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-Chain1
*
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain gene.
Finally, the cytoplasmic 98-kDa form enters the proteasome pathway and
undergoes degradation. In conclusion, different forms of Elf-1 are the
products of posttranslational modifications that determine its
subcellular localization, activity, and metabolic
degradation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (2, 3), IL-2R 
-chain
(4), and CD4 (5)); and cytokines (GM-CSF
(6) and IL-2 (7)). The contribution of Elf-1
to the transcriptional regulation of each gene may be variable. In the
case of the TCR -chain gene, point mutations, introduced into the
two Elf-1 binding sites (-147/-119 and -66/-33) of the TCR
-chain promoter, abolish the basal transcriptional activity of the
-chain promoter in Jurkat cells (2, 3). The Elf-1 cDNA (GenBank accession no. P32519) contains an open reading frame of 1,857 nt and therefore encodes a protein of 619 aa with an expected molecular mass of 68 kDa. However, most of the published work indicates that Elf-1 exists as a protein with an apparent molecular mass of 98 kDa, although 80-kDa and 68-kDa forms have also been mentioned (1, 8). Interestingly, this discrepancy in the apparent molecular mass is not limited to Elf-1 and characterizes other Ets family members. Nerf, an Ets family-related protein, has a calculated molecular mass of 58 kDa, but it migrates as a 69-kDa protein (1). Proteins that contain a high ratio of either positively or negatively charged amino acids are known to display discrepancies between the estimated molecular mass and that observed by SDS gel electrophoresis. However, Elf-1 does not contain a high ratio of charged amino acids, suggesting that charge alone cannot be responsible for the dichotomy between the predicted and observed molecular mass. Proteins undergo one or even multiple types of modifications after translation, which are responsible for the molecular mass added to that calculated from the gene structure. Although phosphorylation is one of the most common posttranslational modifications of proteins, acetylation, sulfation, and glycosylation are also involved (9). The posttranslational modification of a protein is crucial for the expression of its functional aspects and, therefore, it is under strict regulation. Whereas phosphorylation and dephosphorylation have been well recognized to determine the activity of proteins, O-linked glycosylation has been recently recognized to participate in the modification of an increasing number of transcription factors and nuclear proteins (9, 10). The fact that the gene structure of Elf-1 encodes for multiple motifs that have the potential to undergo phosphorylation and glycosylation suggests that such modifications should represent an integral part of the biochemistry and biology of the Elf-1 protein.
Therefore, we have hypothesized that one or multiple posttranslational modifications of Elf-1 are responsible for the large gap between the observed and predicted molecular mass. Our experiments show that both protein kinase C (PKC)-mediated phosphorylation and O-N-acetylglucosamine (GlcNAc) glycosylation are responsible for the formation of the 98-kDa form of Elf-1. We found that these modifications are important in determining the subcellular localization, metabolism, and DNA binding ability of Elf-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The isolation and purification of primary T lymphocytes and culture of Jurkat cells have been recently described (11, 12).
Plasmids
The expression plasmid of Elf-1 was a kind gift of Dr. J. Leiden (Harvard University, Cambridge, MA). The PKC expression plasmids were generously provided by Dr. G. Baier (University of Innsbruck, Innsbruck, Austria).
Reagents
Okadaic acid and bacterial phage 
phosphatase was purchased
from Calbiochem (La Jolla, CA) and New England Biolabs (Beverly, MA),
respectively. Kinase inhibitors PD98059, SB203580, and KT5720 were
purchased from Calbiochem. Proteasome inhibitor LLnL
(N-Ac-Leu-Leu-norleucinal) is a product of Sigma-Aldrich
(St. Louis, MO). The anti-Elf-1 Ab was a kind gift of Dr. J.
Leiden. Anti-Elf-1 Ab was also purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). The other Abs were purchased from various
manufacturers as follows: O-GlcNAc (RL-2) Ab from
Affinity BioReagents (Golden, CO), PKC-
Ab from BD Transduction
Laboratories (Franklin Lakes, NJ), actin and Fli-1 Abs from Santa Cruz
Biotechnology, phosphoserine Ab from Sigma-Aldrich, retinoblastoma (Rb)
Ab from BD PharMingen (San Diego, CA), and p-CREB Ab from
Upstate Biotechnology (Lake Placid, NY).
Protein purification and Western blotting
To prepare cytoplasmic extracts, cells (5 x 106) were incubated on ice in 400 µl of lysis buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA supplemented with freshly added 1 mM DTT, 0.5 mM PMSF, 2 mM aprotinin, 1 mM of leupeptin, 10 mM NaF, and 2 mM Na3VO4) for 15 min. At the end of the incubation, Nonidet P-40 was added to the reaction mixture at a concentration of 0.6%. The reaction mixture was then subjected to centrifugation at 13,000 rpm for 1 min, and the supernatant was saved as cytoplasmic extract. The pellet was first washed once with buffer A to remove the residual cytoplasmic proteins, then resuspended in 40 µl of buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA supplemented with 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 mM aprotinin, and 1 mM of leupeptin) and vortexed in the cold room for 20 min. The supernatant was used as nuclear extract. To prepare whole cellular extract, cells were lysed in buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40 and supplemented with 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 mM aprotinin, and 1 mM of leupeptin on ice for 30 min. After centrifugation, the supernatant was saved as whole cellular extract. Western blotting was performed by following the manufacturer’s instruction (ECL; Amersham, Piscataway, NJ).
Transfection of primary human lymphocytes
The electroporation methods used for the transfection of Jurkat and primary T lymphocytes recently have been described (11, 12, 13, 14).
Immunoprecipitation
Nuclear proteins (150 µg) were precleaned in the Kyriaki’s
buffer (20 mM HEPES (pH 7.4), 50 mM 
-glycerophosphate, 2 mM EGTA, 10
mM NaF, 1%Triton X-100, 10% glycerol supplemented with 1 mM
Na3VO4,1 mM PMSF, 2 mM
aprotinin, and 1 mM leupeptin) and 20 µl of protein A agarose beads
at 4°C for 1 h. The reaction mixture was spun, and the
supernatant was incubated with the protein agarose beads and specific
Ab for another hour. The resulting beads were thoroughly washed with
the same buffer five times. The beads were then resuspended in the SDS
gel-loading buffer and boiled for 3 min, and the supernatant was
subjected to SDS-gel electrophoresis and Western blotting.
EMSAs
Nuclear extracts (4 µg) were incubated with a radiolabeled probe (5'-TCGAGAACCTCCAGGGCTTCCTGCCTGTGAACCA-3') and 1 µg of poly(dI:dC) in the binding buffer (20 mM Tris-HCl (pH 7.5), 50 mM KCl, 2.5 mM DTT, 0.5 mM EDTA, and 7.5% glycerol) for 15 min at room temperature. The reaction mixture was then subjected to separation in 6% nondenaturing gel (Invitrogen, Carlsbad, CA). The dried gel was then autoradiographed. For supershift assays, the nuclear proteins were incubated with specific Abs at room temperature for 30 min before the probe, poly(dI:dC), and binding buffer were added. The reaction was further conducted for another 15 min at room temperature.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first transfected primary human T and Jurkat cells, using a
cell electroporation technique (11, 12, 13, 14), either with the
plasmid-encoding cDNA (pcDNA)/Elf-1 plasmid, which encodes the
full-length Elf-1 cDNA, or with the empty plasmid to establish the
protein products of the Elf-1 gene. The cytoplasmic extracts from
primary (Fig. 1
a) and Jurkat T
cells (data not shown) transfected with the empty plasmid expressed the
80-kDa protein and barely any detectable levels of the 98-kDa form of
Elf-1. Transfection of T cells with the pcDNA/Elf-1 vector resulted in
enhanced expression of both 80- and 98-kDa bands, and the intensity of
the 80-kDa band was 10 times higher than that of the 98-kDa band. These
data suggest that both the 80- and 98-kDa forms of Elf-1 are directly
related to the plasmid-encoded protein and that they do not represent
products of differential mRNA splicing or other proteins homologous to
Elf-1, which may have been recognized by the Ab used.
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b). To ensure the
purity of the cytoplasmic and nuclear preparations, we probed
cytoplasmic and nuclear proteins from either Jurkat or primary T cells
sequentially with Abs against p-CREB and PKC-, two
proteins known to reside in the nuclear and cytoplasmic compartments,
respectively. As shown in Fig. 1b, PKC- colocalized with
the 80-kDa Elf-1 in the cytoplasm, whereas p-CREB did so
with the 98-kDa form of Elf-1 in the nucleus. These data proved that
the preparations of cytoplasmic and nuclear proteins were not
cross-contaminated and that the 80- and 98-kDa forms of the Elf-1
displayed differential subcellular compartment localization.
It has been reported that the Rb protein retains Elf-1 in the cytoplasm
and that, upon its phosphorylation, Elf-1 is free to translocate to the
nucleus (15). To determine which form of Elf-1 binds to
Rb, we immunoprecipitated nuclear extracts from T cells with Abs
against the Rb and Elf-1 proteins. The anti-Elf-1 Ab
immunoprecipitated both the 98- and 80-kDa bands (Fig. 1c)
at a ratio (30:1) comparable to the input of nuclear proteins (see Fig. 1b). In contrast, the anti-Rb Ab immunoprecipitated 98-
and 80-kDa Elf-1 at a ratio of 3:1, which is lower than that of the
input of nuclear proteins, suggesting that Rb interacts better with the
80-kDa form than with the 98-kDa form. Therefore, the conversion of
Elf-1 from its 80-kDa form to the 98-kDa form decreases its interaction
with Rb and contributes to its nuclear migration.
PKC is involved in the phosphorylation of Elf-1
Because PKC- recently has been shown to be involved in the
phosphorylation of several transcription factors involved in the
regulation of the expression of T lymphocyte genes such as AP-1
(16), NF-
B (17), and CREB
(12), we considered that similar PKC isoforms may also be
involved in the phosphorylation of Elf-1 and may be responsible for the
increase in the apparent molecular mass of Elf-1. In addition, sequence
analysis of Elf-1, using the Expert Protein Analysis System proteomics
server from the Swiss Institute of Bioinformatics, revealed the
existence of multiple PKC phosphorylation sites. As shown in Fig. 2a, Jurkat cells cultured in
the presence of PMA and calphostin C, which inhibits all the activities
of PKC isoforms, displayed diminished expression of 98-kDa Elf-1.
Similar results were obtained in the absence of PMA (see below). The
PKC- inhibitor rotterlin also decreased the expression of 98-kDa
Elf-1, although not to the same extent as calphostin C, suggesting that
more than one isoform of PKC is involved in the conversion of the
80-kDa to the 98-kDa form. Interestingly, treatment of Jurkat cells
with calphostin C enhanced the expression of 80-kDa Elf-1, indicating
that the 98-kDa form can be derived directly from the 80-kDa form.
Incubation of Jurkat cells with inhibitors of other kinases, such as
PD98059 (mitogen-activated protein kinase), SB203580 (P38;
stress-activated protein kinase), and KT5720 (protein kinase A), did
not alter the expression of either the 98- or 80-kDa forms of Elf-1
(Fig. 2a). Furthermore, calphostin displays similar effects
on the conversion of Elf-1 from 98- to 80-kDa form in the presence
(Fig. 2a) or absence of PMA (see below).
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b, Jurkat cells transfected with the empty vector
expressed only the 80-kDa Elf-1 in the cytoplasmic extracts. Transient
expression of either PKC- or PKC-
increased the expression of the
98-kDa form, whereas overexpression of PKC- did not affect the
relative levels of 80- and 98-kDa forms of Elf-1. These data show that
the so-called novel PKC isoforms and , but not the conventional
PKC isoform , are involved in the posttranslational modification of
Elf-1.
The fact that the levels of 98-kDa Elf-1 increase after overexpression
of PKC suggests that PKC-mediated phosphorylation is involved in the
formation of the 98-kDa form of Elf-1. To confirm this hypothesis, we
treated Jurkat T cells with different doses of okadaic acid, an
inhibitor of Ser and Thr phosphatases. To determine whether
phosphorylation of multiple PKC Elf-1 phosphorylation sites occurs
gradually, we separated the proteins in 6% instead of 10% SDS gels
(Fig. 2c). The 98-kDa Elf-1 resolved into three bands, and
the 80-kDa Elf-1 resolved into two bands. Treatment of the cells with
okadaic acid enhanced the expression of all three 98-kDa bands in a
dose-dependent (100–500 nM) manner, whereas it minimally enhanced the
intensity of the upper 80-kDa Elf-1 band. These data indicate that in T
cells cultured in FCS, in the absence of other exogenous stimuli, the
98-kDa form and only a small portion of the 80-kDa form of Elf-1 are
phosphorylated.
To further investigate the role of phosphorylation in the expression of
the Elf-1 forms, we performed two additional sets of experiments.
First, we isolated cell lysates in the presence or absence of the
phosphatase inhibitors NaF and
Na3VO4, and we noted that
the absence of these inhibitors results in decreased expression of the
98-kDa but not the 80-kDa Elf-1 form (Fig. 2d). When the
cellular extracts, purified in the absence of phosphatase inhibitors,
were incubated at 37°C for 18 h in the absence of phosphatase
inhibitors, the 98-kDa form almost disappeared, whereas in the presence
of the inhibitors, its disappearance was prevented (data not shown).
Second, we treated total cellular proteins with the bacteriophage
phosphatase. The bands that displayed enhanced intensity after
treatment with okadaic acid decreased in intensity after treatment with
the bacteriophage phosphatase (Fig. 2e). Altogether,
these experiments indicate that the 98-kDa Elf-1 is heavily
phosphorylated and undergoes dephosphorylation in a dynamic manner.
The 80- and 98-kDa forms of Elf-1 are O-GlcNAc glycosylated
Because the okadaic acid-induced hyperphosphorylation of Elf-1
(Fig. 2c) cannot account for the shift in the migration of
Elf-1, we considered that glycosylation contributes to the appearance
of the 98-kDa form of Elf-1. Elf-1 has multiple potential sites for
O-GlcNAc glycosylation, and therefore we investigated
whether Elf-1 is also O-GlcNAc modified. We first
immunoprecipitated nuclear proteins either with an Ab against Elf-1 or
with an Ab (RL-2) against the O-GlcNAc moiety (15, 18, 19) and then blotted the separated immunoprecipitates with the
reverse Ab. Both processes revealed a 98-kDa band that is
O-GlcNAc glycosylated (Fig. 3a). Because the nuclear
proteins contain mainly the 98-kDa form and a barely detectable 80-kDa
form, we repeated the above immunoprecipitation experiments using whole
cell extracts. The whole cellular extract contains comparable amounts
of both 98- and 80-kDa forms and therefore will help to further
determine whether the 80-kDa Elf-1 is also glycosylated. Fig. 3b shows that the 80-kDa form is also recognized by the RL-2
Ab, and therefore it is also decorated with GlcNAc moieties. However,
the interaction between RL-2 Ab and the 98-kDa form is more than eight
times stronger than that between RL-2 and the 80-kDa form, as
determined by densitometry. These experiments indicate that the
interactions among the different forms of Elf-1 and RL-2 are
specifically related to the extent of glycosylation and indicate that
the glycosylation contributes to the difference in the apparent
molecular mass between the 98- and 80-kDa forms.
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-chain promoter is both
glycosylated and phosphorylated
To determine the functional consequences of both the
phosphorylation and glycosylation on Elf-1, we performed EMSA analysis
using nuclear proteins from primary and Jurkat T cells and a labeled
oligonucleotide spanning the Elf-1 binding site (-147/-119) of the
TCR -chain promoter. We observed two major bands resulting from the
binding of nuclear proteins to the Elf-1 oligonucleotide (Fig. 4a). The upper band represents
specific binding because its intensity decreases dramatically in the
presence of an anti-Elf-1 Ab. In contrast, the incubation of
nuclear proteins with Abs against Fli-1 (a member of the Ets family) or
Rb did not affect the binding patterns. Significantly, the presence of
the anti-O-GlcNAc Ab, RL-2, in the EMSA reaction
completely prevented the formation of the upper band, providing
additional evidence that the Elf-1 protein that binds to the TCR
-chain promoter contains O-GlcNAc moieties (Fig. 4a).
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b, lower
panel). Pretreatment of cells with calphostin C decreased Elf-1
binding (Fig. 4b). To further prove that the 98-kDa Elf-1
represents a phosphorylated form of Elf-1, we incubated nuclear
extracts from Jurkat cells with an anti-phosphoserine Ab. This Ab
prevented the formation of the upper band, but it did not affect the
lower band (data not shown). Also, we dialyzed the whole cellular
extracts from cells treated with okadaic acid (Fig. 2c) to
remove the detergent and incubated them with the Elf-1 oligonucleotide.
Okadaic acid enhanced the binding of the cellular proteins to the
-chain promoter-defined Elf-1 site in a dose-dependent manner (Fig. 4c). Taken together, these data indicate that both
glycosylation and phosphorylation are required for Elf-1 to bind the
Elf-1 site on the TCR -chain promoter.
The expression levels of the 98-kDa form, but not the 80-kDa form,
of Elf-1 in T cells correlate with the binding to the Elf-1 site on the
TCR -chain promoter
We showed above (Fig. 4b) that the nuclear proteins
from cells treated with calphostin, which contains more 80-kDa Elf-1
and less 98-kDa Elf-1, display decreased DNA binding to the Elf-1 site
on the TCR -chain promoter. This observation supported the notion
that the 98-kDa form of Elf-1 is the one mainly responsible for its DNA
binding. Because we observed that samples from various donors differ
between themselves in the expression level of the 80- and 98-kDa Elf-1,
we analyzed samples in parallel by immunoblotting, using an
anti-Elf-1 Ab, and with EMSA using a labeled Elf-1 oligonucleotide.
Fig. 5a demonstrates that the
nuclear proteins from individual blood donors have unique expression
patterns of 98/80-kDa forms of Elf-1. In Fig. 5b the same
samples were analyzed by EMSA to determine their ability to bind to
DNA. Specificity of the binding was established by using anti-Elf-1
or control anti-Fli-1 Ab as discussed in Fig. 4a. Fig. 5, a and b, shows that the intensity of the
specific DNA binding bands in different donors reflects the expression
levels of the 98-kDa form, but not the 80-kDa form. Differences in
binding of nuclear proteins to the Elf-1 site of the TCR -chain
promoter are not mirrored in the binding of the same nuclear proteins
to other oligonucleotides, such as the one defined by the IL-2 promoter
(-164/-189) (11) (Fig. 5c).
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Elf-1 is present in the nucleus in its 98-kDa form, which is
phosphorylated and able to bind to the promoter of the TCR -chain.
We considered that the phosphorylated 98-kDa Elf-1 undergoes
degradation to prevent excessive accumulation. Because many
transcription factors are degraded through the proteasome pathway, we
treated Jurkat cells with different doses of the proteasome inhibitor
LLnL for variable time periods. As shown in Fig. 6a, in the presence of LLnL
for 3 h, the cytoplasmic levels of the 98-kDa Elf-1 increased
significantly, indicating that the 98-kDa Elf-1 is constantly degraded
through the proteasome pathway. In contrast, the 80-kDa Elf-1 did not
change in the presence of LLnL, suggesting that this form is not
degraded by the proteasome pathway. Compared with the cytoplasmic
98-kDa Elf-1, the nuclear 98-kDa Elf-1 did not increase rapidly after
treatment with LLnL. Instead, it slowly decreased over time (Fig. 6b).
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Discussion |
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-specific inhibitor, also
had an effect similar to that of calphostin C. The fact that calphostin
C has a more prominent effect than that of rotterlin indicates that
more than one class of PKC is involved in the conversion of Elf-1 from
80 to 98 kDa. Furthermore, transfection of PKC- (a member of the
novel PKC subfamily) into T lymphocytes resulted in the increase of the
98-kDa form at the cost of the 80-kDa form (Fig. 2b). In
contrast, we were unable to demonstrate that transfection of PKC- (a
member of the conventional PKC subfamily) can change the ratio between
these two forms. The other class of PKC, which is able to convert Elf-1
from 80- to 98-kDa form, may contain members of the atypical subfamily,
such as PKC-. Finally, sequence analysis discloses that Elf-1
encodes >10 potential PKC sites.
To confirm that the conversion of Elf-1 from 80- to 98-kDa form is
accompanied by the phosphorylation of Elf-1, we have treated T
lymphocytes with okadaic acid. This phosphatase inhibitor significantly
increased the level of the 98-kDa form, but only minimally increased
that of the 80-kDa form (Fig. 2c). This finding indicates
that compared with the 80-kDa form, the 98-kDa form is more susceptible
to a phosphatase-regulated process. We have further digested the total
cellular proteins with bacteriophage phosphatase. Although the
98-kDa form was extensively digested, the 80-kDa form of Elf-1 was not
affected (Fig. 2e). It is interesting that the decrease in
the intensity of the 98-kDa form is not associated with an increase in
the intensity of the 80-kDa form. This apparently means that
dephosphorylation triggers the degradation of the 98-kDa form of Elf-1
rather than its return to the 80-kDa form. The same mechanism has been
implicated in the degradation of activating transcription factor-2
(20) and the protooncogene Mos (21).
Altogether, these data indicate that the PKC-mediated conversion of Elf-1 from 80- to 98-kDa form involves the phosphorylation of Elf-1. It has not been determined whether PKC phosphorylates Elf-1 protein directly or indirectly. In other data (not shown), we found that transfection of an active form of Raf into T cells also causes phosphorylation of Elf-1. Because Raf can be activated by PKC (22), it is possible that the PKC may be involved in the phosphorylation of Elf-1 indirectly.
In addition to phosphorylation, we have demonstrated that glycosylation
is also responsible in part for the increment in the apparent molecular
mass of Elf-1. Whereas N-linked glycosylation frequently
occurs in cell surface and secreted proteins, O-linked
glycosylation, specifically the addition of O-GlcNAc to the
hydroxyl groups on the side chains of Ser and Thr, represents the
glycosylated form of an increasing number of transcription factors,
such as Sp1, AP-1, AP-2, Hnf-1, c-Myc, p53, v-Erb-A, estrogen receptor,
serum response factor (the protein regulating both gene
expression and DNA replication (SV40 large T Ag)), the nuclear pore
proteins, RNA polymerase II, and various chromatin proteins (9, 10). The results from the immunoprecipitation (Fig. 3a) and the gel shift assays (Fig. 4a) prove that
Elf-1 undergoes O-GlcNAc modification. The number of GlcNAc
moieties attached to each Elf-1 molecule is currently under
investigation in our laboratory. Computer analysis using the Expert
Protein Analysis System proteomics server from the Swiss Institute of
Bioinformatics has indicated the presence of at least 20 residues on
the Elf-1 (11 Thr and 9 Ser) that have high possibility (0.75–0.98,
which is above the threshold) for O-GlcNAc modification.
When total T cell extracts, which contain comparable amounts of 98- and
80-kDa Elf-1, were used for immunoprecipitation with the RL-2 Ab (Fig. 3b), the immunoprecipitates comprise mainly the 98-kDa form
of Elf-1 (98-kDa form:80-kDa form = 8:1, as determined by
densitometry). This finding indicates that 98-kDa Elf-1 is highly
O-GlcNAc modified, which results in the strong interaction
between RL-2 Ab and Elf-1. Phosphorylation and glycosylation have been
shown to coexist in numerous transcription factors (9, 10). The exact relation and hierarchy, inasmuch as they concern
the addition of phospho or glycosyl groups to the Elf-1 protein, are
not clear at this point.
We have found that the conversion of Elf-1 from the 80-kDa to the
98-kDa form has multiple functional consequences. First, we have found
that the 98-kDa form has less affinity to its cytoplasmic tethering
protein Rb than the 80-kDa form (Fig. 1c). It is likely that
the phosphoryl as well as the glycosyl groups block the access of Elf-1
to Rb. This mechanism, in conjunction with the phosphorylation of Rb,
which was previously found to help releasing the bound Elf-1
(15), should be important in the control of the
subcellular distribution of Elf-1. Second, by using EMSA, we found that
the Elf-1 bound to the TCR -chain promoter is glycosylated (Fig. 4a). Also, nuclear proteins from okadaic acid-treated T
cells demonstrated increased DNA binding to the TCR -chain promoter
in a dose-dependent manner (Fig. 4c). Consistently,
treatment of T cells with calphostin C, which should decrease the
PKC-mediated phosphorylation, decreased this binding (Fig. 4b).
Although we have identified both phosphorylation and glycosylation to be involved in the conversion of Elf-1 from 80- to 98-kDa form, it is not known whether other posttranslational modifications are also involved. Considering the huge molecular mass discrepancy between these two forms (18 kDa), it is likely that additional modifications are involved. One potential mechanism that may be involved in the formation of the 98-kDa form of Elf-1 is ubiquitination. Uni- or di-ubiquitination can endow their modified proteins with an increase of 8 and 16 kDa, respectively, because ubiquitin is a peptide of 7.6 kDa. Accumulated evidence in recent years has essentially overthrown the notion that ubiquitination is a mechanism solely involved in targeting proteins for degradation. Ubiquitination has been found to play a role, beyond protein degradation, in receptor endocytosis, signaling transduction, cell cycle progression, and transcription regulation (23). Ligation of ubiquitin to the transactivation domain of transcription factor virion polypeptide 16 (VP16) is essential for its transaction capacity. VP16 loses its transaction capacity in yeast strains, which are defective in the expression of the VP16-responsible ubiquitin-ligase Met30 (24). However, an anti-ubiquitin Ab failed to detect any protein in nuclear lysates immunoprecipitated with an anti-Elf-1 Ab (data not shown), suggesting that ubiquitination is not involved in the posttranslational modification of Elf-1.
We and others (8) have reproducibly found that the 98-kDa form of Elf-1 exists abundantly in the nucleus in a variety of cell types. Our study further reveals that this form has multiple features that characterize active transcription factors: it resides in the nucleus, is highly regulated by phosphorylation, constitutively binds to DNA, and is subject to tight control by the proteasome pathway. The maintenance of high level of this constitutively active form of transcription factor implies that Elf-1-dependent genes have essential functions and are indispensable.In contrast, it is mandatory for the cell homeostasis to tightly control this highly active transcription factor. We have demonstrated that the cytoplasm-located 98-kDa Elf-1 is accumulated rapidly in response to the inhibition of the proteasome pathway, whereas the same treatment resulted in the decrease of the 98-kDa form in the nucleus. LLnL did not promote the nuclear export of the 98-kDa form to the cytoplasm because the addition of leptomycin, a commonly used nuclear export inhibitor (25), failed to inhibit the decrease of the 98-kDa form in the nucleus (data not shown).
In conclusion, we found that in Jurkat and primary T cells, posttranslational phosphorylation and glycosylation of Elf-1 are responsible for the observed two major 98- and 80-kDa forms. The 98-kDa form migrates to the nucleus, binds to DNA, and activates gene transcription. Our findings illustrate the mechanisms that govern the activation and the degradation of Elf-1 and should help the understanding of the pathogenesis of diseases associated with abnormal expression of Elf-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George C. Tsokos, Department of Cellular Injury, Walter Reed Army Institute of Research, Building 503, Room 1A32, 503 Robert Grant Road, Silver Spring, MD 20910-7500. E-mail address: gtsokos{at}usa.net
3 Abbreviations used in this paper: Ets, E 26 specific; PKC, protein kinase C; Rb, retinoblastoma; GlcNAc, N-acetylglucosamine; pcDNA, plasmid-encoding cDNA; VP16, virion polypeptide 16.
Received for publication October 31, 2001. Accepted for publication January 17, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 P. G.P. Andrews, M. W. Kennedy, C. M. Popadiuk, and K. R. Kao Oncogenic Activation of the Human Pygopus2 Promoter by E74-Like Factor-1 Mol. Cancer Res., February 1, 2008; 6(2): 259 - 266. [Abstract] [Full Text] [PDF]
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Y.-T. Juang, L. Sumibcay, M. Tolnay, Y. Wang, V. C. Kyttaris, and G. C. Tsokos Elf-1 Binds to GGAA Elements on the FcR{gamma} Promoter and Represses Its Expression J. Immunol., October 1, 2007; 179(7): 4884 - 4889. [Abstract] [Full Text] [PDF]
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 K. Tenbrock, Y.-T. Juang, V. C. Kyttaris, and G. C. Tsokos Altered signal transduction in SLE T cells Rheumatology, October 1, 2007; 46(10): 1525 - 1530. [Abstract] [Full Text] [PDF]
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 S. S. Andrali, Q. Qian, and S. Ozcan Glucose Mediates the Translocation of NeuroD1 by O-Linked Glycosylation J. Biol. Chem., May 25, 2007; 282(21): 15589 - 15596. [Abstract] [Full Text] [PDF]
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K. Tenbrock, V. C. Kyttaris, M. Ahlmann, J. M. Ehrchen, M. Tolnay, H. Melkonyan, C. Mawrin, J. Roth, C. Sorg, Y.-T. Juang, et al. The Cyclic AMP Response Element Modulator Regulates Transcription of the TCR {zeta}-Chain J. Immunol., November 1, 2005; 175(9): 5975 - 5980. [Abstract] [Full Text] [PDF]
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 T. Shimokawa and C. Ra C/EBP{alpha} functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (Fc{alpha}R, CD89) gene promoter Blood, October 1, 2005; 106(7): 2534 - 2542. [Abstract] [Full Text] [PDF]
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N. E. Zachara, N. O'Donnell, W. D. Cheung, J. J. Mercer, J. D. Marth, and G. W. Hart Dynamic O-GlcNAc Modification of Nucleocytoplasmic Proteins in Response to Stress: A SURVIVAL RESPONSE OF MAMMALIAN CELLS J. Biol. Chem., July 16, 2004; 279(29): 30133 - 30142. [Abstract] [Full Text] [PDF]
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S. Krishnan, M. P. Nambiar, V. G. Warke, C. U. Fisher, J. Mitchell, N. Delaney, and G. C. Tsokos Alterations in Lipid Raft Composition and Dynamics Contribute to Abnormal T Cell Responses in Systemic Lupus Erythematosus J. Immunol., June 15, 2004; 172(12): 7821 - 7831. [Abstract] [Full Text] [PDF]
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 M. Cotman, D. Jezek, K. F. Tacer, R. Frangez, and D. Rozman A Functional Cytochrome P450 Lanosterol 14{alpha}-Demethylase CYP51 Enzyme in the Acrosome: Transport through the Golgi and Synthesis of Meiosis-Activating Sterols Endocrinology, March 1, 2004; 145(3): 1419 - 1426. [Abstract] [Full Text] [PDF]
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 S. A. Whelan and G. W. Hart Proteomic Approaches to Analyze the Dynamic Relationships Between Nucleocytoplasmic Protein Glycosylation and Phosphorylation Circ. Res., November 28, 2003; 93(11): 1047 - 1058. [Abstract] [Full Text] [PDF]
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D. O'Reilly, C. M. Quinn, T. El-Shanawany, S. Gordon, and D. R. Greaves Multiple Ets Factors and Interferon Regulatory Factor-4 Modulate CD68 Expression in a Cell Type-specific Manner J. Biol. Chem., June 6, 2003; 278(24): 21909 - 21919. [Abstract] [Full Text] [PDF]
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Y.-T. Juang, K. Tenbrock, M. P. Nambiar, M. F. Gourley, and G. C. Tsokos Defective Production of Functional 98-kDa Form of Elf-1 Is Responsible for the Decreased Expression of TCR {zeta}-Chain in Patients with Systemic Lupus Erythematosus J. Immunol., November 15, 2002; 169(10): 6048 - 6055. [Abstract] [Full Text] [PDF]
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