(Received for publication, March 25, 1997)
From the Department of Inflammation Research, Merck Research Laboratories, Rahway, New Jersey 07065
Following interleukin (IL)-1 stimulation, the
majority of the cellular interleukin-1 receptor-associated kinase
(IRAK) translocates to a discrete subset of the Type I IL-1 receptor
(IL-1R1) in MRC-5 human lung fibroblasts. As the IRAK becomes
multiphosphorylated, it is degraded by proteasomes at a rate comparable
to that of the degradation of the phosphorylated IB
protein.
Proteasome inhibitors block the degradation of phosphorylated IRAK and
correspondingly increase the amount of IL-1R1 that can be
coimmunoprecipitated with IRAK. The nonspecific kinase inhibitor K-252b
blocks IRAK phosphorylation and degradation, but does not inhibit IRAK
association with the IL-1R1 indicating that translocation of IRAK to
the IL-1R1 and its phosphorylation are independent events. The IL-1
specificity of these effects is indicated by the lack of IRAK
phosphorylation and degradation by IL-1 in the presence of the IL-1
receptor antagonist or by the activation of MRC-5 cells by tumor
necrosis factor
. Long term exposure of MRC-5 cells to IL-1
desensitizes the resynthesized I
B
to IL-1, but not to tumor
necrosis factor
stimulation, but no additional effects on IRAK are
seen.
IL-11 is a master
cytokine responsible for the induction of a number of proteins
associated with inflammation, such as metalloproteinases, cyclooxygenase, nitric-oxide synthetase, and adhesion proteins (1).
Many of these responses are activated by the rapid activation of the
transcription factor NF-B following signal transduction by IL-1
or IL-1
bound to the type I IL-1 receptor (IL-1R1) (see Ref. 2 for
review). Activation of the IL-1R1 leads to the activation of at least
two kinases resulting in the phosphorylation of I
B
and the
activation of NF-
B. Following IL-1 activation, a distinctive Ser/Thr
kinase activity binds to and immunoprecipitates with the IL-1R1 (3, 4).
This purified IL-1 receptor-associated kinase (IRAK) is highly
homologous to the Drosophila kinase Pelle but not to other
mammalian Ser/Thr kinases, and it has only a limited ability to
phosphorylate I
B
(5, 6). A second Ser/Thr kinase has been
identified that is dependent upon ubiquitinylation for its subsequent
phosphorylation of I
B
(7). Presumably this latter kinase is
activated directly by IRAK or indirectly by as yet unidentified
intermediate kinases as well as the TNF receptor associated kinases
(see Refs. 8-11). The specific phosphorylation of I
B
on Ser-32
and Ser-36 by this ubiquitin-dependent kinase leads to the
recognition and destruction of the I
B
by proteasomes, which then
frees the NF-
B to translocate into the nucleus and begin
transcription (8, 12-16).
A similar signal transduction sequence has been observed in
Drosophila. In Drosophila Toll activates a
several-step signal transduction pathway leading to the nuclear
localization and activity of the transcription factor Dorsal, which is
highly homologous to mammalian NF-B (see Refs. 17 and 18)). The
IL-1R1 cytoplasmic domain is homologous to that of the
Drosophila receptor Toll, which provides the essential
signal for embryo ventralization following fertilization (19-21).
These homologous cytoplasmic regions of Toll and the IL-1R1 can be
interchanged so that a chimera of the Toll cytoplasmic domain and the
IL-1R1 extracellular domain is active when stimulated by
IL-1.2 The activity of Dorsal
is controlled by the inhibitor protein Cactus, which like its
homologous counterpart I
B
, is phosphorylated at discreet
N-terminal sites, and is then destroyed by proteasomes, freeing Dorsal
to migrate to the nucleus and begin transcription (22, 23). Key to the
phosphorylation of Cactus in Drosophila is the unique
Ser/Thr kinase Pelle, which is activated by its interaction with the
membrane bound adapter protein Tube via death domains found on the N
terminus of both proteins (24-26). While it appears that Tube recruits
Pelle to the Toll receptor following activation, the molecular details
of how this recruitment occurs and how Pelle is subsequently activated
are currently unclear.
Cao et al. (5) have shown in IL-1R1-transfected HEK cells
that IRAK is rapidly translocated to the IL-1R1 and becomes
multiphosphorylated following IL-1 stimulation. In the present paper we
show that a similar rapid translocation and phosphorylation of IRAK
occurs following IL-1, but not TNF, activation of normal human lung fibroblast MRC-5 cells. Following that activation IRAK largely disappears within minutes, and that destruction is prevented by proteasome inhibitors and by K-252b, a nonspecific kinase
inhibitor.
MRC-5 cells were obtained from the American Type
Culture Collection (ATCC CCL-171; Rockville, MD) and were grown in
Williams medium with 10% fetal bovine serum (JRH Biosciences, Lenexa,
KS). The LLL, IEAL, and LLNV proteasome inhibitors were obtained from Peptides International (Louisville, KY). The PP-2A phosphatase was
obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The
IL-1, TNF
, and soluble IL-1R1 were obtained from R & D Systems (Minneapolis, MN). K-252b was obtained from Alexis Biochemicals (San
Diego, CA). The I
B
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies to the IL-1R1 and IRAK
were raised at Covance Research Products (Denver, PA) against the
soluble IL-1R1 and against synthetic peptides of the indicated IRAK
sequence (see Fig. 1A). The peptides were prepared at R & D
Antibodies (Berkeley, CA) with an N-terminal Cys and coupled to bovine
thyroglobulin as described previously (57). IL-1RA was prepared at
Merck and shown to prevent the high affinity binding of IL-1
and
IL-1
on MRC-5 cells and to prevent the subsequent production of
PGE2.3 All other
reagents not specifically noted were obtained from Sigma.
Cell Growth and Sample Preparation
MRC-5 cells were
subcultured weekly and for experiments they were grown to confluence in
150-cm2 plates with medium supplemented with
penicillin-streptomycin and Mycostatin (Life Technologies, Inc.). One
150-cm2 plate was used for each immunoprecipitation. Prior
to use, the medium was replaced with medium with
penicillin-streptomycin, but no serum, for 16 h. Inhibitors
dissolved in Me2SO at 1000 × final concentration were
added 40 min prior to cell stimulation. IL-1 or TNF
were added
for 2-120 min at various concentrations, typically 10 ng/ml. For
harvesting, the plates were immediately chilled in an ice tray, washed
once with ice-cold PBS, and then scraped in ice-cold PBS containing 1 mM EDTA, 10 mM
-glycerol phosphate, and 1 mM Na orthovanadate. The cells were centrifuged, and the
cell pellet was lysed for 30 min on ice in 300 µl of a buffer (lysis
buffer) containing 50 mM HEPES, pH 7.5, 0.5% Nonidet P-40,
100 mM NaCl, 1 mM EDTA, 10 mM
-glycerol phosphate, 1 mM sodium orthovanadate, 10%
glycerol, and a protease inhibitor mixture containing 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 10 µg/ml
leupeptin. The cells were spun for 20 min at 2500 rpm in a Beckman GPR
centrifuge, and the supernatant was stored frozen at
80 °C.
The IRAK and IL-1R1
proteins were immunoprecipitated from the Nonidet P-40-solubilized
extract by the addition of 1-3 µl of antibody and incubation at
5 °C for 2-3 h on a rotator. Then 20 µl of a 50% slurry of
prewashed protein A-agarose beads was added to each sample, followed by
incubation for an additional 1 h at 5 °C. The samples were spun
in a microcentrifuge and washed twice in lysis buffer without glycerol
or protease inhibitors and then twice in a buffer (kinase buffer)
consisting of 20 mM HEPES, pH 7.5, 20 mM
MgCl2, 20 mM -glycerol phosphate, and 1 mM sodium orthovanadate.
For immunoblotting, the beads were resuspended in 20 µl of 2 × SDS sample buffer, separated on 15-lane, 4-12% SDS Tris/glycine gradient gels (Novex, San Diego, CA) for IRAK or IL-1R1 immunoblots and
10% gels for IB
immunoblots. Transfers were made to
polyvinylidene difluoride membranes with blocking, washing, and
visualization by ECL techniques as described previously (58). Dilutions
used in immunoblotting for the primary antibodies were 1:1000 for IRAK, 1:1000 for I
B
, and 1:3000 for the IL-1R1. For the secondary visualization step horseradish peroxidase-protein A (Amersham Life
Science, Inc.) was used at a 1:5000 dilution. Exposures were made from
5 s to 15 min. Films were scanned with a Quantity One densitometry
system (PDI, Huntington Station, NY).
For multiple immunoblots on the same samples, the membranes were stripped immediately following the immunoblotting. To strip the membranes, they were washed four times with PBS containing 0.05% Tween 20 (PBS/Tween) and incubated for 30 min at 50 °C in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol. The stripped membranes were then washed 6 times with PBS/Tween and immunoblotted for the next antibody. Each membrane was blotted for a maximum of three times.
Kinase AssayProtein A beads containing IRAK or IL-1R1 immunoprecipitated protein washed in kinase buffer were incubated in a 20-µl assay containing 2 µM ATP and 10 µCi of [33P]PO4 (Amersham) for 30 min at 30 °C. The samples were heated with 20 µl of 2 × SDS sample buffer, separated on 4-12% gels, blotted to polyvinylidene difluoride membranes, and exposed overnight to Kodak X-OMAT film. The samples were then immunoblotted as described above.
Phosphatase AssayImmunoprecipitated IRAK-agarose beads were washed twice in a buffer consisting of 50 mM Tris-HCl, pH 7.0, 0.1 mM CaCl2, and 2 mM NiCl2. In a 20-µl assay with the same buffer 0.5 unit of PP-2A was added, the samples were incubated 30 min, 37 °C, and then 20 µl of 2 × SDS sample buffer was added, followed by heating, and separation on SDS 4-12% gels.
IRAK is a multidomain protein containing an N-terminal
death domain, a kinase domain, and an undetermined C-terminal domain, which is absent in Pelle (Fig.
1A). In this diagram the
putative IRAK death domain homologous to that of Pelle is shown with
the six-core -helices (see Refs. 27 and 28), and the locations of
the kinase subdomains are shown together with the locations of the 15 conserved kinase residues (5, 29). The locations of the introns are
depicted by inverted triangles, based upon the sequence of
the human gene within Xq28 as reported in GenBankTM (accession number
U52112, deposited by M. Platzer, D. Bauer, and B. Drescher). Those
peptide regions used for the generation of polyclonal antipeptide IRAK
antibodies in the present report are indicated with a bar. All three
antisera immunoprecipitated a common protein band in unstimulated MRC-5
cells (Fig. 1B). When the MRC-5 cells were activated with
IL-1
, this sharp IRAK band disappeared and a diffuse higher
molecular weight band was formed as detected with the I700-712
antisera (Fig. 1B), analogous to the migration of the
phosphorylated IRAK reported earlier (5) (see below). A similar pattern
was seen when the I504/523 and I364/388 antisera were used for
immunoblotting, but the antisera had substantially weaker
immunoblotting activity (data not shown). No new immunodetectable
IRAK protein fragments were detected in the IL-1
stimulated cells
coincident with the disappearance of the IRAK protein band (data not
shown).
A detailed time course of IL-1 activation of the MRC-5 cells was
performed, and the amount of IRAK associated with the IL-1R1 was
observed by immunoprecipitation and immunoblotting with the corresponding antisera (Fig.
2A) In the absence of IL-1
stimulation, no IRAK was found associated with the IL-1R1. Within
30 s a small amount of IRAK was associated with the IL-1R1, and
this small amount of IRAK migrated at about the same or at just a
slightly slower rate as the unmodified IRAK observed in the IRAK
intraperitoneal (Fig. 2A). This association of IRAK with the
IL-1R1 was specific for IL-1 activation, because it was blocked by a
1000-fold higher concentration (10 µg/ml) of the IL-1 receptor
antagonist (IL-1RA (30); see Fig. 2B). Furthermore, the
IL-1R1/IRAK association was not induced when the MRC-5 cells were
stimulated with TNF
(see below).
The diffuse bands of IRAK observed immediately after IL-1 activation were due to phosphorylation of IRAK; in an in vitro [33P]PO4 kinase assay of IRAK immunoprecipitated from MRC-5 cells before and after IL-1 activation, the same immunoblottable diffuse IRAK bands could be labeled with [33P]PO4, whereas the faster migrating IRAK band found in the unstimulated cells was not labeled with [33P]PO4 (Fig. 2C). Multiple phosphorylations of the IRAK were observed in a time-dependent fashion with an increased phosphorylation observed at later times (Fig. 2A, 4- and 10-min time points versus 0.5 min) leading to a preponderance of the slowest migrating, most heavily phosphorylated forms at later times (see Fig. 2C; cf. Ref. 5). This conversion of IRAK to a phosphorylated form was not observed in the MRC-5 cells in which IL-1RA blocked the IL-1 activation (Fig. 2B). All of the observed phosphorylations on IRAK were on Ser and Thr residues; the elution of all the phosphorylated IRAK protein could be shifted to the more rapid rate observed with the unphosphorylated IRAK following treatment with the Ser/Thr-specific phosphatase PP-2A (Fig. 2D; see Ref. 31).
Scans of the the immunoblots showed that following a short lag the unphosphorylated IRAK disappeared with a t1/2 of about 2 min (bold line, Fig. 2E, top). This decrease probably reflected the conversion of unphosphorylated IRAK to the phosphorylated state, because it was reversed by phosphatase treatment (Fig. 2D). The amount of phosphorylated IRAK reached a maximum amount before 2 min and then began to rapidly decline (Fig. 2E, top panel). This pattern of the phosphorylated IRAK was observed regardless of whether the IRAK was detected by immunoprecipitation with an IRAK or an IL-1R1 antibody (Fig. 2E, top panel). If the amount of phosphorylated IRAK were normalized to that of the total IRAK detected to correct for the disappearance of IRAK, then maximal phosphorylation occurred later between 2 and 4 min (Fig. 2E, bottom). Hence, IRAK began to disappear as it was phosphorylated, so that the maximal amount of phosphorylated IRAK detected was a balance of the formation of phosphorylated IRAK and its degradation. The loss of IRAK observed within the Nonidet P-40-solubilized MRC-5 cells was not due to the translocation of IRAK to a detergent insoluble nuclear pellet, because immunochemical analysis of the detergent-insoluble pellets also showed the absence of IRAK (data not shown).
In contrast to the rapid disappearance of IRAK following IL-1 activation, the MRC-5 cell IL-1R1 is neither phosphorylated nor diminished in total amount (data not shown). If anything, the total amount of IL-1R1 that can be immunoprecipitated by IL-1R1 antibodies following IL-1 stimulation increases in amount during stimulation (see Fig. 2A, short exposure). The IL-1R1 seen by straight immunoblot or after immunoprecipitation of MRC-5 cell extracts by IL-1R1 consists of primarily two hazy bands. The same pattern is also found in the soluble IL-1R1 standard (missing the cytoplasmic tail and transmembrane region) that is expressed in mammalian cells (Fig. 2A). This heterogeneity is most likely a result of variable glycosylation on the IL-1R1, which is known to have multiple glycosylation sites producing receptors with 23-35% total carbohydrate (32). The IL-1R1 coimmunoprecipitated with IRAK comprises roughly 2% of the total IL-1R1 in the MRC-5 cells that can be immunoprecipitated by the IL-1R1. The form of the IL-1R1 associated with IRAK is principally that form that migrates the most slowly, perhaps with the largest amount of carbohydrate (Fig. 2A).
To compare the effects of IL-1 on IRAK activation and disappearance to
that of that of the subsequently activated IB
, aliquots of the
IL-1
-activated samples (Fig. 2A) were immunoblotted with an antibody specific for I
B
. The results showed that the
I
B
, like IRAK, was likewise phosphorylated by 1-2 min, and at
the same time it largely disappeared from the cells by 10 min (Fig. 3, top panel). Comparison of
the total amount of I
B
to the total amount of IRAK as
detected by scans of immunoblots indicated that the
t1/2 of disappearance of both
proteins was about 4 min (Fig. 3, bottom panel).
Hence, the disappearance from cells of both IRAK and I
B
occurred
rapidly and in a parallel fashion following their phosphorylation.
IRAK Phosphorylation Does Not Occur following TNF
MRC-5 cells activated for 5 min with a dose response
of IL-1 produced a graded response of IRAK activation, as measured by the formation of the phosphorylated band and the association with the
IL-1R1, as well as a graded disappearance of IRAK (Fig.
4, top and middle
panels). A comparable extent of IB
phosphorylation and
degradation in the same cells was observed (Fig. 4, bottom panel). In contrast, TNF
activation performed on a parallel
series of plates showed that while the MRC-5 cells were activated as measured by the phosphorylation and degradation of I
B
(Fig. 4,
bottom panel), there was no effect on either the
phosphorylation or the degradation of IRAK (Fig. 4, top
panel). Similarly in the TNF
stimulated cells, there was no
association of IRAK with the IL-1R1 (Fig. 4, middle panel).
Hence, as was noted earlier using IL-1RA (Fig. 2B), the
stimulus that induces IRAK association with the IL-1R1 and IRAK
phosphorylation is specific for IL-1 stimulation and is not a result of
an activated MRC-5 state.
Proteasome Inhibitors Prevent IRAK Disappearance
The addition
to activated cells of various inhibitors of proteasome activity have
enabled the accumulation of phosphorylated IB
and have prevented
its degradation. When effective concentrations of the peptide aldehyde
inhibitors Cbz-Leu-Leu-Leu-H (LLL, 25 µM (33)),
Cbz-Ile-Glu(O-t-butyl)-Ala-leucinal (IEAL, 60 µM (34)), or Cbz-Leu-Leu-norVal-H (LLNV, 50 µM (35)) were added to MRC-5 cells stimulated by IL-1
,
I
B
was retained for up to 40 min (Fig.
5A, bottom panel). The
destruction of IRAK within the same cells is similarly inhibited:
proteasome inhibitors lead to a retention of the phosphorylated form of
IRAK (Fig. 5A, top panel). The effects of the proteasome
inhibitors did not prevent the disappearance of the majority of the
nonphosphorylated IRAK (Fig. 5B, top panel), but rather
prevented the loss of the phosphorylated IRAK (Fig. 5B, bottom
panel). Comparison of changes in the total amount of IRAK in the
cells to that of I
B
showed that the proteasome inhibitors had a
similar effect quantitatively on the retention of both proteins following IL-1
stimulation (Fig. 5C).
Just as the proteasome inhibitors prevented IRAK destruction, they also increased the amount of IL-1R1 found associated with IRAK immunoprecipitates (Fig. 5D). The amount of the IRAK associated IL-1R1 doubled to a level of about 4% of the total immunoprecipitable receptor, but the type of IL-1R1 associating with IRAK (slower migrating form in SDS gels) remained the same. The total amount of IL-1R1 immunoprecipitated by the IL-1R1 antibody over the 40-min incubation period was not significantly changed, indicating that proteasome activity had no effects on IL-1R1 levels during IL-1 stimulation (data not shown).
IRAK Degradation Is Inhibited by the Kinase Inhibitor K-252bThe observation that it is the phosphorylated form of IRAK
that is degraded by proteasomes (Fig. 5B) led to the
prediction that an inhibitor of IRAK phosphorylation should prevent the
degradation of IRAK. Because staurosporine analogs are known to be
nonspecific inhibitors of a number of cellular kinases (36, 37), the
effect of K-252b on IRAK phosphorylation and degradation was determined in IL-1-stimulated MRC-5 cells. The formation of phosphorylated IRAK
(as determined by the band shift on SDS gels) particularly in cells
treated with 25 µM K-252b was delayed relative to the control, and at the same time the disappearance of IRAK was inhibited (Fig. 6, top panel). The
association of IRAK with the IL-1R1 was, however, not inhibited by
K-252b; in fact, the inhibited cells showed a greater amount of IL-1R1
associated with IRAK following activation (Fig. 6, middle
panel) similarly to what was observed when the degradation of IRAK
was inhibited by proteasome inhibitors (Fig. 5). Despite the effects on
IRAK, only modest effects were seen in the inhibition of I
B
degradation (Fig. 6, bottom panel). This suggests that
K-252b differentially inhibits the kinase activity that results in IRAK
hyperphosphorylation from that kinase activity producing I
B
phosphorylation.
Unstimulated I
Previously it had been observed that
prolonged IL-1 or TNF incubation with sensitive cells first led to
the degradation of I
B
, followed by its resynthesis and return
within 60 min (8, 38). To compare the corresponding effects of
continuous IL-1 administration on the presence of IRAK to that of
I
B
, MRC-5 cells were incubated for up to 2 h with IL-1
.
As is shown in Fig. 7 during prolonged
exposure of IL-1, cellular I
B
was quickly lost, but returned to
almost normal levels within 60 min (Fig. 7A, bottom panel;
Fig. 7B, middle panel). If the proteasome inhibitor LLL were
present, the initial loss of I
B
was delayed, and a considerable
amount of the I
B
was retained as the phosphorylated I
B
(Fig. 7A, bottom panel). The resynthesized I
B
,
however, was nonphosphorylated as seen by the absence of the
phosphorylated I
B
band with LLL at 60 and 120 min (Fig. 7A,
bottom panel). That the newly synthesized I
B
was
unstimulated was seen by the subsequent addition of TNF
to dishes of
MRC-5 cells that had been continuously stimulated for 105 min with
IL-1
. After 15 min of TNF
exposure the newly synthesized I
B
disappeared unless LLL was present in the incubation, at which point
much of the I
B
was present as the phosphorylated form (Fig.
7A, bottom right set of panels). In contrast, the readdition
of more IL-1
had no effect on the phosphorylation or disappearance
of I
B
, indicating that the cells were down-regulated with respect
to IL-1-mediated activity.
The long term effects of the IL-1 treatment on IRAK in the MRC-5 cells
were much less dramatic than those of IB
. While most of the IRAK
was phosphorylated and removed by 12-30 min, a small amount of both
phosphorylated and unphosphorylated IRAK persisted for as long as 120 min (Fig. 7, A and B, top panels). Addition of
LLL in the incubation increased the amount of phosphorylated IRAK, but
had no effect on the unphosphorylated IRAK (Fig. 7A, top
panel). It thus appeared that there was a continuous production of
a newly synthesized pool of IRAK that became phosphorylated and
subsequently degraded unless inhibited by the presence of LLL. IL-1R1
levels associated with IRAK gradually declined after 12-60 min unless
LLL was present (Fig. 7A, middle panel; Fig. 7B,
bottom panel; compare with Fig. 7B, top panel). As
expected from the prior presence of IL-1 with these cells and the lack of effects of TNF
on IRAK, analysis of the IRAK in the cells that
had received additional TNF
or IL-1
at 105 min showed no difference from the cells that had continuous IL-1
exposure (Fig. 7A, top right set of panels).
In the present report we confirm in a normal human fibroblast line
the previous observations in IL-1R1-transfected HEK cells that IRAK
associates with the IL-1R1 and becomes phosphorylated after IL-1
stimulation (5). We now find that following its phosphorylation IRAK
becomes a target for cellular proteolysis by proteasomes in a similar
manner to that seen previously with the phosphorylation and subsequent
proteasome destruction of IB
. IRAK phosphorylation and its
destruction is specific for activation of the IL-1R1: prevention of
IL-1R1 activation by the specific IL-1 receptor antagonist IL-1RA or
independent cellular and I
B
activation by other stimulators such
as TNF
have no effect on the phosphorylation and destruction of
IRAK. Inhibition of IRAK phosphorylation, like inhibition of proteasome
activity, does not, however, prevent association of IRAK with the
IL-1R1, indicating that translocation of IRAK to the IL-1R1 occurs
prior to or independently of IRAK phosphorylation. The low
phosphorylation state of IRAK observed when it initially translocates
to the IL-1R1 (see Fig. 2A and Ref. 5) suggests that the
phosphorylation of IRAK occurs subsequent to its translocation. Either
it is the association of IRAK with the IL-1R1 perhaps together with the
IL-1 accessory protein (see Ref. 3) that activates IRAK producing its
autophosphorylation, or there is present another kinase that
cotranslocates to the same IL-1R1·IRAK complexes that can
phosphorylate IRAK in a K-252b-inhibitable fashion.
The model for IRAK behavior following IL-1 activation is diagrammed in
Fig. 8. In the unstimulated cell state
(Fig. 8A) IRAK is a presumably cytoplasmic inactive kinase
(4) that is not associated with the IL-1R1. The IL-1R1 itself exists on
the membrane as a mixture of receptors of differing glycosylation state
(32). In Fig. 8A the IL-1R1 is depicted as containing either
five or two glycosylation chains, although the exact molecular
differences between these forms are unknown. When IL-1 occupies the
IL-1R1, IRAK translocates to the most highly glycosylated form of the IL-1R1 (Fig. 8B), where it is also found associated with the
IL-1 accessory protein (data not shown). As IRAK becomes associated with the IL-1R1, it becomes increasingly phosphorylated and
simultaneously activated (4). Because roughly three fuzzy bands can be
seen following activation (Fig. 2A and Ref. 5), IRAK is
depicted with three phosphates attached, although exactly how many
residues are phosphorylated, and to what extent, is not known. Based
upon the conservation of sequence of IRAK with the
Drosophila Pelle kinase (5) and its known requirement for
its N-terminal death domain for activation (25, 26), we assume that it
is this domain of IRAK that is responsible for its association with the cytoplasmic domain of the IL-1R1.
Both IL-1 and TNF stimulate a ubiquitin-dependent kinase
that is responsible for the phosphorylation of I
B
(7). Presumably this I
B
-kinase is activated directly or indirectly through
unknown intermediary kinases by IRAK as well as by kinases associated with the TNF
Type I (10, 11) and/or Type II receptors (9, 40). The
resultant I
B
phosphorylation tags I
B
for its subsequent destruction by proteasomes (8, 13-15, 41) and enables the subsequent
activation and nuclear translocation of NF-kB (see Ref. 42). The
phosphorylated IRAK is also largely destroyed by proteasome activity at
a rate comparable with that of I
B
. Degradation of both IRAK and
I
B
is retarded by proteasome inhibitors, but proteasome
inhibitors do not prevent the association of IRAK with the IL-1R1 nor
the subsequent conversion of the nonphosphorylated IRAK to the
phosphorylated form. It is not clear at this point how closely
correlated the activation of IRAK is relative to its total
phosphorylation. The addition of 25 µM K-252b inhibits
IRAK phosphorylation to the extent that IRAK is largely undegraded by
10 min, yet it has little effect on the subsequent activation of the
I
B
kinase as measured by the phosphorylation and degradation of
I
B
(Fig. 6). This suggests that IRAK may still be activated (perhaps even phosphorylated on its activation loop, see Ref. 43) to a
sufficient extent to lead to subsequent I
B
phosphorylation, but
not so extensively phosphorylated that it itself is recognized for
proteasome destruction. Alternatively, the signaling pathway may be so
amplified that a small amount of IRAK activity still formed at 25 µM K-252b is sufficient to activate enough of the downstream kinases to phosphorylate I
B
. Unfortunately the amount of IRAK activity in the MRC-5 cells using artificial exogenous substrates is too low to accurately correlate its activity with the
extent of its phosphorylation (data not shown).
Clearly there are at least two forms of IRAK present within the cells. The first and most prominent form of IRAK is that form that associates with the IL-1R1, becomes phosphorylated, and is then degraded. Despite the destruction of the majority of IRAK following IL-1 stimulation, a significant amount of IRAK remains undegraded, replaced perhaps by new synthesis, to enable the prolonged immunoprecipitation of the IL-1R1. But the steady state amount of IRAK during prolonged IL-1 stimulation is probably only 20% of the original amount (see Figs. 5C and 7B, top panels), yet the amount of the IL-1R1 remaining associated with IRAK is almost unchanging (Figs. 5D and 7B, bottom panels). This suggests perhaps that the IL-1R1 comes together in an unknown complex containing a number of IL-1R1 molecules associated with IRAK. Perhaps once formed, this IL-1R1 complex is stable so that only a small amount of IRAK need be present to enable the immunoprecipitation of the whole complex. Proteasome inhibitors do not change this stoichiometry; proteasome inhibitors lead to the retention of double the amount of IRAK as well as double the amount of IL-1R1 that can be coimmunoprecipitated (see Fig. 7B, top and bottom panels).
The number of IL-R1 molecules that we observe to be immunoprecipitated in these experiments is comparable with earlier reports of IL-1R1 molecules that need to occupied for cell activation. MRC-5 cells contain about 3000 high affinity IL-1 receptors/cell (KD ~10 pM (44)), a level which is among the highest of any cell lines (see Ref. 45). Occupation by IL-1 of fewer than 20 receptors/cell is sufficient for activation (46), resulting in only 2-3% of the available receptors that need to be utilized (see Ref. 47). In line with these former observations, we have observed that from a plate containing 5 × 106 MRC-5 cells we immunoprecipitate with anti IL-1R1 antibodies an amount of IL-1R1 comparable with 1 ng of sIL-1R1 (estimated molecular weight: 60,000, (48); see, for example, Fig. 2A). This corresponds to an immunoprecipitation of 2500 IL-1R1 molecules/MRC-5 cell. Following IL-1 stimulation and immunoprecipitation by IRAK antibodies, only 2-4% of these receptors become associated with IRAK. Because apparently only a portion of the most highly glycosylated form of the IL-1R1 becomes associated with IRAK, those IL-1R1 molecules associating with IRAK are a distinct subset of the total IL-1R1 molecules in the MRC-5 cells. It is possible that different glycosylation or other unidentified posttranslational modifications yield IL-1R1 molecules of varying affinity for IL-1 on the extracellular surface or for IRAK on the cytoplasmic surface.
A second form of IRAK comprises that small percentage of IRAK that remains as unphosphorylated, unstimulated IRAK (see e.g. Figs. 5A and 7A, top panels). It is not found associated with IL-1R1 immunoprecipitates (Fig. 2A and data not shown), suggesting that it is not associated with the IL-1R1 at any time during the IL-1 stimulation. The amounts of this form of IRAK do not change with time, nor are they increased in the presence of proteasome inhibitors. It is possible that this unmodified IRAK represents a pool of unphosphorylated IRAK sequestered in a different part of the cell. Alternatively this IRAK form may be structurally different, because it has varying posttranslational modifications or is translated from an alternatively transcribed mRNA.
While TNF clearly activates MRC-5 cells to a similar extent as IL-1,
it is unknown which is the responsible TNF receptor in the MRC-5 cells.
Most likely it is the 55-kDa TNFR1 that is active, since it is known
that the TNFR1 is more widespread among cell types and that the TNFR1
is thought to be associated with the activation of nonlymphoid cells
(see Refs. 49 and 50 for reviews). Furthermore, when cells containing
both types of TNF receptors were activated with TNF muteins specific
for individual TNF receptors (51), only the TNFR1 produced an
activation of NF-
B (52). This kinase has recently been identified as
the receptor interacting protein, which also interacts with the TNFR1 via its death domain sequence (53-56).
Following its initial degradation, IB
is resynthesized and
returns as a nonphosphorylated protein insensitive to further IL-1
activation (see Fig. 8C). Because this new pool of I
B
remains sensitive to activation by TNF
, however, the
ubiquitin-dependent kinase is still functional, whereas the
IL-1/IRAK activation pathway is down-regulated (Fig. 8C).
Prolonged IL-1 stimulation has little overall effect on the levels of
IL-1R1 in the MRC-5 cells nor their ability to be associated with IRAK.
There is present from 30 to 120 min a persistent low level amount of
phosphorylated IRAK as seen by immunoblots as well as a small amount of
the apparently unphosphorylated IRAK (Fig. 7A) that suggests
a steady state resynthesis, activation, and subsequent degradation of
IRAK. The presence of proteasome inhibitors increases the steady state
amount of the phosphorylated IRAK that is detected and the
corresponding amount of the IL-1R1 that is coprecipitated. Neither the
addition of more IL-1
nor of TNF
has any further effect on these
levels.
We thank John Mudgett, Jack Schmidt, Mike Tocci, and Rick Mumford for helpful discussions and comments on this manuscript and John Shockey for the preparation of the figures.