(Received for publication, June 14, 1995)
From the
Cellular responses initiated by tumor necrosis factor (TNF) are mediated by two different cell surface receptors with respective molecular masses of 55 kDa (p55) and 75 kDa (p75). p55 is functional in almost every cell type and can independently transmit most biological activities of TNF. In contrast, TNF signaling via p75 seems so far largely restricted to cells of lymphoid origin, where it can induce proliferation, cytokine production, and/or apoptosis. The mechanisms that regulate TNF receptor activity are largely unknown. Here we report that the p75 of unstimulated p75-responsive PC60 T cells is phosphorylated on serine by a kinase activity present in p75 immune complexes. Several lines of evidence indicate that the latter kinase is casein kinase-1 (CK-1). Previous results have shown that the p75 TNF receptor is constitutively phosphorylated in vivo. Our data show that the latter in vivo phosphorylation is also at least partially due to CK-1. Pretreatment of cells with TNF had no detectable effect on p75 phosphorylation in vitro or in vivo. However, a specific CK-1 inhibitor potentiated TNF-induced apoptosis mediated by p75, suggesting an inhibitory role for phosphorylation by CK-1. Although in vivo p75 phosphorylation could be seen in both p75-unresponsive and p75-responsive cell lines, in vitro p75 phosphorylation in p75 coimmunoprecipitates could not be observed in cell lines that were biologically unresponsive to p75 stimulation. The latter observation further indicates a regulatory role for p75 phosphorylation in p75-mediated signaling. Taken together, our data demonstrate that the p75 TNF receptor is phosphorylated and associated with CK-1, which negatively regulates p75-mediated TNF signaling.
Tumor necrosis factor (TNF) ()is a pluripotent
cytokine inducing an extraordinary wide variety of biological
responses, mainly related to immune regulation and inflammation
(reviewed by Vassalli(1993) and Fiers(1995)). Several signal
transducing mechanisms have been shown to be involved in the action of
TNF, dependent on the cell type and the cell response studied (reviewed
by Beyaert and Fiers(1994)). Cellular signals occurring within minutes
after addition of TNF include generation of cAMP in some fibroblast
lines (Zhang et al., 1988), activation of
phosphatidylcholine-specific phospholipase C and sphingomyelin
degradation in myeloid cell lines (Kim et al., 1991;
Schütze et al., 1992; Dressler et
al., 1992), activation of the transcription factor NF-
B
(Lowenthal et al., 1989), and phosphorylation of several
cellular proteins, including heat shock protein 27, epidermal growth
factor receptor, and eukaryotic initiation factor 4E (Hepburn et
al., 1988; Saklatvala et al., 1991; Marino et
al., 1991). Enhanced phosphorylation of cellular proteins could be
due to the documented activation of a wide variety of kinases (Zhang et al., 1988; Guy et al., 1991; Kim et al.,
1991; Mathias et al., 1991; Van Lint et al., 1992;
Guesdon et al., 1993) and/or to inhibition of phosphatases
(Guy et al., 1993; Tan, 1993). TNF-induced signaling is
initiated by oligomerization of TNF receptors upon binding of the
trimeric TNF ligand (Engelmann et al., 1990; Van Ostade et
al., 1991). Two distinct, high affinity TNF receptors of 55 kDa
(p55) and 75 kDa (p75) have been identified and cloned (reviewed by
Loetscher et al.(1991) and Tartaglia and Goeddel(1992)).
Although both TNF receptor types are expressed by most cell types, p55
is responsible for the majority of TNF activities (reviewed by
Tartaglia and Goeddel(1992)). The contribution of p75 can be explained
in part by the so-called ligand passing model, in which p75 presents
TNF to neighboring p55 molecules, the latter being signal transducing
(Tartaglia and Goeddel, 1992). Direct p75 signaling involves mainly
effects on lymphoid cells, such as proliferation of the CT6 T cell line
and induction of granulocyte/macrophage colony-stimulating factor
secretion by a T cell hybridoma (Gehr et al., 1992;
Vandenabeele et al., 1992; Tartaglia et al., 1993).
In addition, an independent signaling role for p75 has been
demonstrated in TNF-mediated cytotoxicity in some specific cell lines
(Heller et al., 1992; Grell et al., 1993;
Vandenabeele et al., 1995).
The cytoplasmic domains of p55 and p75 lack sequence homology, suggesting that they generate distinct activation signals. To date, it is not clear how TNF receptor aggregation leads to signal transduction. Oligomerization of cytoplasmic domains might unveil discrete peptide motifs, activate receptor-associated enzymes, or create a composite, tertiary binding site to attract signal transduction molecules. The fact that neither TNF receptor contains intrinsic protein kinase activity further suggests that associated proteins, rather than the intracellular domain of either receptor, act as essential elements in signal transduction. Recently some evidence has been presented for the presence of proteins that are specifically interacting with p55 or p75 (Darnay et al., 1994a, 1994b; Rothe et al., 1994; VanArsdale and Ware, 1994; Song et al., 1995). Some of these proteins were shown to phosphorylate one or both TNF receptors. The biological significance as well as the identity of the latter kinase(s) remain unknown. Interestingly, it was demonstrated that the unspecific protein kinase inhibitor staurosporine (STS) could inhibit p55 phosphorylation in U937 cells (VanArsdale and Ware, 1994). The finding that STS can sensitize tumor cells to TNF-induced cell death (Beyaert et al., 1993) suggests that phosphorylation of p55 might negatively regulate its activity. In contrast, the functional role of p75 phosphorylation as well as the identity of p75-associated kinase(s) still must be investigated. In an attempt to gain more insight into this matter, we analyzed p75 immune complexes from a rodent hybridoma PC60 T cell line, engineered to overexpress the human p55 and p75 (in this paper referred to as PC60p55p75). We show that p75 becomes phosphorylated in vitro and in vivo by a kinase activity present in p75 immune complexes. We identified this kinase as CK-1, and we present evidence that phosphorylation by CK-1 negatively regulates p75-mediated signaling to TNF-induced apoptosis.
Phosphorylation of the CK-1-specific peptide (1 mg/ml) was analyzed as described above, except that phosphorylation reactions were stopped by spotting an aliquot of the supernatant of the incubation mixture onto square papers of Whatman P-81 and subsequent immersion and washing in 75 mM phosphoric acid (Agostinis et al., 1987). Controls with either substrate or immunoprecipitates alone were performed at the same time and subtracted from the peptide phosphorylation data.
Figure 1:
In vitro phosphorylation of a
75-kDa protein in utr1 immunoprecipitates of PC60 cells. A,
immunoprecipitates (protein G-Sepharose 4 Fast Flow was used as
carrier) of control PC60 transfectants (PC60 control) or PC60 cells
expressing either full-length (PC60p55p75) or intracellularly truncated
(PC60p55p75CD) human p75 were made with (+) or
without(-) utr1 and subjected to in vitro phosphorylation as described under ``Materials and
Methods.'' Reaction mixtures were then analyzed on a 10%
SDS-polyacrylamide gel followed by autoradiography. The arrowhead points to the position of the 75-kDa phosphoprotein. B,
phosphoamino acid analysis of the in vitro phosphorylated p75
protein band shown in A. Radioactivity on the thin layer
plates was detected by phosphorimaging. The migration positions of
phosphoserine, phosphothreonine, and phosphotyrosine are
indicated.
As
immunoprecipitates of control transfectants did not contain the 75-kDa
phosphoprotein, it is very likely that the latter is the p75 TNF
receptor itself. This conclusion was substantiated by the following
observations. First, p75 immunoprecipitated from PC60p55p75 cells
metabolically labeled with P
in vivo comigrated and showed a largely overlapping proteolytic
fingerprint with the 75-kDa protein phosphorylated in vitro (Fig. 2A and data not shown). In addition, the
75-kDa phosphoprotein generated in vitro was found to be
reactive with antibodies to p75. This was demonstrated as follows.
Immunoprecipitates were made with antibodies to p75 covalently coupled
to an agarose matrix. Following its in vitro phosphorylation
and SDS-driven, mild release from the immobilized antibodies in the
immunoprecipitate, the 75-kDa protein could be re-immunoprecipitated
with p75 TNF receptor-specific antibodies (Fig. 2B).
From these data we conclude that the 75-kDa phosphoprotein generated in
p75 immune complexes is the p75 TNF receptor itself, and the associated
kinase activity will be further referred to as p75 kinase.
Figure 2:
The 75-kDa phosphoprotein generated in
vitro in p75 immunoprecipitates comigrates with p75 labeled in
vivo with P
and is reactive with
antibodies to p75. A, immunoprecipitates of PC60p55p75 cells
made without(-) or with (+) utr1 were subjected to in
vitro phosphorylation, followed by analysis on a 10%
SDS-polyacrylamide gel and autoradiography. A similar analysis was
performed on lysates of PC60p55p75 cells in vivo labeled with
P
and immunoprecipitated with (+) or
without(-) utr1. The arrowhead points to the position of
the 75-kDa phosphoprotein. B, immunoprecipitates of PC60p55p75
cells were made with AvidGel, which was either untreated (lane1) or coupled to utr4 (lanes2-4)
and subjected to in vitro phosphorylation. In lanes1 and 2, the phosphorylation reaction was
stopped by adding Laemmli loading buffer and directly analyzed on an
SDS-polyacrylamide gel. In lanes3 and 4,
the phosphorylation reaction was stopped by the addition of
SDS-containing lysis buffer, and after a 30-min rotation at room
temperature, the supernatant was diluted in Nonidet P-40 and
protein-containing lysis buffer as detailed under ``Materials and
Methods.'' Following a 45-min rotation at room temperature, this
supernatant mixture was again subjected to immunoprecipitation now
using protein G-Sepharose without (lane3) or with (lane4) utr1. The arrowhead points to the
position of p75.
To
investigate the effect of TNF stimulation in vivo on p75
kinase activity in vitro, PC60p55p75 cells expressing
full-length human p75 were treated with human TNF, after which p75
immune complexes were prepared and assayed for p75 kinase activity.
Immunoprecipitates were made with the utr4 monoclonal antibody, which
binds human p75, even when the latter is occupied by TNF (as assessed
by flow fluorimetric analysis) (data not shown; see also Brockhaus et al.(1990)). TNF doses, varying between 0.1 and 500 ng/ml,
were given for different time periods (30 s up to 4 h). In some
experiments, cells starved by a 24-h incubation in the absence of serum
were used. Under none of these conditions a TNF-stimulated enhancement
of p75 kinase activity could be demonstrated, as judged from the
absence of an increase of P incorporated in p75 on
SDS-polyacrylamide gels (analyzed by phosphorimaging; data not shown).
This finding is reminiscent of the absence of an effect of TNF on in vivo phosphorylation of p75 (Pennica et al.,
1992).
Figure 3: Effect of protein kinase inhibitors on p75 kinase activity in vitro. p75 immunoprecipitates (Trisacryl GF-2000 protein A was used as carrier) of PC60p55p75 cells were pretreated for 10 min with or without the indicated drugs, after which an in vitro kinase assay was performed. Immunoprecipitates were then resolved on SDS-polyacrylamide gels as described under ``Materials and Methods.'' The position of p75 is indicated by an arrowhead. A, effect of STS; B, effect of CKI-7 and heparin.
Figure 4:
Effect of CKI-7 on p75 kinase activity in vivo. 2 10
PC60p55p75 cells were
metabolically labeled with 100 µCi of
P
for 2.5 h. Treatment with CKI-7 started 30 min before the
labeling. At the end of the incubation period, cells were lysed and p75
was immunoprecipitated with utr1 as described under ``Materials
and Methods.'' Immunoprecipitates were then resolved on an 8%
SDS-polyacrylamide gel. The position of p75 is indicated by an arrowhead.
Figure 5:
Absence of in vitro p75
phosphorylation in p75 immunoprecipitates of U937 and HL60 cell lines. A, immunoprecipitates of the indicated cell lines were made
without (lanes1, 3, and 5) or with (lanes2, 4, and 6) utr1, subjected
to in vitro phosphorylation in the presence of labeled ATP and
analyzed as described under ``Materials and Methods.''
Trisacryl GF-2000 protein A was used for immunoprecipitation. The arrowhead points to the position of p75 from PC60p55p75 cells. B, immunoblotting of p75 from PC60p55p75 and U937 cell lines.
Lysates of PC60p55p75 (lane1) or U937 (lane2) were immunoprecipitated with utr1 and immunoblotted as
described under ``Materials and Methods.'' C,
lysates of U937 (lane1) or PC60p55p75 (lane2) labeled with P
in vivo were immunoprecipitated with utr1 and analyzed by
SDS-polyacrylamide gel electrophoresis and
autoradiography.
To date, receptor-associated mechanisms involved in TNF
signal transduction, as well as the mechanisms determining whether the
signal is initiated by p55 or p75, are largely unknown. Covalent
modification of proteins by phosphorylation-dephosphorylation plays a
central role in cellular regulation, including signal transduction. The
cytoplasmic domains of p55 and p75 TNF receptors, like that of other
members of the cytokine receptor superfamily, lack any protein kinase
motifs. However, there is increasing evidence that signal transduction
by TNF receptors involves tyrosine and serine/threonine phosphorylation
of proteins. The p75 receptor has been shown to be constitutively
phosphorylated on serine residues in vivo (Pennica et
al., 1992). Recent results have provided evidence for physical
association of a protein kinase with the cytoplasmic domain of p75
(Darnay et al., 1994a). However, the identity of the kinase as
well as the TNF response in which it is involved was not stated.
Therefore, we have performed biochemical analyses of p75
imunoprecipitates in vitro and in vivo, with the aim
to characterize the p75 kinase and its biological role. In this study,
we made use of rodent PC60 T cells transfected with human p55 and p75
receptors. Specific triggering of the transfected p75 receptor (either
by species-specific antisera or receptor-specific mutants of human TNF)
has been shown to induce granulocyte/macrophage colony-stimulating
factor production in these cells, especially in the presence of
interleukin-1 (Vandenabeele et al., 1992). Recently, we found
that selective p75 triggering was also capable of inducing apoptosis in
PC60 cells, but, surprisingly, only in cells that were also transfected
with human p55 (Vandenabeele et al., 1995). Here we report
that p75 receptors from these p75-responsive PC60 cells became
phosphorylated in vitro by a kinase activity that was present
in p75 immunoprecipitates. Serine was the only detectable phosphoamino
acid identified in p75. At present, we have no indication that p75
kinase activity is affected by TNF treatment of cells. This finding is
reminiscent of the absence of a detectable effect of TNF on in vivo p75 phosphorylation (Pennica et al., 1992). However, the fact that the intracellular domain of p75 contains a
very high number of potential phosphoamino acids (32 serines and 11
threonines, but no tyrosine residues, out of 174 amino acids), renders
this a very complex substrate to study possible minor TNF-induced
alterations in phosphorylation.
Several lines of evidence point to
CK-1 as the p75 kinase identified in the present study. First, CKI-7,
an inhibitor with relative specificity for CK-1 (Chijiwa et
al., 1989), completely inhibited p75 kinase activity that could be
coimmunoprecipitated with p75. Second, casein and a CK-1-specific
peptide substrate are both phosphorylated by the p75 kinase in
vitro. Third, a partially purified CK-1 phosphorylates p75 in
vitro. The p75 kinase could be further distinguished from CK-2 by
its inability to use GTP as phosphate donor (Hathaway and Traugh, 1979)
and its insensitivity to heparin (Hathaway et al., 1980). In
addition, the involvement of a whole range of other kinases could be
excluded by the absence of an effect of the broad spectrum protein
kinase inhibitor STS. Similar to the in vitro phosphorylation,
CKI-7 also inhibited p75 phosphorylation in vivo. Although we
cannot exclude that CKI-7 only reaches suboptimal concentrations in
cultured cells, the residual in vivo phosphorylation of p75
that is still observed in cells treated with CKI-7 might be due to
other kinases for which multiple potential phosphorylation sites can be
found in the intracellular domain of p75. CK-1 prefers substrate sites
with acidic residues N-terminal to the modified residue, of which eight
can be found in the intracellular domain of human p75. In addition, the
phosphorylated sequence -S(P)-X-X-S- (where
S(P) indicates a phosphorylated serine) is also a substrate for CK-1,
suggesting CK-1 can act in a process termed hierarchical protein
phosphorylation (Kennelly and Krebs, 1991). If one takes the latter in
consideration, a total of 22 serine residues are potential
phosphorylation sites for CK-1. Recently it was found that a C-terminal
region of 78 amino acids within the cytoplasmic domain of p75,
mediating the interaction with TRAF proteins, was indispensable for
signal transduction toward cell proliferation and NF-B activation
in a T cell line (Rothe et al., 1994). The latter stretch of
amino acids contains 13 serine residues as potential acceptor sites for
CK-1-mediated phosphorylation. Preliminary results suggest that p75 is
mainly phosphorylated in this region. (
)
Recently, a
p75-associated kinase activity that phosphorylated p75 in U937 cells
has been described (Darnay et al., 1994a). In these studies
p75 was phosphorylated on serine and threonine residues, and a 59-kDa
p75-binding protein also served as a substrate. These properties
distinguish it from the p75 kinase activity described in the present
study. It has also been reported that cell extracts of TNF- or
IL-1-treated cells contain an activated kinase that specifically
phosphorylates -casein on serine and threonine residues, with
little activity on
-casein (Guesdon et al., 1993). The
latter observation, together with the sensitivity of
-casein
kinase to STS, makes it unlikely that the latter kinase is involved in
p75 phosphorylation.
Several cDNA clones for CK-1 s have been
isolated and sequenced (reviewed by Issinger(1993)). They have been
found in the cytosol, associated with membranes, and in the nucleus. A
large number of proteins become phosphorylated by CK-1. Some of these
substrates undergo defined functional changes when phosphorylated by
CK-1. For example, the regulatory M-subunit (inhibitor-2) of protein
phosphatase-1 appears to be phosphorylated by CK-1 and this inhibits
the phosphatase activity (Agostinis et al., 1987). CK-1 has
also been shown to phosphorylate the insulin receptor and the
progesterone receptor (Rapuano and Rosen, 1991; Chauchereau et
al., 1992). The latter could also be copurified with CK-1.
Interestingly, the p75 kinase as well as CK-1 could also phosphorylate
the cytoplasmic domain of the p55 receptor, providing a
mechanism for functional cross-talk between p55 and p75 TNF receptors.
No previous studies have linked CK-1 or p75 phosphorylation with
biological activities of TNF. Our observation that inhibition of CK-1
increases p75-mediated apoptosis indicates that CK-1 negatively
regulates TNF-induced signaling. The level at which this regulation
takes place is still unclear. Based on our observations of a
correlation between p75 phosphorylation in vitro and p75
signaling capacity in vivo (in vitro phosphorylation
and signaling in PC60p55p75 cells, not in U397 or HL60), it is tempting
to speculate that the phosphorylation level of p75 regulates its
signal-transducing capacity. p75 is constitutively phosphorylated in vivo (Pennica et al., 1992; Fig. 3A) and the turnover of this phosphorylation seems
to be high. For example, in PC60p55p75 cells, P
was found to be incorporated in p75 even after short (e.g. 15 min) in vivo labeling with
P
.
In addition, mild permeabilization of these cells (with 0.01%
digitonin) and incubation in the presence of
[
-
P]ATP resulted even after 5 s in
P labeling of p75. (
)Possibly, the p75 of
p75-responsive cells is associated with (a) constitutively active
phosphatase(s), the action of which results in the formation of an
effective substrate for in vitro phosphorylation. A role for
phosphatase activity in TNF signaling has already been suggested by the
observation that phosphatase inhibitors can inhibit TNF cytotoxicity in
certain cell lines (Totpal et al., 1992).
Taken together,
our data demonstrate that p75 is phosphorylated by a coprecipitating
kinase, which has been identified as CK-1. In addition, we have
obtained evidence that CK-1 negatively regulates TNF-induced signaling
to apoptosis. These findings provide a framework for future studies.
Ongoing studies with PC60 cells expressing intracellular p75 deletion
mutants of varying length have already indicated that phosphorylation
takes place in a C-terminal region of 78 amino acids within the
cytoplasmic domain of p75 that has been shown to be required for signal
transduction and association of TRAF proteins (Rothe et al.,
1994). Mutational analysis of the many intracellular serine
residues in this region may provide more insight regarding the
importance of p75 kinase activity in p75-mediated TNF signaling.