
are different from the published GRIF-1 sequence (52).
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Fig. 3.
A, in vitro expression and JH
3286 analysis of GRIF-1 and KIAA1042/OIP106 in a mammalian expression
system. GRIF-1 and KIAA1042/OIP106 cDNAs were expressed in in
vitro transcription and translation reactions in rabbit
reticulocyte lysates and synthesized as [35S]Met-labeled
proteins. Following the reactions, the lysates were analyzed by 7.5%
SDS-PAGE and autoradiography (left panel). In a
separate reaction, expressed proteins were purified via their S-tags
and analyzed by S-protein HRP (middle
panel) and JH 3286 Western blot (right
panel). JH 3286 reacted equally well with both GRIF-1 and
KIAA1042/OIP106. B, JH 3286 Western blot analysis of GRIF-1
and KIAA1042/OIP106. JH 3286 reactivity is observed in all tissues
examined. H, heart; B, brain; S,
spleen; L, lung; Li, liver; Sm, smooth
muscle; K, kidney; Te, testis; Thy,
thymus; HeLa, HeLa whole cell extracts. The top
and bottom asterisks indicate the ~106- and
~98-kDa proteolytic fragments, respectively. C, SAI1
antibody is specific for KIAA1042/OIP106. HeLa whole cell extract was
analyzed by SAI1 antibody in the presence (right
panel) or absence (left panel) of
KIAA1042 N20 peptide antigen. SAI1 reactivity is competed away in the
presence of N20 peptide, indicating that SAI1 specifically reacts with
KIAA1042/OIP106 in HeLa cells. The numbers to the
left represent molecular weight marker positions.
D, SAI1 is specific for KIAA1042/OIP106 and not GRIF-1.
Purified recombinant Xpress-tagged GRIF-1 and KIAA1042/OIP106 were
probed with anti-Xpress Omniprobe antibody (right
panel) and SAI1 (left panel). SAI1
only reacted with KIAA1042/OIP106 and not GRIF-1, indicating that it is
specific for GRIF-1. E, KIAA1042/OIP106 is expressed in all
human cell lines that were examined. Cruz-Blot B (Santa Cruz
Biotechnology) containing 50 µg each of extracts from various human
cell lines (as indicated at the top) was probed with SAI1
and -tubulin antibodies. The top two
panels represent short and long exposures (respectively) of
the SAI1 blot, and the bottom panel shows the
-tubulin protein loading control blot (Con). As is seen
in the SAI1 blots, KIAA1042/OIP106 reactivity was observed in all human
cell line extracts that were examined.
-Xpress blot (right panel) shows that
recombinant purified Xpress-tagged GRIF-1 or KIAA1042 was
immunopurified by
-Xpress Omniprobe antibody from reticulocyte
lysates expressing the cDNA for each protein. However, the SAI1
blot (Fig. 3D, left panel) clearly
shows that only recombinant KIAA1042, and not GRIF-1, reacted with
SAI1, indicating that SAI1 is specific for KIAA1042. Since we now had
an KIAA1042-specific antibody with SAI1, we wanted to confirm its
expression in human tissue, since SAI1 is specific for human KIAA1042.
However, since human tissue was not easy to obtain, we decided to
examine the expression of KIAA1042 on a blot containing cell lysates
from a diverse range of human cell lines containing both excitable
(IMR-32) and nonexcitable (HL-60, HeLa, etc.) cell lines. As seen in
Fig. 3E, SAI1 signal was observed in every tissue, as seen
in both short and long exposures (top and middle
panels, respectively). The
-tubulin blot (Fig.
3E, bottom panel) shows the protein
loading control. Thus, KIAA1042 expression was noticed in all cell
types that were examined. This is consistent with its reported mRNA
expression.3
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Fig. 4.
A, OGT co-immunoprecipitates with
KIAA1042(OIP106)/GRIF-1 from rat brain extracts in a reciprocal manner.
KIAA1042(OIP106)/GRIF-1 were immunoprecipitated with
affinity-purified chicken anti-GRIF-1 polyclonal antibody, separated on
10% SDS-PAGE, and blotted with affinity-purified JH 3286, anti-OGT,
anti-tubulin, and anti-actin. B, KIAA1042(OIP106)/GRIF-1
co-immunoprecipitate with OGT from rat brain extracts in a reciprocal
manner. OGT was immunoprecipitated with affinity-purified AL28 anti-OGT
antibody and blotted with affinity-purified anti-OGT (AL28) and JH
3286. C, OGT co-immunoprecipitates with overexpressed GRIF-1
and KIAA1042/OIP106. GRIF-1 and KIAA1042/OIP106 were separately
expressed in HEK 293 cells as Xpress-tagged proteins. Transfected cell
lysates were subjected to anti-Xpress Omniprobe immunoprecipitation to
IP Xpress-tagged proteins. Omniprobe IPs were then analyzed by
Omniprobe (top left panel) and
anti-OGT AL28 (bottom left panel).
GRIF-1 and OIP106 are modified by O-GlcNAc. Omniprobe IPs were probed
by anti-O-GlcNAc antibody CTD 110.6, either in the absence
(middle panel) or presence of 50 mM
GlcNAc. As is seen in the middle panel, CTD 110.6 reacted well with both GRIF-1 and OIP106. The CTD 110.6 reactivity is
competed away by probing in the presence of 50 mM GlcNAc
(right panel), indicating that the reactivity
observed is due to the O-GlcNAc modification and not due to
nonspecific binding.
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Fig. 5.
GRIF-1 and OIP106 interact with the TPR
domain of OGT. Recombinant full-length OGT, TPR domain, and the
C-termOGT (C) or BSA ( ) control were blotted
on polyvinylidene difluoride, renatured en blot, and probed
with 35S-labeled GRIF-1 (A) or OIP106
(B) synthesized in vitro in rabbit reticulocyte
lysates. Following probing, the blots were washed extensively, dried,
and subjected to autoradiography. C, GRIF-1 and OIP106
binding to OGT appears to be stoichiometric. S-tagged GRIF-1 and OIP106
were synthesized in vitro in rabbit reticulocyte lysates and
purified by S-protein-agarose. Beads were washed extensively and
analyzed by SDS-PAGE and silver stain.
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Fig. 6.
Other high affinity substrates such as
nucleoporin p62 do not interact with OGT. Nucleoporin p62 and
OIP106 were synthesized in vitro in rabbit reticulocyte
lysates as S-tagged proteins and incubated with anti-OGT antibody AL28
to immunoprecipitate endogenous OGT. Immune complexes were washed, and
bound proteins were analyzed via S-protein HRP and anti-OGT Western
blots. The right and left panels
represent short and long exposures of the same blots. The
top panel shows that S-tagged OIP106
co-immunoprecipitates with OGT (lane 5), but p62
does not (lane 2). Anti-OGT Western blot in the
bottom panel shows that equal amounts of OGT were
immunoprecipitated by anti-OGT in each IP.
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Fig. 7.
Subcellular localization of OIP106 in HeLa
cells. A, HeLa cells were biochemically fractionated
into nuclear (N) and cytoplasmic (C) extracts and
immunoblotted with JH 3286 or anti-OGT AL28 (control). Native OIP106
immunofluorescence in HeLa cells. B, OIP106 co-localizes
with RNA polymerase IIA in vivo via confocal microscopy.
HeLa cells were stained with affinity-purified JH 3286 alone
(a) or co-stained with anti-hypophosphorylated polymerase II
(IIA) 8WG16 antibodies (b). Specimens were visualized with a
Bio-Rad MRC-1042 laser-scanning confocal microscope. The compound image
(c) shows their co-localization in the nucleus. The
inset (d) shows the co-localization of the two
proteins in yellow. C, OIP106 and RNA polymerase
IIA co-localize in vivo via EM. a, HeLa cells
were fixed and stained with affinity-purified JH 3286 and 8WG16
antibodies. Staining was observed using a JEM 1010 electron microscope.
The inset (b) shows the co-localization of both
polymerase IIA and OIP106 via their differently sized colloidal gold
particles. D, RNA polymerase II co-immunoprecipitates with
OIP106. OIP106 immune complexes were immunoprecipitated from HeLa
nuclear extracts with chicken anti-OIP106 IgY or preimmune IgY.
Immunoprecipitates were separated on 7.5% SDS-PAGE and analyzed with
JH 3286 (top panel), 8WG16 (anti-polymerase II;
second panel), AL28 (anti-OGT; third
panel), and anti-Rb p107 (control)
(bottom panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
) that have been shown to be substrates of various
kinases (protein kinase A, protein kinase C,
Ca2+/calmodulin-dependent protein kinase II)
(42). GRIF-1 interacts with the intracellular loop of the
2 receptor subunit, which is phosphorylated at
Ser410 (42). Thus, OGT could be recruited to this domain
for its potential O-GlcNAc modification and for potential
O-GlcNAc modification of associated scaffolding proteins.
O-GlcNAc and O-phosphate modifications often
occur on the same or adjacent Ser/Thr residues (15), so potential
O-GlcNAc modification of GABAA receptor
(complexes) may be a way of regulating its function. GRIF-1 would
mediate this regulation by targeting OGT to these complexes.
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ACKNOWLEDGEMENTS |
---|
We thank Katie Sackstedter, Brian Geisbrecht, and Dr. Steve Gould for the pJL59 vector and helpful advice with the yeast two-hybrid screen. We thank Dr. Peter Agre for the PJ69-4A yeast strain and Dr. Natasha Zachara for careful reading of the manuscript.
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FOOTNOTES |
---|
* 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/EBI Data Bank with accession number(s) AF474163.
To whom correspondence should be addressed: Dept. of
Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-614-5993; Fax: 410-614-8804; E-mail: gwhart@jhmi.edu.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209384200
2 Available on the World Wide Web at genome.ucsc.edu/cgi-bin/ hgTracks?position=chr3:48370736-48542321&hgsid=223215.
3 Available on the World Wide Web at www.kazusa.or.jp/huge/ gfpage/KIAA1042/.
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ABBREVIATIONS |
---|
The abbreviations used are:
O-GlcNAc, -O-linked N-acetylglucosamine;
OGT, uridine
diphospho-N-acetylglucosamine:polypeptide
-N-acetylglucosaminyltransferase;
O-GlcNAcase, N-acetyl-
-D-glucosaminidase;
TPR, tetratricopeptide repeat;
HBS, Hepes-buffered saline;
PBS, phosphate-buffered saline;
CTD, carboxyl-terminal domain;
IP, immunoprecipitation;
SD, synthetic dropout medium;
AD, activation
domain;
BD, DNA binding domain;
HRP, horseradish peroxidase;
OIP, OGT-interacting protein;
BSA, bovine serum albumin.
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