Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha -activated neutrophils

Laurie E. Kilpatrick, Young-Han Song, Michael W. Rossi, and Helen M. Korchak

Departments of Pediatrics and Biochemistry/Biophysics, University of Pennsylvania School of Medicine, and the Joseph Stokes Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNF-alpha ) triggers degranulation and oxygen radical release in adherent neutrophils. The p60TNF receptor (p60TNFR) is responsible for proinflammatory signaling, and protein kinase C (PKC) is a candidate for the regulation of p60TNFR. Both TNF-alpha and the PKC-activator phorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR. Receptor phosphorylation was on both serine and threonine but not on tyrosine residues. The PKC-delta isotype is a candidate enzyme for serine phosphorylation of p60TNFR. Staurosporine and the PKC-delta inhibitor rottlerin inhibited TNF-alpha -triggered serine but not threonine phosphorylation. Serine phosphorylation was associated with receptor desensitization, as inhibition of PKC resulted in enhanced degranulation (elastase release). After neutrophil activation, PKC-delta was the only PKC isotype that associated with p60TNFR within the correct time frame for receptor phosphorylation. In vitro, only PKC-delta , but not the alpha -, beta I-, beta II-, or zeta -isotypes, was competent to phosphorylate the receptor, indicating that p60TNFR is a direct substrate for PKC-delta . These findings suggest a selective role for PKC-delta in negative regulation of the p60TNFR and of TNF-alpha -induced signaling.

cytokines; serine/threonine kinases; signal transduction; inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CYTOKINE TUMOR NECROSIS factor-alpha (TNF-alpha ) plays an important role in the pathology of inflammatory diseases such as sepsis, rheumatoid arthritis, and asthma. This proinflammatory cytokine is capable of exerting profound effects on neutrophil function that, if not adequately controlled, can lead to host tissue damage (21). In adherent neutrophils, this proinflammatory cytokine triggers the release of oxygen radicals, degradative granule enzymes such as elastase, and cytoskeleton reorganization. These cellular responses to TNF-alpha are dependent on neutrophil adherence to a surface and the activation of beta 2-integrins. When neutrophils are in suspension, TNF-alpha is an incomplete secretagogue and only acts as a priming agent. Tight regulation of TNF-alpha -induced responses is required to prevent potentially destructive stimulation of neutrophils. One mechanism of control is at the level of receptor function.

Two receptors for TNF-alpha , one of 55-60 kDa (p60TNFR) and the other of 75-80 kDa (p80TNFR), have been cloned and sequenced and are members of the TNF-nerve growth factor receptor superfamily (16, 25). Neutrophils possess both TNF receptors, and the p60TNFR is responsible for proinflammatory cellular responses (20). Receptor phosphorylation and desensitization are important mechanisms in the regulation of receptor function. A role for protein kinase C (PKC) as a negative modulator has been demonstrated for multiple receptors, including, for example, the Fcepsilon , prostacyclin, GABA, rhodopsin, and transferrin receptors (5, 7, 11, 29, 30). Activators of PKC have also been shown to downregulate cellular responses to TNF mediated through the p60TNFR, suggesting a role for PKC in the regulation of this TNF receptor function (1, 26, 27, 31).

PKC, a phospholipid-dependent family of serine/threonine kinases, is implicated in multiple signal transduction pathways. PKC exists as a family of multiple isotypes having closely related structures, including four calcium/phosphatidylserine/diglyceride (Ca2+/PS/DG)-dependent isotypes (PKC-alpha , -beta I, -beta II, and -gamma ), four Ca2+-independent but PS/DG-dependent isotypes (PKC-delta , -epsilon , -eta , and -theta ), and three Ca2+/DG-independent, PS-dependent isotypes (PKC-lambda , -iota , and -zeta ) (9). Phagocytic cells such as neutrophils and monocytes possess multiple PKC isotypes, including Ca2+-dependent PKC-alpha , -beta I, and -beta II, Ca2+-independent PKC-delta , and atypical PKC-zeta (15, 18, 19).

Activation of PKC isotypes requires the generation of specific lipid mediators such as DG and phosphatidylinositol 3,4,5-trisphosphate (PIP3) and the elevation of cytosolic Ca2+. TNF-alpha activates phosphatidylcholine-specific phospholipase to cleave phosphatidylcholine and generate DG, an activator of PKC-alpha , -beta I, -beta II, and -delta . TNF-alpha also activates phosphatidylinositol 3-kinase and the generation of PIP3 from phosphatidylinositol 4,5-bisphosphate, an activator of PKC-delta and PKC-zeta . Thus TNF-alpha triggers the generation of DG and PIP3, selective cofactors for activation of different PKC isotypes (24, 28). A positive role for PKC is proposed in activation of O2- generation but not in degranulation (15); however, PKC can also act as a negative regulator of cell functions (9). In this study, we established that p60TNFR is phosphorylated selectively by PKC-delta and demonstrated that phosphorylation of p60TNFR on serine residues was associated with receptor downregulation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of human neutrophils. Neutrophils were isolated from heparinized venous blood (10 U/ml). We used standard isolation techniques (14), employing Ficoll-Hypaque centrifugation, followed by dextran sedimentation and hypotonic lysis to remove residual erythrocytes. Cells were suspended in a HEPES buffer (pH 7.3) with the following composition (in mM): 150 Na+, 5 K+, 1.29 Ca2+, 1.2 Mg2+, 155 Cl-, and 10 HEPES.

Elastase release. The extracellular release of the granule-associated enzyme elastase (an azurophil granule marker) from surface-adherent neutrophils was monitored by determining the cleavage of the substrate MeOSuc-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (13). Neutrophils were incubated at 37°C for 30 min before the addition of TNF-alpha or buffer. The kinetics of elastase release was monitored using a microplate fluorometer (Packard Instruments) at an excitation wavelength of 360 nm and emission wavelength of 460 nm. When appropriate, cells were preincubated with either 100 nM staurosporine (Stau, 10 min) or 5 µM myristoylated PKC pseudosubstrate (amino acid residues 19-27), a cell-permeable PKC inhibitor (myrPSS, 30 min), before the addition of stimulus.

Immunoprecipitation of p60TNFR. Immunoprecipitation (IP) buffer was added to 50 × 106 neutrophils and vortexed for 20 min at 4°C to solubilize the membrane fraction. The IP buffer consisted of 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1.0 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.2% NP-40, 0.027 TIU/ml aprotinin, 2 µg/ml leupeptin, and 5 mg/ml BSA. The supernatant was collected after centrifugation for 5 min. Three micrograms of monoclonal antibody to p60TNFR were then added to the 50 × 106 cells, which were then incubated overnight with shaking at 4°C. Anti-mouse IgG agarose (300 µl/tube) was added, and incubation continued for 1 h at 4°C with shaking. The reaction tubes were then centrifuged for 30 s, and the supernatants were discarded. The IgG agarose pellet was washed four times with IP buffer, and the sample was eluted by incubation for 15 min at 65°C in 2× SDS-PAGE sample buffer.

Western blots. Immunoprecipitated p60TNFR samples were run on a 4-12% gradient SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked for 1 h at room temperature with Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 and 1% BSA-3% casein. For Western blotting procedures that used mouse antibodies, the membrane was incubated with either mouse monoclonal anti-p60TNFR, mouse monoclonal anti-TyrP (PY20), or mouse monoclonal anti-PKC-zeta followed by incubation with peroxidase-conjugated goat anti-mouse IgG. Other PKC isotypes (PKC-alpha , -beta I, -beta II, and -delta ) were identified with a panel of rabbit polyclonal antipeptide antibodies. For analysis of protein phosphorylation, membranes were initially blocked for 2 h with a membrane blocking solution obtained from Zymed (San Francisco, CA) and then incubated with either a rabbit polyclonal anti-phosphoserine or anti-phosphothreonine antibody. The membranes were then incubated with peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive bands were visualized by Pierce SuperSignal ULTRA chemiluminescence substrate. Serine and threonine phosphorylation of p60TNFR was quantitated by densitometry analysis by Scan Pro of Western blots. Values are expressed in arbitrary densitometry units (ADU).

Protein phosphorylation in activated neutrophils. For measurement of phosphorylation of endogenous p60TNFR, neutrophils (100 × 106 cells/ml) were incubated for 60 min at 37°C with 32P-labeled Pi (200 µCi [32P]orthophosphoric acid/ml). 32P-labeled cells were plated onto a six-well plate (50 × 106 neutrophils/well) and incubated at 37°C for 30 min. Cells were then treated with buffer alone, TNF-alpha (10 ng/ml), or phorbol 12-myristate 13-acetate (PMA; 1 µg/ml) and incubated for an additional 5 min. Where appropriate, cells were pretreated with the inhibitor Stau (100 nM). Cells were harvested, the p60TNFR was immunoprecipitated as detailed above, and samples were run on 4-12% gradient SDS-PAGE, transferred to a PVDF membrane, and subjected to autoradiography.

Phosphorylation of p60TNFR by recombinant human PKC isotypes. p60TNFR was immunoprecipitated from nonactivated neutrophils as described above and used as substrate for recombinant human PKC (rhPKC) isotypes. The reaction mixture contained 20 mM HEPES (pH 7.4), 10 mM MgCl2, and 100 µM [gamma -32P]ATP. Reactions were carried out in the absence or presence of the cofactors PS (200 µg/ml) and DG (40 µg/ml). For Ca2+-dependent PKC isotypes (alpha , beta I, and beta II), 100 µM CaCl2 was added to the appropriate samples. All other samples contained 100 µM EGTA. Specific activity of the different rhPKC isotypes was normalized using a peptide based on the pseudosubstrate region of PKC-zeta {[Ser119]PKC ()} as substrate. Equivalent activities of the different PKC isotypes (7.5 units) were added to the reaction mixture, in which one unit was equal to one picomole of [Ser119]PKC(113-130) phosphorylated per minute. Incubations were carried out for 15 min at 30°C, and the reaction was stopped by the addition of 4× SDS-PAGE sample buffer. Samples were run on SDS-PAGE, transferred to a PVDF membrane, and subjected to autoradiography.

Statistical analysis. Results are expressed as means ± SE of n (no. of experiments). Data were analyzed by Student's t-test.

Reagents. Recombinant human TNF-alpha was obtained from R&D Systems (Minneapolis, MN). The monoclonal mouse anti-human p60TNFR was a kind gift from Immunex (Seattle, WA). Anti-phosphotyrosine PY20 was purchased from ICN (Costa Mesa, CA). Anti-peptide polyclonal antibodies to PKC-alpha , PKC-beta I, PKC-beta II, and PKC-delta were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal anti-PKC-zeta was obtained from Transduction Laboratories (Lexington, KY). Polyclonal rabbit anti-phosphoserine and anti-phosphothreonine were obtained from Zymed Laboratories (San Francisco, CA). rhPKC isotypes PKC-alpha , PKC-beta I, PKC-beta II, PKC-delta and PKC-zeta were obtained from Pan Vera (Madison, WI). Rottlerin was obtained from BIOMOL (Plymouth Meeting, PA), and myrPSS (amino acid residues 19-27) was purchased from Calbiochem (La Jolla, CA). PS was purchased from Avanti Polar Lipids (Alabaster, AL). BSA, EGTA, PMA, Stau, DG, goat anti-mouse IgG agarose, protease inhibitors (leupeptin and aprotonin), and PMSF were obtained from Sigma Chemical.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo phosphorylation of p60TNFR in activated neutrophils. To determine whether the p60TNFR was phosphorylated during neutrophil activation, cells were prelabeled with [32P]Pi, allowed to adhere to six-well plates for 30 min, and then exposed to buffer alone, TNF-alpha (10 ng/ml), or PMA (1 µg/ml) for 5 min at 37°C. The p60TNFR was then immunoprecipitated, and the immunoprecipitated samples were run on 4-15% gradient SDS-PAGE. Autoradiography of the gel demonstrated that phosphorylation of p60TNFR was low in nonactivated cells but was enhanced in cells stimulated with either TNF-alpha or PMA (Fig. 1A). To confirm that equal amounts of p60TNFR were obtained for each condition, the samples were probed by Western blotting with the antibody to p60TNFR that was used for the immunoprecipitation (Fig. 1B). The immunoprecipitate was also immunoreactive to p60TNFR antibodies obtained from R&D Systems and Biosource International (Camarillo, CA) (results not shown). Equivalent amounts of receptor were observed in all conditions, indicating that neutrophil activation with either TNF-alpha or PMA for 5 min did not cause a loss of cell-associated p60TNFR. Thus the p60TNFR is phosphorylated during neutrophil activation.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1.   Phosphorylation of p60 tumor necrosis factor-alpha receptor (p60TNFR) during neutrophil activation. Effect of staurosporine (Stau) is shown. 32P-labeled surface-adherent neutrophils were activated with buffer alone, TNF-alpha (10 ng/ml), or phorbol 12-myristate 13-acetate (PMA; 1 µg/ml) for 5 min at 37°C. Where indicated, Stau (100 nM) was added 5 min before addition of stimuli. p60TNFR was immunoprecipitated from each sample, run on 4-12% gradient SDS-PAGE, and followed by autoradiography (representative of 3 experiments). A: autoradiogram. B: Western blot using anti-p60TNFR.

Phosphorylation of the p60TNFR triggered by either TNF-alpha or PMA was inhibited by pretreatment of the cells with the kinase inhibitor Stau (Fig. 1A). The decreased phosphorylation of p60TNFR in the presence of Stau (100 nM) was not the result of varying receptor concentrations. As shown in Fig. 1B, equivalent amounts of receptor were present for each condition when the gels were probed for immunoreactivity to p60TNFR.

TNF-alpha triggers both serine and threonine phosphorylation of p60TNFR. The p60TNFR contains numerous potential phosphorylation sites in the cytoplasmic region of the receptor (16). To determine whether TNF-alpha triggers phosphorylation on serine, threonine, and/or tyrosine residues, p60TNFR was immunoprecipitated from stimulated and nonactivated adherent neutrophils and Western blots were probed with phosphoamino acid antibodies. Low-level immunoreactivity to TyrP was observed in p60TNFR immunoprecipitated from nonactivated cells (buffer alone) as well as in cells stimulated with either TNF-alpha (10 ng/ml) or PMA (1 µg/ml) (Fig. 2). Preincubation of samples with 100 nM Stau before addition of stimuli did not decrease the level of immunoreactivity to this anti-phosphotyrosine antibody (Fig. 2). Therefore, activation of neutrophils with TNF-alpha or PMA did not elicit phosphorylation of p60TNFR on tyrosine residues. Thus the enhanced 32P-labeling of the p60TNFR triggered by TNF-alpha and PMA is concordant with phosphorylation of the receptor by a Stau-sensitive serine/threonine kinase such as PKC.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2.   Tyrosine phosphorylation of the p60TNFR. Surface-adherent neutrophils were treated with either buffer alone, PMA (1 µg/ml), or TNF-alpha (10 ng/ml) for 5 min at 37°C (n = 3). Where indicated, Stau (100 nM) was added 5 min before addition of stimuli. p60TNFR was immunoprecipitated from each sample and run on 4-12% SDS-PAGE followed by Western blotting. A: Western blot using an anti-phosphotyrosine antibody (PY20). B: Western blot using anti-p60TNFR.

When Western blots were probed with antibodies to phosphoserine and phosphothreonine, there was low-level immunoreactivity in p60TNFR immunoprecipitated from untreated adherent neutrophils (Fig. 3). The addition of TNF-alpha (25 ng/ml) to adherent neutrophils resulted in a large increase in both serine and threonine phosphorylation (Fig. 3). Serine phosphorylation of the p60TNFR was significantly decreased when adherent neutrophils were pretreated with Stau (100 nM) compared with those treated with TNF-alpha alone (Fig. 3A). Densitometry analysis revealed that pretreatment with Stau decreased TNF-alpha -mediated serine phosphorylation to 58% (P < 0.05, n = 3) of those treated with TNF-alpha alone. In contrast, Stau pretreatment had no statistically significant effect on TNF-alpha -mediated threonine phosphorylation [Fig. 3B; P = not significant (NS), n = 3]. Increased immunoreactivity to both phosphoserine and phosphothreonine antibodies was sometimes observed with the addition of Stau to adherent neutrophils. This, however, was not a consistent finding nor was it statistically significant (P = NS, n = 3). Thus TNF-alpha triggers phosphorylation of both serine and threonine residues of the p60TNFR, but only serine phosphorylation is inhibitable by Stau.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Serine and threonine phosphorylation of the p60TNFR. Effect of Stau is shown. Surface-adherent neutrophils were treated with either buffer alone or TNF-alpha (25 ng/ml) for 5 min at 37°C. Where indicated, Stau (100 nM) was added 5 min before addition of stimuli. p60TNFR was immunoprecipitated from each sample and run on 4-12% SDS-PAGE as described in Fig. 2. A: Western blot using an anti-phosphoserine antibody. B: Western blot using anti-phosphothreonine antibody (representative experiment of 3).

Stau enhances TNF-alpha -elicited elastase release. We next examined the functional consequences of Stau-inhibitable serine phosphorylation of p60TNFR. The effect of Stau on TNF-alpha -triggered degranulation was examined by monitoring elastase release from azurophil granules. As shown in Fig. 4, adherent neutrophils stimulated with 25 ng/ml of TNF-alpha released elastase at a rate of 330 ± 38 arbitrary fluorescence units (AFU)/min, a significant increase over basal levels (180% of buffer alone, P < 0.01). When adherent neutrophils were pretreated with 100 nM Stau before the addition of TNF-alpha , elastase release was enhanced to 125 ± 3% of TNF-alpha alone (P < 0.01, n = 3). Stau alone did not significantly enhance elastase release compared with buffer alone (P = NS, Fig. 4). To ascertain whether the enhanced elastase release in the presence of Stau was the result of PKC inhibition, we next examined the effects of the more specific cell-permeant PKC inhibitor myrPSS (amino acid residues 19-27) on TNF-alpha triggered degranulation. myrPSS is based on the pseudosubstrate domain of PKC-beta and is a highly specific PKC inhibitor (6). When adherent neutrophils were preincubated in the presence of 5 µM myrPSS before the addition of TNF-alpha , elastase release was enhanced to 153 ± 14% of TNF-alpha alone (P < 0.01, n = 3, Fig. 4). myrPSS alone had no significant effect on elastase release compared with buffer alone (P = NS, Fig. 4). In summary, pretreatment of adherent neutrophils with the PKC inhibitors Stau and myrPSS was associated with enhanced TNF-alpha -elicited degranulation.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Stau enhances release of elastase triggered by TNF-alpha from adherent neutrophils. Surface-adherent neutrophils were stimulated with 25 ng/ml TNF-alpha in the absence or presence of 100 nM Stau or 5 µM myristoylated protein kinase C (PKC) pseudosubstrate (myrPSS). Release of elastase from azurophil granules was monitored at 37°C using the fluorescent substrate MeOSuc-Ala-Ala-Pro-Val-MCA and measured as arbitrary fluorescence units (AFU)/min. Representative experiment of 3 is shown.

Association of different PKC isotypes with the p60TNFR. To determine whether specific PKC isotypes play a role in TNF-alpha -triggered serine phosphorylation of the p60TNFR, we ascertained which PKC isotypes associated with the p60TNFR on neutrophil activation. Surface-adherent neutrophils were exposed to TNF-alpha for 0, 5, 15, and 30 min. The p60TNFR was immunoprecipitated, and the resulting immunoprecipitate was run on SDS-PAGE. As shown in Fig. 5, the level of cell-associated p60TNFR remained constant over the first 5 min of cell activation with TNF-alpha but declined thereafter as shown at the 15-min and 30-min time points. This decrease in cell-associated p60TNFR is presumably the result of receptor shedding and has been reported previously (2, 23). Western blotting using antibodies to different isotypes of PKC demonstrated that, in activated cells, there was selective association of p60TNFR with PKC isotypes, whereas, in nonactivated cells, there was little or no association (Fig. 5). In either nonactivated or TNF-alpha -activated cells, there was no evidence of PKC-alpha or PKC-zeta association with the p60TNFR (Fig. 5). In samples obtained 15 and 30 min after TNF-alpha addition, both PKC-beta I and PKC-beta II were found to associate with the p60TNFR. In contrast, the Ca2+-independent PKC-delta isotype associated with the p60TNFR at the earliest time point taken after TNF-alpha activation (i.e., 5 min).


View larger version (99K):
[in this window]
[in a new window]
 
Fig. 5.   Association of PKC isotypes with p60TNFR during activation of neutrophils by TNF-alpha . Surface-adherent neutrophils were stimulated with TNF-alpha for 0, 5, 15, and 30 min. p60TNFR was immunoprecipitated from each sample, run on 4-12% gradient SDS-PAGE, and followed by Western blotting (representative of 4 separate experiments). Western blotting by an anti-p60TNFR identified the p60TNFR. PKC-alpha , -beta I, -beta II, and -delta isotypes were identified by Western blotting using a panel of rabbit polyclonal antipeptide antibodies. PKC-zeta was identified using a mouse monoclonal anti-PKC-zeta . Molecular mass markers are indicated on left and p60TNFR and PKC isotypes on right.

It was also noted that two other proteins with a molecular mass of 40 and 60 kDa also associated with the p60TNFR at low levels in nonactivated cells and at greater concentrations in TNF-alpha -activated cells (Fig. 5). These proteins were immunoreactive to a PKC-delta antibody prepared against a peptide-derived from the COOH terminus of PKC-delta (residues 657-676). Incubation of anti-PKC-delta with the peptide used to prepare the antibody completely blocked reactivity to all three bands [40, 60, and 78 kDa (full-length PKC-delta ); data not shown], suggesting that the lower molecular mass bands were cleavage products of PKC-delta . On binding to p60TNFR, TNF-alpha activates caspase 3, an enzyme that cleaves PKC-delta and produces a cofactor-independent, enzymatically active PKC-delta (8). Thus TNF-alpha -mediated activation of caspase 3 is the most likely source of cleaved PKC-delta in our studies. Cleavage products of other PKC isotypes were not associated with the p60TNFR in either nonactivated or TNF-alpha -activated neutrophils (data not shown). In summary, full-length and cleaved PKC-delta were the only PKC isotypes that associated with the p60TNFR within 5 min of cell activation, the same time period in which the p60TNFR was phosphorylated and elastase release was triggered and before p60TNFR shedding.

Rottlerin inhibits serine phosphorylation of p60TNFR. To ascertain the role of PKC-delta in TNF-alpha -triggered phosphorylation of p60TNFR in vivo, we used the PKC-delta inhibitor rottlerin (8). Rottlerin is a relatively specific inhibitor of PKC-delta and inhibits PKC-delta 5- to 10-fold more potently than PKC-alpha and PKC-beta and 13- to 33-fold more potently than PKC-zeta , with an IC50 of 3-6 µM. Surface-adherent neutrophils were pretreated with rottlerin (5 µM) for 10 min before the addition of TNF-alpha or buffer alone. After a 5-min incubation, the receptor was immunoprecipitated and Western blots were probed with either phosphoserine or phosphothreonine antibodies. Similar to Stau, rottlerin markedly inhibited TNF-alpha -induced serine phosphorylation of the p60TNFR (Fig. 6A, Table 1) with little or no effect on TNF-alpha -triggered threonine phosphorylation of the p60TNFR (Fig. 6B). Inhibition of TNF-alpha -triggered serine phosphorylation was even greater than with Stau in the presence of the more specific PKC-delta inhibitor rottlerin, which decreased serine phosphorylation to 18% compared with TNF-alpha alone (P < 0.05, n = 3; Table 1, Fig. 6A). In contrast to serine phosphorylation, rottlerin, similar to Stau, did not have any significant effect on TNF-alpha -induced threonine phosphorylation of p60TNFR (P = NS, n = 3; Table 1, Fig. 6B). Pretreatment with rottlerin had no statistically significant effect on either serine or threonine phosphorylation compared with buffer alone (P = NS, n = 3; Table 1). These results provide additional evidence of a role for PKC-delta in TNF-alpha -mediated serine phosphorylation of the p60TNFR.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Serine and threonine phosphorylation of the p60TNFR. Effect of rottlerin is shown. Surface-adherent neutrophils were treated with either buffer alone or TNF-alpha (25 ng/ml) for 5 min at 37°C. Where indicated, rottlerin (5 µM) was added 10 min before addition of stimuli. p60TNFR was immunoprecipitated from each sample and run on SDS-PAGE as described in Fig. 3. A: Western blot using an anti-phosphoserine antibody. B: Western blot using anti-phosphothreonine antibody (representative experiment of 3).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Phosphorylation of p60TNFR

p60TNFR is a substrate for PKC-delta in vitro. To determine whether the p60TNFR is a direct substrate for PKC-delta , the p60TNFR was immunoprecipitated from nonactivated neutrophils and incubated with different recombinant PKC isotypes in the absence and presence of their appropriate cofactors. Samples were incubated in the presence of [gamma -32P]ATP and run on SDS-PAGE gels followed by autoradiography. The different PKC isotypes were capable of autophosphorylation (Fig. 7) and of both histone IIIS and [Ser119]PKC (residues 113-130) peptide phosphorylation (data not shown), confirming their enzymatic activity. However, when used at concentrations of equivalent peptide phosphorylating activity, only the rhPKC-delta isotype, but not PKC-alpha , -beta I, -beta II, or -zeta , was able to phosphorylate the immunoprecipitated p60TNFR in a cofactor-dependent manner (Fig. 7). Densitometry analysis revealed that p60TNFR phosphorylation by PKC-delta was significantly increased (72%) from 32.36 ± 8.76 to 55.63 ± 8.13 (SE) ADU (n = 3) in the presence of PS and DG compared with phosphorylation in the absence of cofactors (P < 0.05). When PKC-delta was incubated in the presence of cofactors but in the absence of added p60TNFR, there was no detectable radioactivity at the 60-kDa band, indicating that p60TNFR was indeed the substrate (Fig. 7). Conversely, when immunoprecipitated p60TNFR was incubated in the absence of rhPKC isotypes but in the presence of cofactors, no radioactive bands were observed (results not shown). Thus p60TNFR is a direct substrate for PKC-delta in vitro, and PKC-delta , but not PKC-alpha , -beta I, -beta II, or -zeta , selectively phosphorylates the p60TNFR in a cofactor-dependent manner.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 7.   Phosphorylation of p60TNFR by recombinant human PKC (rhPKC) isotypes. p60TNFR immunoprecipitated from nonactivated neutrophils was used as substrate for rhPKC isotypes and incubated with MgCl2 and [gamma -32P]ATP for 15 min at 30°C. Reactions were carried out in the absence or presence of appropriate cofactors [calcium, phosphatidylserine, and diglyceride (Ca, PS, DG, respectively)]. Reactions were terminated by the addition of Laemmli buffer, and the samples were subjected to SDS-PAGE followed by autoradiography (representative experiment of 4). Molecular mass markers are indicated on left and p60TNFR and PKC isotypes on right.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies demonstrate that the p60TNFR is phosphorylated in adherent neutrophils after cell activation triggered by TNF-alpha . Phosphorylation of p60TNFR was also triggered by the PKC activator PMA (Fig. 1) and by the chemotactic peptide fMet-Leu-Phe (data not shown). The finding that p60TNFR was phosphorylated in the absence of TNF-alpha suggests that phosphorylation of the receptor is independent of the activation state of the receptor and can be initiated by activation of other receptors. TNF-alpha triggered serine/threonine phosphorylation of the p60TNFR but not tyrosine phosphorylation, indicating the involvement of serine/threonine kinases. PKC was implicated in TNF-alpha -triggered phosphorylation of the p60TNFR because 1) the PKC activator PMA triggered phosphorylation of the p60TNFR, 2) PMA downregulated cellular responses to TNF-alpha , and 3) phosphorylation of the receptor was attenuated by the relatively nonspecific PKC inhibitor Stau and the more specific PKC inhibitor rottlerin.

PKC consists of a family of related isotypes; association of a specific PKC isotype with the p60TNFR is critical for phosphorylation of the receptor following neutrophil activation. Only PKC-delta and its cleaved product associated with the p60TNFR within 5 min of cell activation, which is coincident with TNF-alpha -induced phosphorylation of p60TNFR and is in the correct time frame to allow PKC-delta -catalyzed phosphorylation of p60TNFR. In contrast, association of the PKC-beta II and PKC-beta I isotypes with the p60TNFR was not apparent until 15-30 min after TNF-alpha addition, a time well after the observation of TNF-alpha -mediated phosphorylation of p60TNFR. The different time frames of recruitment of PKC-delta and PKC-beta isotypes suggests different roles in TNF-alpha -mediated signaling.

The ability of different PKC isotypes to phosphorylate p60TNFR in vitro was examined to determine whether the p60TNFR was a direct substrate for PKC-delta and whether it was isotype specific. Only PKC-delta , but not PKC-alpha , PKC-beta I, PKC-beta II, or PKC-zeta , was able to phosphorylate the immunoprecipitated p60TNFR, indicating PKC isotype selectivity. Furthermore, PKC-delta phosphorylated the p60TNFR in a cofactor-dependent manner, suggesting that the receptor is a direct substrate for PKC-delta . Cleaved PKC-delta also phosphorylated p60TNFR in vitro (Kilpatrick, Rossi, and Korchak, unpublished observations). Thus both full-length and cleaved PKC-delta are candidates for the in vivo phosphorylation of p60TNFR. These results do not rule out the possibility that PKC-delta may not phosphorylate the receptor directly in vivo but may act indirectly through alterations in signaling.

Elongation factor eEF-1alpha , small heat shock protein HSP25/27, Ser-23/Ser-24 in troponin I, and the gamma -chain of the high-affinity receptor for immunoglobulin E have previously been identified as PKC-delta -specific substrates (7, 10, 12, 17). Although all PKC isotypes favor phosphorylation sites with strong basic environments, PKC-delta is unique in that it will phosphorylate substrates with either basic or hydrophobic residues (12, 17, 22). This may account for its relatively distinct substrate specificity.

The functional consequence of PKC-delta -mediated phosphorylation of the p60TNFR is downregulation of receptor function. The ability of PKC to downregulate TNF-alpha -induced signaling has been demonstrated by PMA-induced receptor downregulation in numerous cell types (1, 26, 27, 31); this was confirmed by our finding in neutrophils in that the PKC inhibitors Stau and myrPSS enhanced TNF-alpha -induced elastase release. Degranulation does not involve PKC as a signal and is not triggered by PMA; therefore, any effect of a PKC inhibitor represents an effect at the receptor level. Phosphorylation of p60TNFR was not temporally associated with receptor shedding, an important long-term means of negatively regulating TNF-alpha -induced responses (23). Phosphorylation of p60TNFR and elastase release were observed within 5 min of cell activation, whereas loss of cell-associated p60TNFR, measured as immunoreactivity, was noted between 15 and 30 min after TNF-alpha addition (Fig. 5). Thus receptor shedding occurred later than receptor phosphorylation. Therefore, inhibition of PKC and inhibition of p60TNFR phosphorylation are associated with enhanced TNF-alpha -elicited responses of degranulation. These findings indicate that activation of PKC elicits a desensitization of the TNF-alpha receptor.

This study also demonstrated that multiple serine/threonine kinases are involved in phosphorylation of the p60TNFR. TNF-alpha triggered both serine and threonine phosphorylation of the p60TNFR. However, Stau, and the more specific PKC-delta inhibitor rottlerin, inhibited serine phosphorylation and not threonine phosphorylation. Therefore, PKC-delta is implicated as having a major role in TNF-alpha -triggered serine phosphorylation of the p60TNFR. Another serine kinase that also interacts with the p60TNFR was previously reported by Van Arsdale and Ware (32) and may in fact be PKC-delta . In contrast, TNF-alpha -triggered threonine phosphorylation of the p60TNFR is not PKC mediated, as phosphorylation was not inhibited by preincubation with either Stau or rottlerin. Possible candidates responsible for threonine phosphorylation include p42MAPK/ERK2, recently shown to phosphorylate the p60TNFR between residues 207 and 425 in the cytoplasmic domain, and TNFR-associated kinase, which phosphorylates between residues 344 and 397 (3, 4). Thus multiple kinases are involved in phosphorylation of the p60TNFR after TNF-alpha binding to the receptor. The precise serine/threonine phosphorylation sites on the p60TNFR have yet to be identified for any of these kinases. The cytoplasmic domain of the p60TNFR is rich in serine and threonine residues, and whether these different kinases show site specificity or are redundant remains to be determined.

In summary, TNF-alpha triggers both serine and threonine phosphorylation of the p60TNFR. Multiple kinases are involved in phosphorylation of the receptor and may mediate different functional effects. Serine phosphorylation of the receptor by PKC is associated with desensitization of cell responses such as degranulation, since inhibition of serine phosphorylation by PKC inhibitors resulted in enhanced elastase release. Our studies demonstrate that PKC-delta has a major role in TNF-alpha -triggered serine phosphorylation of the p60TNFR on the basis of the following lines of evidence. 1) TNF-alpha -triggered serine phosphorylation of the p60TNFR is inhibited by the PKC-delta inhibitor rottlerin. 2) Only PKC-delta and the PKC-delta cleavage products associated with p60TNFR within the correct time frame for receptor phosphorylation. 3) In vitro, only PKC-delta , but not PKC-alpha , -beta I, -beta II, or zeta -PKC, was competent to phosphorylate the receptor in a cofactor-dependent manner, indicating that the p60TNFR is a direct substrate for PKC-delta . These results suggest that, during TNF-alpha -initiated cell activation, PKC-delta initiates a negative feedback loop by catalyzing the phosphorylation and downregulation of the p60TNFR, leading to inhibition of the release of proinflammatory mediators. Downregulation of the p60TNFR on phagocytic cells such as the neutrophil could act as an important anti-inflammatory mechanism.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-24840.


    FOOTNOTES

Present address of Y.-H. Song: Molecular Genetics, Room 7119, Massachusetts General Hospital Cancer Center CNY-7, Building 149, 13th St., Charlestown, MA 02129.

Present address of M. W. Rossi: Astra-Zeneca Pharmaceuticals, Wayne, PA 19087.

Address for reprint requests and other correspondence: L. E. Kilpatrick, Immunology Section, Rm. 1207J Abramson Bldg., Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104 (E-mail: kilpatrick{at}emailchop.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 March 2000; accepted in final form 12 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aggarwal, BB, and Eessalu TE. Effect of phorbol esters on down-regulation and redistribution of cell surface receptors for tumor necrosis factor-alpha . J Biol Chem 262: 16450-16455, 1987[Abstract/Free Full Text].

2.   Bemelmans, MHA, Gouma DJ, and Buurman WA. LPS-induced sTNF-receptor release in vivo in a murine model. Investigation of the role of tumor necrosis factor, IL-1, leukemia inhibiting factor, and IFN-gamma . J Immunol 151: 5554-5562, 1993[Abstract/Free Full Text].

3.   Cottin, V, Van Linden A, and Riches DWH Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42mapk/erk2 induces changes in its subcellular localization. J Biol Chem 274: 32975-32987, 1999[Abstract/Free Full Text].

4.   Darnay, BG, Singh S, Chaturvedi MM, and Aggarwal BB. The p60 tumor necrosis factor (TNF) receptor-associated kinase (TRAK) binds residues 344-397 within the cytoplasmic domain involved in TNF signaling. J Biol Chem 270: 14867-14870, 1995[Abstract/Free Full Text].

5.   Davis, RJ, Johnson GL, Kelleher DJ, Anderson JK, Mole JE, and Czech MP. Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C. J Biol Chem 261: 9034-9041, 1986[Abstract/Free Full Text].

6.   Eichholtz, T, de Bont DBA, de Widt J, Liskamps RMJ, and Ploehg HL. A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J Biol Chem 268: 1982-1986, 1993[Abstract/Free Full Text].

7.   Germano, P, Gomez J, Kazanietz MG, Blumberg PM, and Rivera J. Phosphorylation of the gamma  chain of the high affinity receptor for immunoglobulin E by receptor-associated protein kinase C-delta . J Biol Chem 269: 23102-23107, 1994[Abstract/Free Full Text].

8.   Gschwendt, M. Protein kinase Cdelta . Eur J Biochem 259: 555-564, 1999[Abstract/Free Full Text].

9.   Hug, H, and Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J 291: 329-343, 1993[ISI][Medline].

10.   Jideama, NM, Noland TA, Jr, Raynor RL, Blobe GC, Fabbro D, Kazanietz MG, Blumberg PM, Hannun YA, and Kuo JF. Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties. J Biol Chem 271: 23277-23283, 1996[Abstract/Free Full Text].

11.   Kellenberger, S, Malherbe P, and Sigel E. Function of the alpha 1beta 2gamma 2S gamma -aminobutyric acid type A receptor is modulated by protein kinase C via multiple phosphorylation sites. J Biol Chem 267: 25660-25663, 1992[Abstract/Free Full Text].

12.   Kielbassa, K, Muller HJ, Meyer HE, Marks F, and Gschwendt M. Protein kinase C delta -specific phosphorylation of the elongation factor eEF-alpha and an eEF-1alpha peptide at threonine 431. J Biol Chem 270: 6156-6162, 1995[Abstract/Free Full Text].

13.   Kilpatrick, LE, Jacabovics E, McCawley LJ, Kane LH, and Korchak HM. Cromolyn inhibits assembly of the NADPH oxidase and superoxide anion generation by human neutrophils. J Immunol 154: 3429-3436, 1995[Abstract/Free Full Text].

14.   Korchak, HM, Kane LH, Rossi MW, and Corkey BE. Long chain acyl coenzyme A and signaling in neutrophils: an inhibitor of acyl coenzyme A synthetase, Triacsin C, inhibits superoxide anion generation and degranulation by human neutrophils. J Biol Chem 269: 30281-30287, 1994[Abstract/Free Full Text].

15.   Korchak, HM, Rossi MW, and Kilpatrick LE. Selective role for beta -protein kinase C in signaling for O2- generation but not degranulation or adherence in differentiated HL60 cells. J Biol Chem 273: 27292-27299, 1998[Abstract/Free Full Text].

16.   Loetscher, H, Pan YE, Lahm H, Gentz R, Brockhars M, Tabuchi H, and Lesslauer W. Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell 61: 351-359, 1990[ISI][Medline].

17.   Maizels, ET, Peters CA, Kline M, Cutler RE, Jr, Shanmugam M, and Hunzicker-Dunn M. Heat shock protein-25/27 phosphorylation by the delta  isoform of protein kinase C. Biochem J 332: 703-712, 1998[ISI][Medline].

18.   Majumdar, S, Kane LH, Rossi MW, Volpp BD, Nauseef WM, and Korchak HM. Protein kinase C isotypes and signal transduction in human neutrophils: selective substrate specificity of calcium dependent beta -PKC and novel calcium independent nPKC. Biochim Biophys Acta 1176: 276-286, 1993[ISI][Medline].

19.   Majumdar, S, Rossi MW, Fujiki T, Phillips WA, Disa S, Queen CF, Johnston RB, Jr, Rosen OM, Corkey BE, and Korchak HM. Protein kinase C isotypes and signalling in neutrophils: Differential substrate specificities of a translocatable, calcium and phospholipid-dependent beta -PKC and a novel calcium independent phospholipid-dependent protein kinase which is inhibited by long chain fatty acyl CoA. J Biol Chem 266: 9285-9294, 1991[Abstract/Free Full Text].

20.   Menegazzi, R, Cramer R, Patriarca P, Scheurich P, and Dri P. Evidence that tumor necrosis factor alpha  (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor. Blood 84: 287-293, 1994[Abstract/Free Full Text].

21.   Nathan, CF, and Sporn M. Cytokines in context. J Cell Biol 113: 981-986, 1991[ISI][Medline].

22.   Nishikawa, K, Toker A, Johannes FJ, Songyang Z, and Cantley LC. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J Biol Chem 272: 952-960, 1997[Abstract/Free Full Text].

23.   Porteu, F, and Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med 172: 599-607, 1990[Abstract].

24.   Reddy, SAG, Huang JH, and Liao WSL Phosphatidylinositol 3-kinase as a mediator of TNF-induced NF-kappa B activation. J Immunol 164: 1355-1363, 2000[Abstract/Free Full Text].

25.   Schall, TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GHW, Gatanaga T, Granger GA, Lentz R, Raab H, Kohr WJ, and Goeddel DV. Molecular cloning and expression of a receptor for human TNF. Cell 61: 361-370, 1990[ISI][Medline].

26.   Scheurich, P, Kobrich G, and Pfizenmaier K. Antagonistic control of tumor necrosis factor receptors by protein kinases A and C. Enhancement of TNF receptor synthesis by protein kinase A and transmodulation of receptors by protein kinase C. J Exp Med 170: 947-958, 1989[Abstract].

27.   Schleiffenbaum, B, and Fehr J. The tumor necrosis factor receptor and human neutrophil function. Deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation. J Clin Invest 86: 184-195, 1990[ISI][Medline].

28.   Schutze, S, Machleidt T, and Kronke M. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J Leukoc Biol 56: 533-541, 1994[Abstract].

29.   Smyth, EM, Hong Li W, and Fitzgerald GA. Phosphorylation of the prostacyclin receptor during homolgous desensitization. J Biol Chem 273: 23258-23266, 1998[Abstract/Free Full Text].

30.   Udovichenko, IP, Newton AC, and Williams DS. Contribution of protein kinase C to the phosphorylation of rhodopsin in intact retinas. J Biol Chem 272: 7952-7959, 1997[Abstract/Free Full Text].

31.   Unglaub, R, Maxeiner B, Thoma B, Pfizenmaier K, and Scheurich P. Downregulation of tumor necrosis factor (TNF) sensitivity via modulation of TNF binding capacity by protein kinase C activators. J Exp Med 166: 1788-1797, 1987[Abstract].

32.   Van Arsdale, TL, and Ware CF. TNF receptor signal transduction. Ligand-dependent stimulation of a serine protein kinase activity associated with (CD120a) TNFR60. J Immunol 153: 3043-3050, 1994[Abstract/Free Full Text].


Am J Physiol Cell Physiol 279(6):C2011-C2018
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society