Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis.

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.

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 phospho-rylated and associated with CK-1, which negatively regulates p75-mediated TNF signaling.
Tumor necrosis factor (TNF) 1 is a pluripotent cytokine inducing an extraordinary wide variety of biological responses, mainly related to immune regulation and inflammation (reviewed by Vassalli (1993) and ). 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;Schü tze 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 TNFmediated 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(Darnay et al., , 1994bRothe 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.

MATERIALS AND METHODS
Reagents-All reagents were from Sigma unless otherwise stated. STS was from Boehringer (Mannheim, Federal Republic of Germany). CKI-7 was from Seikagaku Corp. (Tokyo, Japan). The synthetic peptide DDDEESITRR, referred to as CK-1-specific peptide in this paper, was obtained from Cambridge Research Biochemicals (Cambridge, United Kingdom). CK-1 was purified from pig spleen essentially as described (Agostinis et al., 1989); 1 unit of CK-1 incorporates 1 nmol of phosphate/ min in casein (2 mg/ml) at 37°C. Recombinant human TNF and the muteins R32WS86T and D143F, specific for human p55 and human p75, respectively, have been described (Van Ostade et al., 1992. Monoclonal antibodies to human p55 and p75 were from the htr and utr series, all of which are directed to the extracellular part of the p55 or p75 TNF receptor, respectively Espevik et al., 1990).
Cell Lines-The rat/mouse T cell hybridoma PC60 (Conzelmann et al., 1982) and derivatives , and the human U937 histiocytoma and HL60 promyelocytic leukemia cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc./BioCult, Paisley, United Kingdom) supplemented with 10% (v/v) fetal bovine serum, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol, and antibiotics. PC60p55p75 cells are derived from transfection of rodent PC60 cells with the human p55 and p75 genes as reported and have been described elsewhere . Human p55 expression in these cells was found to be lower than 50 molecules/cell. Human p75 was expressed at 5000 receptors/cell, numbers that are in the same range as endogenous p75 levels found in human U937 and HL60 cells. We reported previously that PC60p55p75 cells coexpressing the two types of human TNF receptors undergo TNF-mediated apoptosis, in contrast to cells expressing only one kind of TNF receptor (Vandenabeele et al., 1995). We further showed that, although the presence of both receptor types was required, triggering of only one type was sufficient to induce intermediate levels of apoptosis. Cells expressing a truncated p75 lacking its intracellular domain were obtained after transfection with a p75 expression vector in which the codons 293 and 294 (encoding Cys and Leu, respectively) of the human p75 were mutated to stop codons (TGA and TAG, respectively) according to Morinaga et al. (1984).
Cell Stimulation, Cell Lysis, and Immunoprecipitation of p75-Immunoprecipitation of p75 from cells labeled with 32 P i in vivo was performed essentially as described (Beyaert et al., 1990). Isolation of p75 immune complexes for in vitro phosphorylation reactions was as follows. After harvesting of the cells at room temperature, conditioned medium was collected for subsequent cellular stimulations. Prewarmed (37°C) conditioned medium (8 ml) with or without human TNF was then added to a 2-ml suspension of 10 8 cells in 200-ml tubes. After various incubation periods at 37°C, an excess of ice-cold phosphate buffer containing 50 M Na 3 VO 4 was added, followed by pelleting of the cells at 350 ϫ g for 10 min in a cooled (0°C) centrifuge. The number of cells used per immunoprecipitate varied from 2 ϫ 10 7 for PC60p55p75 up to 10 9 in case of U937 and HL60 cell lines. After one additional wash in ice-cold phosphate buffer, the cell pellet was resuspended at 2-5 ϫ 10 7 cells/ml in lysis buffer and rotated for 15 min at 4°C. The lysis buffer consisted of 1% (w/v) digitonin, 10 mM triethanolamine-HCl, pH 7.8, 139 mM NaCl, 10 mM NaF, 1 mM Na 3 VO 4 , 400 nM microcystin-LR, 180 g/ml phenylmethylsulfonyl fluoride, 0.27 trypsin inhibitory units/ml aprotinin, and 10 g/ml leupeptin. Following 10 min of centrifugation at 31,000 ϫ g, the supernatant was rotated for 2 h at 4°C in the presence of antibodies directed against human p75 (5 g/10 8 cells). The lysates were then mixed with 100 l of a 50% (v/v) slurry of either protein G-Sepharose 4 Fast Flow (Pharmacia LKB Biotechnology, Uppsala, Sweden), TSK-protein A (Affiland, Liège, Belgium), or Trisacryl GF-2000 protein A (Pierce). After a 90-min rotation at 4°C, immune complexes were spun down and washed three times in 4 ml of lysis buffer.
In Vitro Kinase Assay-Washed immunoprecipitates were resuspended in 60 l of 2 ϫ phosphorylation buffer (40 mM Tris-HCl, pH 7.4, 20 mM MgCl 2 , 200 M Na 3 VO 4 , 40 mM ␤-glycerophosphate, 200 M ATP, and 80 Ci/ml (27 nM) of [␥-32 P]ATP (3000 Ci/mmol; Amersham International, Amersham, United Kingdom)). Reaction mixtures (in some cases containing 1 mg/ml whole casein) were incubated for 10 min at 30°C and stopped by the addition of Laemmli sample buffer containing 2-mercaptoethanol (Laemmli, 1970). After heating, the mixtures were separated on a 10% SDS-polyacrylamide gel. For inhibition studies with protein kinase inhibitors, immunoprecipitates were pretreated for 10 min at 4°C in phosphorylation buffer without cold ATP and containing 100 g/ml ovalbumin as a stabilizing protein. Inhibitors remained present during the subsequent phosphorylation reaction, which was also performed in the absence of cold ATP. Quantification of phosphorylated bands was done by phosphorimaging analysis or by excising and counting the corresponding bands in a scintillation counter.
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.
Re-immunoprecipitation of Phosphoproteins Generated by in Vitro Phosphorylation-In some experiments, p75 was immunoprecipitated using utr4 antibody covalently coupled to Hydrazide AvidGel (BioProbe International, Tustin, CA). After washing and phosphorylation as described above, the phosphorylation reaction was now stopped by addition of 1 ml of lysis buffer supplemented with 1% SDS. After a 30-min rotation at room temperature, the supernatant was harvested and diluted with 12 ml of lysis buffer supplemented with 1.5% (w/v) Nonidet P-40 and 1 ml of lysate of untransfected PC60 cells, prepared at 5 ϫ 10 7 cells/ml. Following a 45-min rotation at 4°C, this supernatant mixture was again subjected to immunoprecipitation now using protein G-Sepharose and utr1 monoclonal antibodies.
Phosphoamino Acid Analysis-The 32 P-labeled p75 bands in SDSpolyacrylamide gels were localized by exposure of the gels to film, excised from the gel, and analyzed for phosphoamino acid content by two-dimensional electrophoresis as described (Hunter and Sefton, 1980). Partial Proteolytic Peptide Mapping-Peptide mapping was performed by limited proteolysis in SDS using Staphylococcus V8 protease, followed by analysis via gel electrophoresis as described (Van Roy et al., 1981).

Phosphorylation of the p75 TNF Receptor by a Kinase Present in p75
Immunoprecipitates-Using protein G-Sepharose and utr1, a species-specific antibody directed to the extracellular domain of human p75, immunoprecipitates were prepared from unstimulated rodent PC60 cells engineered to express human p55 and p75 (referred to as PC60p55p75). Following short term incubation of these precipitates with [␥-32 P]ATP, a major phosphorylated protein with a M r of approximately 75,000 was revealed on SDS-polyacrylamide gels (Fig. 1A). This 75-kDa phosphoprotein was absent in immunoprecipitates of control PC60 transfectants not expressing human p75, or expressing a human p75 lacking its cytoplasmic domain (p75⌬CD). Expression levels of wild-type and truncated p75 were similar in both transfectants, as judged by immunoblotting and flow cytometry (data not shown). The 75-kDa phosphoprotein could also be generated in immune complexes using the monoclonal antibodies utr2 and utr4, all of which are reactive with the extracellular part of human p75 (data not shown). Phosphoamino acid analysis of the 75-kDa phosphoprotein demonstrated that phosphorylation was exclusively on serine (Fig. 1B). Phosphate distribution found in p75 immunoprecipitated from cells that have been metabolically labeled with 32 P i is also mainly on serine, with a very low level of incorporation (3%) in threonine residues (Pennica et al., 1992). 2 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 32 P i 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.
To investigate the effect of TNF stimulation in vivo on p75 kinase activity in vitro, PC60p55p75 cells expressing fulllength 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 32 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). 2 Characterization of p75 Kinase Activity-To characterize the p75 kinase, we examined the effect of various drugs on in vitro p75 phosphorylation. We first tested STS, a very broad spectrum protein kinase inhibitor blocking, among others, most 2 R. Beyaert, unpublished data. FIG. 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 (PC60p55p75⌬CD) human p75 were made with (ϩ) or without (Ϫ) 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. members of the protein kinase C family, cAMP-dependent kinase, cGMP-dependent kinase, Ca 2ϩ /calmodulin-dependent protein kinase, and myosin light chain kinase (Beyaert et al., 1993). Under none of the conditions tested was STS found to affect p75 kinase activity (Fig. 3A), which means that a contribution of the above-mentioned kinases in the in vitro p75 phosphorylation is unlikely. The observation that, in contrast to p75 phosphorylation, STS did inhibit the phosphorylation of an unknown, faster migrating doublet, which was unspecifically bound by the Trisacryl GF-2000 protein A matrix, shows that STS is active under the conditions tested. In addition, the protein kinase C inhibitors H-7 and calphostin C, as well as the cAMP-dependent kinase inhibitor H-8 had no effect on p75 phosphorylation (data not shown). Although STS inhibits a broad spectrum of kinases, it has no effect on casein kinases. We therefore tested the contribution of the latter by testing p75 kinase activity in the presence of CKI-7 and heparin, which inhibit CK-1 and CK-2, respectively (Hathaway et al., 1980;Chijiwa et al., 1989). CKI-7 clearly inhibited p75 phosphorylation, while heparin had no effect (Fig. 3B). The inhibition of p75 phosphorylation was quantitated by phosphorimager analysis and found to be 100%, 80% and 45%, respectively at a CKI-7 concentration of 500 M, 100 M and 20 M. Similarly, in vivo phosphorylation of p75 from 32 P i -labeled PC60p55p75 cells was also diminished by CKI-7 pretreatment (75%, 45% and 24% inhibition, at CKI-7 concentrations of 500, 100, and 20 M, respectively; Fig. 4). In contrast, STS pretreatment had no effect on in vivo p75 phosphorylation (data not shown). The above observations, together with our finding that the p75 kinase can not use GTP as phosphate donor (further excluding CK-2; data not shown), indicate that p75 is phosphorylated by CK-1, or a CK-1-like enzyme, which is also present in p75 immunoprecipitates. The observation that p75 is a good substrate for a partially purified CK-1 also points in the same direction (data not shown). To further demonstrate that the p75 kinase has CK-1 like properties, we tested whether whole casein and the CK-1-specific peptide DDDEESITRR (Agostinis et al., 1989), are substrates for the p75 kinase identified in p75 immunoprecipitates of PC60p55p75 cells. A partially purified preparation of CK-1 that clearly phosphorylated casein and the specific peptide substrate was taken as a positive control ( Table  I). As expected, both substrates were also phosphorylated by the p75 kinase. However, casein, but not the CK-1-specific peptide, was also highly phosphorylated when incubated with control precipitates that were prepared without an antibody recognizing p75. The latter observation suggests that in addition to CK-1 which is specifically retained by p75, p75 immunoprecipitate still contains some aspecifically sticking kinase(s) that also phosphorylate(s) casein, thereby masking the effect of the p75 kinase on the latter substrate. Attempts to identify and eliminate this aspecifically binding kinase have so far been unsuccessful.
Inhibition of CK-1 Increases p75-mediated Apoptosis-Selective triggering of either the human p55 or p75 with receptorspecific muteins of human TNF has been shown to induce apoptosis in PC60 cells expressing human p55 and p75 (Vandenabeele et al., 1995). To investigate a possible correlation between phosphorylation by CK-1 and TNF responsiveness, we analyzed the effect of the specific CK-1 inhibitor CKI-7 on p55-mediated and p75-mediated apoptosis in PC60p55p75. Triggering was either with human TNF, which binds both TNF receptors, or by R32WS86T and D143F, which are specific for p55 and p75, respectively. TNF-induced apoptosis was maximal when both receptors were stimulated (Table II). Interestingly, CKI-7 significantly potentiated both p55-mediated and p75-mediated apoptosis, suggesting a negative regulation of the TNF response by CK-1. The above increase in apoptosis could not be explained by enhanced TNF binding, since the latter was not affected by CKI-7 treatment of the cells (data not shown).
p75 Phosphorylation in Vitro Correlates with Cellular p75 Responsiveness-To further analyze the role of CK-1-mediated phosphorylation in p75-mediated signaling, we examined p75 kinase activity in p75 immunoprecipitates prepared from human U937 and HL60 myeloid cell lines which, in contrast to PC60p55p75, are not responsive to selective p75 triggering (analyzed for apoptosis, cytokine production, NF-B activation, and hsp27 phosphorylation; data not shown). As shown in Fig.  5A (lanes 2 and 4), no in vitro phosphorylated 75-kDa protein was observed in p75 immunoprecipitates of U937 or HL60 cells, even when 20 times more cells were used (data not shown). The absence of in vitro phosphorylation of p75 was not due to differences in molecular forms of p75 in PC60p55p75 versus U937 or HL60 cells as similar p75 bands were revealed on immunoblots ( Fig. 5B; data for HL60 are not shown). Moreover, p75 phosphorylation in PC60p55p75 immune complexes was found to be unaltered after mixing with U937-derived p75 immunoprecipitates, excluding the presence in the latter of a p75-directed protease or phosphatase activity (data not shown). Also in the latter mix, p75 immunoprecipitates from U937 cells were not phosphorylated by the p75 kinase present in p75 immunoprecipitates from PC60p55p75 cells. In contrast to in vitro p75 phosphorylation, there was no obvious difference in phosphorylation of p75 immunoprecipitated from PC60p55p75 and U937 cells labeled with 32 P i in vivo (Fig. 5C). The above results might be explained by assuming that, in contrast to p75-unresponsive U937 and HL60 cells, p75-responsive PC60p55p75 cells regulate p75 phosphorylation to a level at FIG. 2. The 75-kDa phosphoprotein generated in vitro in p75 immunoprecipitates comigrates with p75 labeled in vivo with 32 P i and is reactive with antibodies to p75. A, immunoprecipitates of PC60p55p75 cells made without (Ϫ) or with (ϩ) 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 32 P i and immunoprecipitated with (ϩ) or without (Ϫ) 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 (lane 1) or coupled to utr4 (lanes 2-4) and subjected to in vitro phosphorylation. In lanes 1 and 2, the phosphorylation reaction was stopped by adding Laemmli loading buffer and directly analyzed on an SDSpolyacrylamide gel. In lanes 3 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 (lane 3) or with (lane 4) utr1. The arrowhead points to the position of p75. which some of the 43 possible phosphorylation sites are not phosphorylated, as reflected by its ability to be further phosphorylated in vitro, and resulting in a p75 that is able to transduce signals.

DISCUSSION
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 phosphorylationdephosphorylation 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 FIG. 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.
FIG. 4. Effect of CKI-7 on p75 kinase activity in vivo. 2 ϫ 10 6 PC60p55p75 cells were metabolically labeled with 100 Ci of 32 P i 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.

TABLE I
Phosphorylation of casein and a CK-1-specific peptide by p75 kinase and CK-1 p75 immunoprecipitates from 3 ϫ 10 7 PC60p55p75 cells prepared with utr1, as well as CK-1 (0.1 unit/ml) were assayed for in vitro kinase activity toward 1 mg/ml casein or 1 mg/ml CK-1-specific peptide as described under "Materials and Methods." A control immunoprecipitate was prepared in the absence of utr1. Results are the mean of two experiments (S.D. Ͻ 10%).

inhibitor CKI-7 on TNF-induced apoptosis in
PC60p55p75 cells Cells (5 ϫ 10 4 /well) were incubated for 20 h in the presence of medium (none), hTNF, the p55-specific mutein R32WS86T, the p75-specific mutein D143F, or combinations. The concentrations used induced maximal levels of apoptosis (data not shown). Where indicated, 150 M CKI-7 was added 1 h before the start of TNF treatment. After staining with propidium iodide, the number of apoptotic cells was analyzed as described under "Materials and Methods." Values are the mean of triplicate cultures with S.D. in parentheses. Induction of apoptosis by receptor-specific agonists and their combination have been described previously (Vandenabeele et al., 1995). effect of TNF on in vivo p75 phosphorylation (Pennica et al., 1992). 2 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. 3 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, 2 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, 32 P i was found to be incorporated in p75 even after short (e.g. 15 min) in vivo labeling with 32 P i . In addition, mild permeabilization of these cells (with 0.01% digitonin) and incubation in the presence of [␥-32 P]ATP resulted even after 5 s in 32 P labeling of p75. 4 Possibly, the p75 of p75-responsive cells is associated with (a) constitutively active phosphatase(s), the action of which results in the forma-3 W. Declercq, unpublished data. 4 B. Vanhaesebroeck, unpublished data.
FIG. 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 (lanes 1, 3, and 5) or with (lanes 2, 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 (lane 1) or U937 (lane 2) were immunoprecipitated with utr1 and immunoblotted as described under "Materials and Methods." C, lysates of U937 (lane 1) or PC60p55p75 (lane 2) labeled with 32 P i in vivo were immunoprecipitated with utr1 and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. tion 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). 4 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.