From the
The p60 form of the tumor necrosis factor (TNF) receptor lacks
motifs characteristic of tyrosine or serine/threonine protein kinases.
Our recent observations have indicated that a p60 TNF
receptor-associated kinase (p60-TRAK) from U-937 cells physically
interacts with and causes the phosphorylation of the cytoplasmic domain
of the TNF receptor. To define which region of the cytoplasmic domain
is necessary for physical interaction with p60-TRAK, we constructed a
series of deletions (grouped into three sets
In spite of the conserved features in the extracellular domains
of the p60 (p55, Type I) and the p80 (p75, Type II) forms of the tumor
necrosis factor (TNF)
Efforts to identify receptor domains
critical for cellular signaling have relied on mutational analysis.
Brakebusch et al.(7) indicated that truncation of at
least the C-terminal half of the cytoplasmic domain of p60 abolished
the ability of TNF to signal for cytotoxicity. Additionally, they
concluded that a mutant receptor lacking most of its cytoplasmic domain
interfered with the endogenous wild-type receptor, suggesting that
receptor clustering is necessary for signal transmission. Similarly,
Tartaglia et al.(8) demonstrated that the expression
of a truncated human p60 receptor in mouse cells suppressed the
signaling of the endogenous mouse TNF receptors in response to the
ligand. A mutational study has shown that a region residing near the
C-terminal end of the cytoplasmic domain (termed the ``death
domain'') is necessary for transmitting the cytotoxic effect of
TNF(9) . Interestingly, this death domain shares weak homology
with a region found in the cytoplasmic domain of the Fas antigen that
is necessary for apoptotic signal transduction(10) .
The
identification of molecules associated with the intracellular region of
both TNF receptors would improve understanding of the signal
transduction mechanisms elicited by the binding of TNF to its
receptors. Both the yeast-based two-hybrid screen and affinity
precipitation with glutathione S-transferase (GST) fusion
proteins has led to identification of several proteins that bind to the
cytoplasmic domains of the p80 and p60 TNF receptors designated TNF
receptor-associated factors (TRAFs) (11, 12) and TNF
receptor-associated proteins (TRAPs)(13) , respectively.
Recently, we and others found that the cytoplasmic domain of the p60
form of the TNF receptor physically and functionally interacts with a
serine/threonine protein kinase (TRAK)(14, 15) .
Therefore, we initiated a study to define the region of the cytoplasmic
domain that is involved in binding to p60-TRAK using a series of
deletion mutants in the p60 cytoplasmic domain. Our data demonstrate
that a short segment (residues 344-397) is sufficient to bind
p60-TRAK activity and undergo phosphorylation and that the C-terminal
30 residues(397-426) inhibit the interaction of p60-TRAK with the
cytoplasmic domain.
Since residues
397-426 appeared to inhibit binding of the kinase activity, we
constructed a set of deletions that had the same N terminus as
Our data demonstrate that residues
344-397 of the cytoplasmic domain of the p60 receptor is
sufficient to bind p60-TRAK activity. These results are consistent with
the report that the site-directed mutants of residues 345, 347, 351,
369, 378, and 390 attained by alanine-scanning mutagenesis (9) rendered the p60 receptor nonfunctional for signaling
cytotoxicity. Additionally, Boldin et al.(17) observed
that expression of only the death domain (residues 328-414) in
HeLa cells triggered TNF-independent signaling for cytotoxicity and
interleukin-8 gene induction. In contrast, expression of only the
N-terminal half (residues 206-326) or residues 367-426 did
not cause ligand-independent signaling(17) . Again, the
importance of the residues found within the death domain in binding
p60-TRAK in our study suggests that the kinase activity correlates with
signaling by TNF.
The regions of the cytoplasmic domain necessary
for binding receptor-associated molecules is depicted in Fig. 4.
Residues found within the cytotoxicity domain have also been noted to
be involved in the aggregation of the receptor(13, 17) .
The cell-surface binding of trimeric TNF to its receptor would cause
trimerization of the receptor to initiate the signaling
mechanism(18, 19) . Ligand-induced conformational
changes within the intracellular domain may bring about the unmasking
of the aggregation domain and provide the surface for binding of the
kinase as described here. As we have observed, the C-terminal 30
residues impede the binding of p60-TRAK. The recent identification of
TRAP-1 as an hsp90-like molecule that binds to the N-terminal half of
the cytoplasmic domain (20) may suggest that different domains
of the TNF receptor are involved in different responses mediated by
TNF. The identification of p60-TRAK, its downstream targets, and its
regulation and the function of the TRAPs and TRAFs may facilitate the
understanding of signal transduction by TNF.
We thank Polly Lee from the Dept. of Neuro-Oncology of
M. D. Anderson Cancer Center for synthesis of oligonucleotides and
Klara Totpal and Ruthie LaPushin for providing cells.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1-
5,
6-
12, and
13-
16) of the p60 cytoplasmic
domain, expressed them as glutathione S-transferase (GST)
fusion proteins, and used them in affinity precipitations, followed by in vitro kinase assays. Our detailed analysis indicated that a
serine-, threonine-, and proline-rich region (residues 243-274,
2) and the N-terminal half of the cytoplasmic domain (residues
243-323,
3) neither associated with p60-TRAK nor underwent
phosphorylation. We found that out of 222 residues(205-426) in
the cytoplasmic domain, only 54 (344-397,
12) were
sufficient for binding p60-TRAK and for phosphorylation of the
cytoplasmic domain. A region of approximately 30
residues(397-426) at the C-terminal end was found to interfere
with optimal binding of the p60-TRAK activity. Thus, our results
indicate that the minimal region of the cytoplasmic domain necessary
for interacting with p60-TRAK and for phosphorylation resides within
the domain previously reported to be needed for signaling the cytotoxic
effect of TNF.
(
)receptor, it has been
difficult to identify common motifs in their intracellular regions
(1-5). Like other members of the TNF/nerve growth factor receptor
family, the p60 and p80 forms of the TNF receptor do not contain
consensus sequences characteristic of tyrosine or serine/threonine
kinases or any other signal transduction motifs. However, ligand
binding to the TNF receptor activates a wide variety of putative second
messenger events, including a rapid increase in protein phosphorylation
(6). It is at present unclear which of these processes forms the link
between ligand binding at the cell surface and the profound effect that
TNF has upon cell function.
Materials
All chemicals were reagent grade or
higher and obtained from previously cited sources(14) . The
histiocytic lymphoma cell line U-937 (CRL 1593) was obtained from the
American Type Culture Collection (ATCC). Cells were grown in RPMI 1640
medium supplemented with 10% fetal bovine serum and 100 µg/ml
streptomycin at 37 °C in a 5% CO incubator. Escherichia coli strain BL21 and plasmids pGEX-2TH and
pCMVXVBpL4-p60 have been described elsewhere(14) .
Construction, Expression, and Purification of GST Fusion
Protein
All subsequent DNA manipulations were carried out as
described by Sambrook et al.(16) . The construction of
the plasmid encoding GST-p60CD1 has been described
elsewhere(14) . Fig. 1illustrates the fusion proteins
that were made for this study. Specific primers for each deletion
construct were used to amplify fragments with unique restriction sites
by polymerase chain reaction using pCMVXVBpL4-p60 as the template. The
polymerase chain reaction products were digested with either EcoRI/HindIII or BamHI/HindIII and
inserted into digested pGEX-2TH to yield the appropriate expression
vectors. All expression vectors were sequenced to ensure that
amplification was correct. Expression and purification of the GST
fusion proteins from BL21 cells harboring the appropriate expression
vector were carried out as described previously(14) . The fusion
proteins were stored at 4 °C on glutathione-agarose beads as a 50%
slurry in Buffer A (20 mM Tris, pH 8.0, 200 mM NaCl,
10% glycerol, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.1%
2-mercaptoethanol). The amount of fusion protein was estimated by
Coomassie Blue staining of SDS-PAGE gels.
Figure 1:
Schematic diagram of the deletion
mutants of the cytoplasmic domain of the p60 TNF receptor. The entire
cytoplasmic domain (residues 205-426) of the p60 TNF receptor is
shown as p60CD. Each deletion mutant was expressed as a fusion protein
linked to GST as described under ``Experimental Procedures.''
The shaded region indicates the death domain
(9).
In Vitro Binding of GST Fusion Protein to Cell
Extracts
U-937 cells were lysed in 600 µl of lysis buffer
(20 mM HEPES, pH 7.4, 0.1% Nonidet P-40, 250 mM NaCl,
10 mM NaF, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2
µg/ml leupeptin) on ice for 30 min followed by 10 min of
centrifugation in a Microfuge. The supernatant was adjusted to 125
mM NaCl by addition of lysis buffer without NaCl and
precleared with 25 µg of GST in 50 µl of 50% (v/v) glutathione
agarose for 1 h at 4 °C. The precleared supernatant was mixed with
approximately 5-10 µg of GST or the appropriate fusion
protein attached to glutathione-agarose beads for 1 h at 4 °C. The
beads were collected by centrifugation and washed extensively with
lysis buffer (4 500 µl) and with kinase buffer (3
500 µl: 20 mM HEPES, pH 7.4, 10 mM NaF, 0.2
mM sodium orthovanadate, and 0.1% 2-mercaptoethanol). The
pellets were then used for in vitro kinase assays.
In Vitro Kinase Assays
Standard kinase assays were
carried out for 20 min at 37 °C in 30 µl containing 20 mM HEPES, pH 7.4, 10 mM MgCl, 0.2 mM ATP, 0.2 mM NaF, 0.1 mM sodium orthovanadate,
and 10 µCi of [
-
P]ATP. Reactions were
stopped with 15 µl of SDS-sample buffer, boiled for 5 min, and
subjected to SDS-PAGE. Protein bands were visualized by staining with
Coomassie Blue, and the dried gels were analyzed by a PhosphorImager
(Molecular Dynamics) and quantitated by ImageQuant Software (Molecular
Dynamics). In vitro kinase assays with 5 µg of histone
(Type VI-S, Sigma) followed the standard kinase assay.
Design of Deletion Mutants of the Cytoplasmic Domain of
the p60 Receptor
We have previously shown that residues
243-426 bind p60-TRAK activity from U-937 cell lysate and also
serve as a substrate for the associated kinase activity(14) .
The intracellular region of p60 contains 222 aminoacyl
residues(205-426) with a large proportion of serine, threonine,
proline, and acidic residues. Essentially, the cytoplasmic domain can
be divided into two parts: an N-terminal half (residues 205-323),
which is rich in serine, threonine, and proline, and a C-terminal half,
which contains a region necessary for signaling the cytotoxic response
of TNF(9, 17) . To determine the minimal region of the
cytoplasmic domain necessary for interacting with p60-TRAK activity and
the potential sites of phosphorylation, we designed a series of
deletion mutants of the cytoplasmic domain of the p60 TNF receptor (Fig. 1). For analysis, the deletions were grouped into three
sets based on our observations (see below): set 1
(1-
5), set 2 (
6-
12), and set 3
(
13-
16).
Binding and Phosphorylation by the Receptor-associated
Kinase Occurs within the C-terminal Half
The first set of
deletions constructed (Fig. 1, 1-
5) were to
ascertain if the kinase activity would bind to and phosphorylate the
domain shown to be important for TNF-dependent
cytotoxicity(9, 17) . Essentially, precleared cell
lysates were bound to the desired fusion protein, washed extensively,
used for in vitro kinase reactions, and analyzed by Coomassie
Blue staining of SDS-PAGE gels (Fig. 2B) and
autoradiographed (Fig. 2A). In the first set of
deletions, fusion proteins
1,
4, and
5 were capable of
binding kinase activity and serving as substrates for the associated
kinase activity (Fig. 2). GST neither bound any kinase activity
nor served as a substrate in this or subsequent kinase reactions (Fig. 2). Kinase activity did not associate with the N-terminal
half of the cytoplasmic domain (i.e. residues 243-323,
3). The phosphorylation of
4 and
5 was approximately 50%
of the phosphorylation of
1. In this set of deletions, the
smallest region that bound kinase activity was contained in residues
324-426 (
5).
Figure 2:
In
vitro kinase assays of U-937 cell lysates bound to each of the GST
fusion proteins. Cell lysates from 2 10
cells were
used for each binding reaction, after which kinase assays were
performed as described under ``Experimental Procedures.'' The
samples were subjected to 9% SDS-PAGE, and the Coomassie Blue-stained
gel was dried (B) and then subjected to autoradiography (A). Molecular mass standards are expressed in kilodaltons.
The arrows indicate the positions of pp55 and pp58, and hp in the lanes of
13-
15 represents
hyperphosphorylated forms. The arbitrary units represent the relative
amount of phosphorylation of each GST fusion protein when quantitated
by a PhosphorImager and ImageQuant Software. Images were essentially
identical in at least 5 other identical
experiments.
The Minimal Region Necessary for Binding Kinase Activity
Is Contained within Residues 344-397
To further narrow the
region necessary for binding the kinase activity, 5 was deleted
more extensively (Fig. 1,
6-
12). Kinase activity
was associated with deletion mutants
8 and
12 more strongly
than
6,
7, and
9-
11 (Fig. 2).
Surprisingly, the phosphorylation of deletion mutants
8 and
12 was also greater than with
1 (Fig. 2). The minimal
region found to be phosphorylated and to associate with the kinase
activity was contained within 54 amino acids that reside within the
death domain, namely residues 344-397, corresponding to
12.
Phosphoamino acid analysis performed on
12 indicated
phosphorylation of both serine and threonine residues (data not shown).
There are two serines (Ser-352 and Ser-373) and three threonines
(Thr-377, Thr-382, and Thr-388) in this region that could be possible
sites of phosphorylation by the associated kinase. The amount of kinase
activity associated with
12 was comparable to that of
8 (Fig. 2); however, a few more proteins were found to be
phosphorylated and associated with
12, either directly or
indirectly. The identity of these associated molecules remains to be
determined.
The C-terminal 30 Residues Contain a Region That Inhibits
Binding of Kinase Activity
That deletion mutants
9-
11 did not bind kinase activity even though they had
the same N terminus as
12 suggests that residues 397-426
serve an inhibitory function. This could arise from the binding of a
protein to residues 397-426, thus inhibiting the binding of the
associated kinase. Another possibility is that residues 397-426
bind a protein that regulates kinase activity. The possibility that
deletions
9-
11 were poor substrates for the associated
kinase was ruled out since the kinase associated with
8 could
phosphorylate
9-
11 (data not shown).
1
and the same C terminus as deletion mutants
10-
12 (Fig. 1,
13-
15). In addition, we constructed a
deletion mutant,
16, which contained residues 394-426. We
hypothesized that if residues 397-426 affect optimal binding of
the kinase activity to
1, then we should observe increased kinase
activity associated with deletion mutants
13-
15. In
fact, we did observe increased phosphorylation using these deletions (Fig. 2). We also observed that deletions
13-
15
were hyperphosphorylated as indicated by slower migrating bands. As was
expected,
16 did not bind any significant kinase activity. When
cell lysate was bound to
16 and mixed with the
8-associated
kinase prior to an in vitro kinase reaction, kinase activity
was not inhibited, thus failing to demonstrate that a protein bound to
16 regulates the kinase activity associated with
8 (data not
shown).
Deletion Mutants
To determine the amount of
kinase activity associated with each of the deletions, we used histone
as an exogenous substrate in the kinase reactions. The histone kinase
activity was highest with deletion 8,
12, and
15 Contain the
Highest Histone Kinase Activity
8,
12, and
15 (Fig. 3). This is consistent with the observation that the
C-terminal 30 residues confer an inhibitory function upon binding of
the kinase to the minimal region, residues 344-397.
Figure 3:
Histone kinase activity associated with
the deletion mutants of the p60 TNF receptor. The receptor-associated
kinase activity was measured using 5 µg of histone as an exogenous
substrate as described under ``Experimental Procedures.''
Images were essentially identical in at least 3 other identical
experiments.
Additional Proteins Are Phosphorylated in Precipitates
with the Deletion Mutants
Interestingly, after in vitro kinase assays, two additional proteins, of approximately 58 kDa
(pp58) and 55 kDa (pp55), were found to be phosphorylated in reactions
containing deletion mutants 1,
4,
5,
7,
8, and
12-
15 (Fig. 2). Both pp58 and pp55 were highly
phosphorylated by the kinase activity bound to
8,
12, and
15, all of which end in the same C-terminal residue (Fig. 1). Since residues found within deletion mutants
8,
12, and
15 are known to be necessary for receptor
aggregation(13, 17) , it may be that either pp58 or pp55
is the endogenous p60 TNF receptor. Immunoblotting with antibodies
directed against the p60 TNF receptor failed, however, to detect
proteins of this size after precipitation of cell lysates with
8,
12, and
15. A few additional proteins were phosphorylated in
the kinase reaction with deletion mutant
12; however, we could not
determine whether these proteins bound directly or indirectly to
12 (Fig. 2).
Figure 4:
Summary of the p60 TNF receptor-associated
molecules. A diagram of the entire cytoplasmic domain is shown with
regions mapped for binding TRAP-1 and TRAP-2 (20), p60-TRAK, the kinase
inhibitory domain, and the aggregation (13, 17) and death domain (9).
The open circles represent alanine substitutions that render
the receptor nonfunctional for cytotoxicity (9). The closed circles represent potential phosphorylation sites by
p60-TRAK.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.