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
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ABSTRACT |
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Tumor necrosis factor-
(TNF-
) 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-
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-
isotype is a candidate enzyme for serine
phosphorylation of p60TNFR. Staurosporine and the PKC-
inhibitor
rottlerin inhibited TNF-
-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-
was the only PKC isotype that associated with p60TNFR within the
correct time frame for receptor phosphorylation. In vitro, only
PKC-
, but not the
-,
I-,
II-, or
-isotypes, was
competent to phosphorylate the receptor, indicating that p60TNFR is a
direct substrate for PKC-
. These findings suggest a selective role
for PKC-
in negative regulation of the p60TNFR and of
TNF-
-induced signaling.
cytokines; serine/threonine kinases; signal transduction; inflammation
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INTRODUCTION |
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THE CYTOKINE
TUMOR NECROSIS factor- (TNF-
) 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-
are dependent on neutrophil adherence to
a surface and the activation of
2-integrins. When
neutrophils are in suspension, TNF-
is an incomplete secretagogue
and only acts as a priming agent. Tight regulation of TNF-
-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-, 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 Fc
,
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-, -
I, -
II,
and -
), four Ca2+-independent but PS/DG-dependent
isotypes (PKC-
, -
, -
, and -
), and three
Ca2+/DG-independent, PS-dependent isotypes (PKC-
, -
,
and -
) (9). Phagocytic cells such as neutrophils and
monocytes possess multiple PKC isotypes, including
Ca2+-dependent PKC-
, -
I, and -
II,
Ca2+-independent PKC-
, and atypical PKC-
(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- activates phosphatidylcholine-specific phospholipase to cleave
phosphatidylcholine and generate DG, an activator of PKC-
, -
I,
-
II, and -
. TNF-
also activates phosphatidylinositol 3-kinase and the generation of PIP3 from phosphatidylinositol
4,5-bisphosphate, an activator of PKC-
and PKC-
. Thus TNF-
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-
and demonstrated that phosphorylation of p60TNFR on serine residues was associated with receptor downregulation.
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MATERIALS AND METHODS |
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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- 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- followed by
incubation with peroxidase-conjugated goat anti-mouse IgG. Other PKC
isotypes (PKC-
, -
I, -
II, and -
) 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- (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 [-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 (
,
I, and
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-
{[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- 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-
,
PKC-
I, PKC-
II, and PKC-
were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). A mouse monoclonal anti-PKC-
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-
, PKC-
I, PKC-
II, PKC-
and PKC-
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.
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RESULTS |
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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-
(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-
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-
or PMA for 5 min did not cause a loss of cell-associated p60TNFR. Thus the p60TNFR
is phosphorylated during neutrophil activation.
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TNF- 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-
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-
(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-
or PMA did
not elicit phosphorylation of p60TNFR on tyrosine residues. Thus the
enhanced 32P-labeling of the p60TNFR triggered by
TNF-
and PMA is concordant with phosphorylation of the receptor by a
Stau-sensitive serine/threonine kinase such as PKC.
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Stau enhances TNF--elicited elastase release.
We next examined the functional consequences of Stau-inhibitable serine
phosphorylation of p60TNFR. The effect of Stau on TNF-
-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-
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-
,
elastase release was enhanced to 125 ± 3% of TNF-
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-
triggered degranulation. myrPSS is based on the pseudosubstrate
domain of PKC-
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-
, elastase
release was enhanced to 153 ± 14% of TNF-
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-
-elicited degranulation.
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Association of different PKC isotypes with the p60TNFR.
To determine whether specific PKC isotypes play a role in
TNF--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-
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-
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-
-activated cells, there was no evidence of
PKC-
or PKC-
association with the p60TNFR (Fig. 5). In samples
obtained 15 and 30 min after TNF-
addition, both PKC-
I and
PKC-
II were found to associate with the p60TNFR. In contrast, the
Ca2+-independent PKC-
isotype associated with the
p60TNFR at the earliest time point taken after TNF-
activation
(i.e., 5 min).
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Rottlerin inhibits serine phosphorylation of p60TNFR.
To ascertain the role of PKC- in TNF-
-triggered phosphorylation
of p60TNFR in vivo, we used the PKC-
inhibitor rottlerin (8). Rottlerin is a relatively specific inhibitor of
PKC-
and inhibits PKC-
5- to 10-fold more potently than PKC-
and PKC-
and 13- to 33-fold more potently than PKC-
, with an
IC50 of 3-6 µM. Surface-adherent neutrophils were
pretreated with rottlerin (5 µM) for 10 min before the addition of
TNF-
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-
-induced serine phosphorylation of
the p60TNFR (Fig. 6A, Table
1) with little or no effect on
TNF-
-triggered threonine phosphorylation of the p60TNFR (Fig.
6B). Inhibition of TNF-
-triggered serine phosphorylation
was even greater than with Stau in the presence of the more specific
PKC-
inhibitor rottlerin, which decreased serine phosphorylation to
18% compared with TNF-
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-
-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-
in TNF-
-mediated serine phosphorylation of the p60TNFR.
|
|
p60TNFR is a substrate for PKC- in vitro.
To determine whether the p60TNFR is a direct substrate for PKC-
, 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 [
-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-
isotype, but not PKC-
,
-
I, -
II, or -
, was able to phosphorylate the
immunoprecipitated p60TNFR in a cofactor-dependent manner (Fig. 7).
Densitometry analysis revealed that p60TNFR phosphorylation by PKC-
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-
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-
in vitro, and
PKC-
, but not PKC-
, -
I, -
II, or -
, selectively
phosphorylates the p60TNFR in a cofactor-dependent manner.
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DISCUSSION |
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Our studies demonstrate that the p60TNFR is phosphorylated in
adherent neutrophils after cell activation triggered by TNF-. 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-
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-
triggered serine/threonine phosphorylation of
the p60TNFR but not tyrosine phosphorylation, indicating the
involvement of serine/threonine kinases. PKC was implicated in
TNF-
-triggered phosphorylation of the p60TNFR because 1)
the PKC activator PMA triggered phosphorylation of the p60TNFR,
2) PMA downregulated cellular responses to TNF-
, 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- and its cleaved
product associated with the p60TNFR within 5 min of cell activation,
which is coincident with TNF-
-induced phosphorylation of p60TNFR and
is in the correct time frame to allow PKC-
-catalyzed phosphorylation
of p60TNFR. In contrast, association of the PKC-
II and PKC-
I
isotypes with the p60TNFR was not apparent until 15-30 min after
TNF-
addition, a time well after the observation of TNF-
-mediated
phosphorylation of p60TNFR. The different time frames of recruitment of
PKC-
and PKC-
isotypes suggests different roles in
TNF-
-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- and whether it was isotype specific. Only PKC-
, but not
PKC-
, PKC-
I, PKC-
II, or PKC-
, was able to phosphorylate the
immunoprecipitated p60TNFR, indicating PKC isotype selectivity.
Furthermore, PKC-
phosphorylated the p60TNFR in a cofactor-dependent
manner, suggesting that the receptor is a direct substrate for PKC-
.
Cleaved PKC-
also phosphorylated p60TNFR in vitro (Kilpatrick,
Rossi, and Korchak, unpublished observations). Thus both full-length
and cleaved PKC-
are candidates for the in vivo phosphorylation of
p60TNFR. These results do not rule out the possibility that PKC-
may
not phosphorylate the receptor directly in vivo but may act indirectly
through alterations in signaling.
Elongation factor eEF-1, small heat shock protein HSP25/27,
Ser-23/Ser-24 in troponin I, and the
-chain of the high-affinity receptor for immunoglobulin E have previously been identified as
PKC-
-specific substrates (7, 10, 12, 17). Although all
PKC isotypes favor phosphorylation sites with strong basic environments, PKC-
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--mediated phosphorylation of the
p60TNFR is downregulation of receptor function. The ability of PKC to
downregulate TNF-
-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-
-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-
-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-
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-
-elicited responses of degranulation.
These findings indicate that activation of PKC elicits a
desensitization of the TNF-
receptor.
This study also demonstrated that multiple serine/threonine kinases are
involved in phosphorylation of the p60TNFR. TNF- triggered both
serine and threonine phosphorylation of the p60TNFR. However, Stau, and
the more specific PKC-
inhibitor rottlerin, inhibited serine
phosphorylation and not threonine phosphorylation. Therefore, PKC-
is implicated as having a major role in TNF-
-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-
. In contrast, TNF-
-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-
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- 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-
has a
major role in TNF-
-triggered serine phosphorylation of the p60TNFR
on the basis of the following lines of evidence. 1) TNF-
-triggered serine phosphorylation of the p60TNFR is inhibited by
the PKC-
inhibitor rottlerin. 2) Only PKC-
and the
PKC-
cleavage products associated with p60TNFR within the correct
time frame for receptor phosphorylation. 3) In vitro, only
PKC-
, but not PKC-
, -
I, -
II, or
-PKC, was competent to
phosphorylate the receptor in a cofactor-dependent manner, indicating
that the p60TNFR is a direct substrate for PKC-
. These results
suggest that, during TNF-
-initiated cell activation, PKC-
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.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-24840.
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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.
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