©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The p60 Tumor Necrosis Factor (TNF) Receptor-associated Kinase (TRAK) Binds Residues 344397 within the Cytoplasmic Domain Involved in TNF Signaling (*)

Bryant G. Darnay , Sanjaya Singh , Madan M. Chaturvedi (§) , Bharat B. Aggarwal (¶)

From the (1)Cytokine Research Laboratory, Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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)()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.

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS AND DISCUSSION

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).

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 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 8, 12, and 15 Contain the Highest Histone Kinase Activity

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 (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).

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.


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.




FOOTNOTES

*
This research was conducted, in part, by The Clayton Foundation for Research and was supported, in part, by new program development funds from The University of Texas M. D. Anderson Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Zoology, Banaras Hindu University, Vararnasi, 22015 India.

To whom all correspondence and reprint requests should be addressed: Cytokine Research Laboratory, Department of Molecular Oncology, Box 41, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030. Tel.: 713-792-3503; Fax: 713-794-1613.

The abbreviations used are: TNF, tumor necrosis factor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; TRAFs, TNF receptor-associated factors; TRAPs, TNF receptor-associated proteins; TRAK, TNF receptor-associated kinase.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.