From the Department of Cell Biology, University Medical Center Utrecht and Institute of Biomembranes, Heidelberglaan 100, AZU-G02.525, 3584 CX Utrecht, The Netherlands
Received for publication, April 28, 2000, and in revised form, December 18, 2000
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ABSTRACT |
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The growth hormone receptor (GHR)
intracellular domain contains all of the information required for
signal transduction as well as for endocytosis. Previously, we
showed that the proteasome mediates the clathrin-mediated endocytosis
of the GHR. Here, we present evidence that the proteasomal inhibitor
MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably
through stabilization of GHR and JAK2 tyrosine phosphorylation. If
proteasomal inhibitor was combined with ligand in an
endocytosis-deficient GHR mutant, the same phenomenon occurred
indicating that proteasomal action on tyrosine dephosphorylation is
independent of endocytosis. Experiments with a GHR-truncated tail
mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by
the loss of a phosphatase-binding site. This raised the question of
what happens to the signal transduction of the GHR after its
internalization. Co-immunoprecipitation of GH·GHR complexes
before and after endocytosis showed that JAK2 as well as other
activated proteins are bound to the GHR not only at the cell surface
but also intracellularly, suggesting that the GHR signal transduction
continues in endosomes. Additionally, these results provide
evidence that GHR is present in endosomes both in its full-length and
truncated form, indicating that the receptor is down-regulated by the proteasome.
The growth hormone receptor
(GHR)1 is a member of the
cytokine/hematopoietin receptor superfamily (for review, see Ref. 1). Cytokines regulate different aspects of cellular growth,
differentiation, and activation and play a critical role in immune and
inflammatory responses. In response to GH, two GHR polypeptides
dimerize, turning on a cascade of events leading to signal transduction
by activating gene transcription in the cell nucleus and, at the same
time, down-regulation and degradation of the receptor (2, 3).
One major characteristic of the cytokine receptor family is the absence
of an intrinsic tyrosine kinase activity. Upon dimerization, the GHR
recruits and activates JAK2, a member of the Janus family of cytosolic
kinases (4-6). Once bound, the two JAK2 molecules are in opposition
and can transphosphorylate each other. Subsequently, the receptor
chains become tyrosine phosphorylated allowing them to interact with
other intracellular signaling components (6). JAK2 acts via special
signal transducers and activators of transcription proteins (STATs),
which dimerize and translocate to the nucleus to convey the appropriate
signal to specific regulatory DNA-response elements (7, 8). In
addition, JAK2 activation by GH facilitates initiation of various
pathways including the Ras, mitogen-activated protein kinase (MAPK),
the insulin receptor substrate (IRS) and the phosphatidylinositol
3-kinase (PI3K) pathway (9, 10). GH-induced activation of the JAK-STAT
signal transduction pathway is both rapid and transient. The molecular
mechanism of JAK deactivation is still poorly understood. Part of the
dephosphorylation of the GHR has been previously attributed to the
activation of the tyrosine phosphatase SHP-1 (11). This enzyme was
found to interact with JAK2, and GH stimulates the catalytic activity
of SHP-1 (11). Another candidate could be SHP-2 because it associates
with the GHR and binding to JAK2 has also been reported. However, no
dephosphorylation of JAK2 by SHP-2 could be demonstrated (12).
Recently, another negative regulatory pathway of the GH receptor
signaling involving the SOCS (suppressor of cytokine signaling)
proteins has been identified (13, 14). The SOCS proteins appear to form
part of a negative feedback loop that regulates cytokine signal
transduction. Their expression is rapidly induced by activation of the
JAK/STAT pathway (15).
Another important system which down-regulates the GHR is the
ubiquitin-proteasome system. This system regulates the degradation of
nuclear and cytosolic proteins via the proteasome (16). The target
proteins are first tagged with ubiquitin molecules to form a
polyubiquitin chain, which is specifically recognized by the multisubunit proteasome complex, leading to their degradation. Proteasomes were found to be involved in regulating JAK/STAT pathways upon interleukin-2, -3, and erythropoietin stimulation (17-19). In the
presence of specific proteasomal inhibitors, activation of both JAK and
STAT molecules was sustained, although neither STAT nor JAK appeared to
be ubiquitinated. These data indicate that proteasomes are involved in
the down-regulation of the activation signals of specific cytokine receptors.
An important factor in GHR down-regulation is its endocytosis. In the
presence of ligand, GHR endocytoses rapidly via clathrin-coated pits
(20), and its degradation occurs at least partially within the lysosome
(21). The ubiquitin system is required for ligand-induced GHR
internalization (23). In particular, the UbE motif within the GHR
cytosolic tail is involved in both GHR ubiquitination and
ligand-induced endocytosis (22). In a Chinese hamster cell line
carrying a temperature-sensitive E1 enzyme (ts20 cells), inactivation
of E1 results in accumulation of nonubiquitinated GHRs at the plasma
membrane, whereas internalization of the transferrin receptor is
unaffected (23). Recently, we showed that the proteasome is also
involved in GHR down-regulation (24). GHR internalization requires
proteasomal action in addition to an active ubiquitin conjugation
system. Specific proteasomal inhibitors block GH uptake of the
full-length GHR, whereas a truncated receptor can endocytose undisturbed.
In this report, we address the role of proteasome-mediated protein
degradation in modulating GHR/JAK2 activity following GH stimulation.
We show that the proteasomal inhibitor MG132 prolongs the GH-induced
activity of both GHR and JAK2, presumably through stabilization of GHR
and JAK2 tyrosine phosphorylation. Furthermore, we observe that JAK2 is
not only bound to the GHR at the cell-surface but also intracellularly,
suggesting that the receptor and other signal transducing molecules are
still active in endosomes.
Cells and Materials--
Chinese hamster ts20 cells were stably
transfected as described previously (23). Truncated receptors were
constructed by introducing stop codons at various positions within the
rabbit cDNA (25). These truncated GHR cDNAs were cloned in
pcDNA3 (In Vitrogene Inc.) and transfected into ts20 cells,
resulting in cell lines stably expressing receptors truncated at amino
acid residues 399 and 369. The internalization-deficient mutant GHR F327A was constructed by site-directed mutagenesis, cloned and transfected into ts20 cells as described before (25). Stable, geneticin-resistant transfectants were grown in Eagle's minimal essential medium (MEM-
Antibody to GHR was raised against amino acids 271-381 of the
cytoplasmic tail (anti-GHR(T)) as previously described (23). Antibody
(Mab5) recognizing the luminal part of the GHR was from AGEN Inc.,
Parsippany, NJ. Antiserum against JAK2 was raised in rabbits against a
synthetic peptide corresponding to the hinge region (amino acids
758-777) between domains 1 and 2 of murine JAK2. Polyclonal antibody
against JAK2 and phosphotyrosine (4G10, anti-PY) were obtained from
Upstate Biotechnologies Inc. (Lake Placid, NY). Antiserum against human
GH was raised in rabbits. Commercial anti-GH was from DAKOPATTS. hGH
was a gift of Lilly Research Labs, Indianapolis, IN. Culture medium,
fetal calf serum and geneticin were purchased from Life Technologies,
Inc. MG132 was from CalBiochem.
Cell Lysis, Immunoprecipitation, and Immunoblotting--
Cells,
grown in 10-cm dishes, were first incubated for 2 h at 30 °C in
fetal calf serum-free MEM- Coimmunoprecipitation of Internalized Proteins--
Cells, grown
on 10-cm dishes, were incubated 1 h on ice in MEM- Effect of Specific Proteasomal Inhibitors on GHR
Phosphorylation--
To determine whether the proteasome is involved
in modulating GHR activity, we examined the effects of the proteasomal
inhibitor MG132 on GH-induced GHR phosphorylation. Using ts20 cells
stably transfected with wtGHR, the time course of tyrosine
phosphorylation of the GHR was determined following GH stimulation in
the presence and absence of MG132 (Fig.
1). In the absence of GH, no
phosphorylated GHR was visible (Fig. 1A). Upon GH
stimulation, a broad 130-kDa band appeared, indicating the GHR tyrosine
phosphorylation. The activity was maximal within 15 min and decreased
thereafter. If MG132 was present, virtually no decrease of the GHR
phosphorylation signal was observed even after 2 h. Reblotting
with an anti-GHR antibody showed equivalent amounts of total
immunoreactive protein in all samples, indicating that MG132 had little
effect on the steady-state level of the GHR (Fig. 1B).
Because GH stimulation was continuous, it was not possible to
discriminate the population of down-regulated receptors from newly
synthesized receptors as done previously by a metabolically labeled
pulse-chase experiment (24). Thus, prolonged phosphorylation of the
receptor caused by the presence of MG132 could account for the
sustained presence of the receptor at the cell surface.
The MG132 Effect Is Not Caused by Inhibition of Internalization of
GHR--
Our previous results have shown that MG132 prevents
internalization of the GHR (24). It is anticipated that a prolonged stay at the cell surface might result in a prolonged phosphorylated state of the GHR and of JAK2. To test this, we used the GHR
F327A-transfected cells, which express receptors defective in
internalization (25, 26). The kinetics of tyrosine
phosphorylation/dephosphorylation of the GHR F327A were similar to the
wtGHR (Fig. 1C), reaching a maximum after 15 min and
decreasing to basal levels after 2 h of GH treatment. However, in
the presence of MG132, the level of tyrosine phosphorylation of the GHR
F327A remained the same. Thus, down-regulation of the GHR
phosphorylation depends on proteasomal action and is not related to the
GH-induced endocytosis.
GHR Sustained Activation Is Caused by Prolonged JAK2
Phosphorylation--
Proteasomal inhibitors prolong signaling of the
interferon- Possible Role of Proteasomes in Modulating Phosphatase
Activity--
Previous reports have implicated JAK proteins in
dephosphorylation by interaction with specific phosphatases (12,
27-29). Hackett et al. (11) using FDP-C1 cells,
demonstrated that the region in the GHR cytosolic tail between 521 and
540 is required for inactivation of the JAK/STAT signaling cascade,
possibly via the protein tyrosine phosphatase SHP-1 that acts as a
negative regulator. However, SHP-1 does not seem to associate with the GHR. Also the tyrosine phosphatase SHP-2, another member of the protein-tyrosine phosphatase family that, unlike SHP-1, is ubiquitously expressed in vertebrate cells, was shown to form a complex with both
the tyrosine phosphorylated receptor (GHR cytoplasmic domain residues
485-620) and JAK2 protein (30). We determined whether MG132 would also
induce prolonged JAK2 phosphorylation upon GH treatment in a
C-terminally truncated GHR. JAK2 was immunoprecipitated from
GHR-(1-399)- and GHR-(1-369)-expressing cells and immunoblotted with
antiphosphotyrosine antibodies for various times of GH treatment (Fig.
3, A and C). In
both cell lines, JAK2 showed tyrosine phosphorylation with no change
over time. In accordance with the above mentioned studies, delayed
dephosphorylation of the kinase was observed both in the presence and
absence of MG132. Reprobing the blots with JAK2 antibody confirmed the
presence of equal amounts of JAK2 protein in each sample (Fig. 3,
B and D). These results suggest that the
activation of a negative regulator (SHP-1 or SHP-2) through distal GHR
tail domains and further association with JAK2 might be the important
factor responsible for down-regulating the GHR/JAK2 phosphorylation in
a proteasome-dependent way. Thus, inhibition of the
proteasome by MG132 inhibits the dephosphorylation of JAK2, resulting
in prolonged activity of both JAK2 and GHR. However, MG132 had no
effect on SHP-2 phosphorylation upon GH induction, or on SHP-2 binding
to both GHR and JAK2 (results not shown).
As shown previously, proteasomal inhibitors do not affect
internalization of GH via the GHR-(1-369) but effectively blocked endocytosis of GHR-(1-399, Ref. 24). As JAK2 phosphorylation is
similar in both cell lines, the data implicate that signaling might
continue after endocytosis.
JAK2 Protein Is Bound to GHR in Endosomes--
Signaling via the
GHR begins at the cell-surface. As demonstrated above using the
endocytosis-defective GHR F327A cell line, the activation/deactivation
(tyrosine phosphorylation/dephosphorylation) cycle can be initiated and
completed at the cell-surface. The next question is whether signal
transduction can continue after endocytosis. To address this, the
activity of GHR had to be established after endocytosis. To accomplish
this, we isolated GH·GHR complexes using anti-GH immunoprecipitation
after acid treatment (Fig. 4). Dissociation of GH·GHR complexes does not occur at (endosomal) pH 5.5 (20), indicating that GH remains complexed to its receptor, independent
of its intracellular routing, unless it is localized to the lysosome.
In that case, the ligand as well as the receptor is rapidly degraded
(21). Treating living cells with buffers of pH higher than 2.5 showed
that GH was not removed from GHR at the cell surface, and only upon
treatment with a buffer of pH 2.5 did GH detach from the receptor
without interfering with the already internalized GH·GHR (results not
shown). If no acidic treatment was performed, the total amount of wtGHR
bound to GH coimmunoprecipitated during the different periods of time
(Fig. 4A, lanes 1-4). The same was observed for
the truncated GHR-(1-369) (Fig. 4B, lanes 9-12)
and GHR F327A (Fig. 4D, lanes 26-29). If the
cells were kept on ice, acid treatment removed virtually all the GH
from the cell surface and hardly any GHR was detectable (Fig. 4,
lanes 5, 13, 21, and 30). Upon incubation at
30 °C, GH became acid-resistant indicating that GH·GHR complexes
had entered the cells. Within 15 min, both wtGHR and GHR-(1-369) were
detectable inside the cells. Longer periods of GH treatment resulted in
a decrease of GH-bound internalized receptors in the endosomes
(lanes 8 and 16). The GHR F327A was not observed
inside the cells because internalization of this receptor was inhibited
(lanes 30-33). In addition to the 130-kDa band of the GHR,
a smear of bands (60-80 kDa) appeared, which only reacted with Mab5
(Fig. 4A) and anti-GHR(T) (not shown) and not with anti-GHR
C-terminal tail antibody (Fig. 4C). No degradation products
were visible if GHR-(1-369) was analyzed (not shown). These
observations show that the partial degradation of the wtGHR starts from
the C terminus very soon after GH binding.
To exclude the possibility that during or after lysis GH is free to
rebind endocytosed or cell-surface receptor, the same experiment for
wtGHR was performed but excess of unstimulated GHR-(1-369) lysate was
added during lysis (Fig. 5). If free GH is available to react with wtGHR at the cell surface or in endosomes, then an excess of GHR-(1-369) will compete for binding to free GH. If
this would be the case, then GH immunoprecipitates blotted with
anti-GHR (Mab5) should present GH complexes with both wtGHR and
GHR-(1-369). As these receptors have different sizes but the same GH
binding affinity, they can easily be distinguished by immunoblot. As
observed on Fig. 5A, wtGHR cells were treated with GH for 15 min and acid-treated. Addition of nonstimulated GHR-(1-369) lysate in
different concentrations, did not result in GH complexes containing the
truncated receptor. Addition of excess of lysate of untransfected ts20
cells to the wtGHR-expressing cells was also tested, with the same
result. Fig. 5B shows total cell lysates blotted with
anti-GHR (Mab5), indicating the amount of the truncated GHR-(1-369) in
the incubations. Performing the same experiment by lysing GH-treated
GHR-(1-369) cells in the presence of unstimulated wtGHR lysate, no
GH·wtGHR complexes were detected (results not shown).
We then addressed the question of whether internalized GHR is able to
bind JAK2. The same time course experiment was performed as in Fig. 4
and analyzed for JAK2 molecules (Fig.
6A). To measure the total
amount of JAK2 bound to the GH·GHR complex, no acidification was
performed (lanes 1-3). In wtGHR-transfected cells upon acid wash, JAK2 coimmunoprecipitated with GH·GHR complexes after
internalization (lanes 5 and 6). The same was
observed in the GHR-(1-369) mutant (lanes 7-9). A faster
migrating background band reacted with anti-JAK2 after cell
acidification, presumably caused by proteolysis. As expected, the GHR
F327A mutant did not show JAK2 binding after acidification. JAK2 was
neither detectable in the anti-GH immunoprecipitates from untransfected
ts20 cells (lane 13) nor unstimulated cells expressing the
wtGHR, the GHR-(1-369), and the GHR F327A (lanes 1, 4, 7, and 10), indicating the efficiency of the acid-wash
procedure. Similar amounts of JAK2 were found for the different cell
lines as seen in Fig. 6B. These results show that JAK2 is
bound to the GHR inside the cell, suggesting that the receptor is
capable of signaling in endosomes.
GHR Signaling Continues Inside the Cell--
To determine whether
other proteins attached to the GH·GHR in endosomes are
phosphorylated, cells expressing wtGHR and GHR-(1-369) were treated as
described above and analyzed for phosphotyrosine-positive proteins. As
seen in Fig. 7A, GH induced
the phosphorylation of a set of medium and high molecular weight
proteins. Upon GH removal from the cell surface, the major
phosphorylated protein (with apparent Mr above
187,000) was also present in the internalized GH·GHR complex, both in
the wtGHR and GHR-(1-369). Untransfected ts20 cells only resulted in a
background pattern (not shown). In the higher molecular weight range
both wtGHR and JAK2 proteins are possible candidates, consistent with
the results presented above. Surprisingly, internalized wild-type as
well as truncated receptors presented roughly similar patterns of
phosphorylated proteins. This can be explained by the fact that
signaling proteins mainly interact with the membrane proximal region of
the cytosolic tail of the GHR via JAK2. For the wtGHR (Fig.
7A, left panel) interpretation of the pattern is
difficult, because of the large signal of tyrosine phosphorylation by
the receptor itself. This is not the case for the truncated receptor,
as it contains only a few tyrosine residues. As apparent from Fig.
7B, showing the amounts of receptor present in Fig.
7A, acid treatment reduces the GHR signals considerably. It
is also clear that, especially in the wtGHR, massive degradation had
occurred upon endocytosis.
The ubiquitin-proteasome system plays an essential role in many
cellular regulatory processes including cell cycle progression, DNA
repair, transcriptional control, and cell surface-associated receptor
endocytosis. In all these processes the ubiquitin-conjugating system
targets ubiquitinated proteins to the proteasome for degradation. For
the GHR, the ubiquitin system was found to be involved in GH-dependent endocytosis (Ref. 31; for review, see Ref.
32). In the present study, we demonstrate that the ubiquitin-proteasome system is involved in the down-regulation of GHR signal transducing events. Others have demonstrated that in several cytokine receptors the
JAK/STAT pathway was down-regulated by the proteasome. Both interleukin-2 and -3 and interferon receptor showed a prolonged JAK/STAT activation as well as other signaling molecules like MAP
kinases, upon treatment with specific proteasomal inhibitors (18, 17).
The studies described in this work demonstrate a similar effect for the
GHR. Using ts20 cells stably transfected with wtGHR, we show that, in
the presence of the specific proteasomal inhibitor MG132, the
phosphorylation/activation of both receptor and tyrosine kinase JAK2
are prolonged for long periods of GH induction. Furthermore, our data
indicate that proteasomal action on signal transduction occurs at the
cell surface because signaling by the GHR F327A endocytosis-deficient
mutant still depends on the proteasome for its down-regulation. These
results support the notion that GHR/JAK2 signal down-regulation is not
determined by endocytosis per se.
Which mechanisms underlie the down-regulation of the GHR and JAK2
proteins? Several reports have shown that tyrosine phosphatases are
involved in the dephosphorylation of JAK proteins. Ligand-induced tyrosine phosphorylation/activation of JAK2 by erythropoietin receptor,
induces binding of the protein tyrosine phosphatase SHP-1 to the
cytoplasmic domain of the receptor. The recruitment of SHP-1 is
accompanied by dephosphorylation of JAK2 and subsequent termination of
erythropoietin-induced cellular proliferation (33, 34). A
similar role for SHP-1 in mediating the down-regulation of JAK2
following stimulation of cells with GH has been proposed (11). Our
results with GHR-(1-399) and GHR-(1-369) indicate that partial
deletion of the C-terminal GHR tail leads to a prolonged JAK2
phosphorylation presumably because of loss of a negative regulator
binding site and its consequent activation. This pattern of prolonged
phosphorylation is similar to that of JAK2 in full-length wtGHRs
treated with MG132. One explanation might be that the phosphatase activity is modulated by proteasome function, perhaps by degrading an
inhibiting complex in a similar manner as it occurs for the inhibitor
of the transcription factor NF- Until now there is clear evidence that tyrosine kinase receptors, like
the epidermal growth factor and the insulin receptor, continue to
signal after endocytosis (42, 43). Our data with the GHR show that
initiation as well as termination of its phosphorylation as well as of
JAK2 can occur at the cell surface. No evidence is available about
signal transduction inside the cells. Combining an acid-wash procedure
with anti-GH immunoprecipitation we show that the GHR can induce a
second round of signal transduction intracellularly. This is not
unexpected because obviously GH keeps the two GHRs complexed after
endocytosis. In this configuration JAK2 has high affinity for the
complex and will either rebind (if it was removed during passage of the
coated pits) or will keep its position on the dimerized tails once
internalized. Although the amount of undegraded, endocytosed wtGHR is
very small, the Western blot signal of JAK2 complexed to the GH·GHR
complexes is significant as compared with control (non acid-washed)
cells. This indicates that the signaling capacity of GH·GHR complexes in endosomes is significant. The relevance for signal transduction in
endosomes is not clear. It is possible that the signaling GHR complexes
in endosomes differ from those at the cell surface. This is not obvious
from our data, because the SDS-polyacrylamide gel electrophoresis
patterns of phosphotyrosine-containing proteins of total and
endocytosed GH complexes look very similar. Together, these
observations indicate that GHR signal transduction continues or resumes
after endocytosis and that the signals, regenerated at the two cellular
locations, do not substantially differ.
Another point of discussion is the presence of truncated GHR
originating from the wtGHR, not from the truncated GHR-(1-369). First,
it is not clear where this process starts. Experiments with the GHR
F327A show that it is ubiquitin system- (UbE motif) independent,
because it also occurs in this mutant GHR, and in the presence of
proteasome inhibitors its formation can still occur.2 Thus, the GHR is
already C-terminally truncated at the cell surface, and the truncated
GHR can endocytose, complexed to GH. It remains to be determined,
whether this truncated GHR plays a role in signal transduction.
Both JAK2 and a multitude of other, mostly high molecular weight,
proteins are activated and interact with both wtGHR and GHR-(1-369)
after GH induction in acid wash-treated cells. Coimmunoprecipitation of
GH·GHR·JAK2 complexes, after uptake by the cells, shows that JAK2
is not only bound to the GHR at the cell surface but also intracellularly, suggesting that the receptor and some of its signal
transducing molecules might still be active in endosomes. A smear of
phosphorylated proteins attached to the GH·GHR complex inside the
cells confirmed the receptor capacity for signaling. Intracellularly,
the pattern of phosphorylated proteins in wtGHR and GHR-(1-369) is
similar, providing both receptors comparable signaling capabilities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) supplemented with 10% fetal bovine serum, penicillin and streptomycin, 4.5 g/liter glucose, and 0.45 mg/ml geneticin. For experiments, cells were grown in the absence of geneticin at ~70% confluence.
in the presence or absence of 20 µM MG132. After hGH (8 nM) incubation, cells
were lysed on ice in 0.6 ml of lysis mix containing 1% Triton X-100, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 50 mM NaF in PBS. The
immunoprecipitations were performed in 1% Triton X-100, 0.5% SDS,
0.25% sodium deoxycholate, 0.5% bovine serum albumin, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 50 mM NaF in PBS. The
lysates were cleared by centrifugation and incubated with GHR antiserum
or JAK2 antiserum for 2 h on ice. Protein A-agarose beads
(Repligen Co., Cambridge, MA) were used to isolate the immune
complexes. The immunoprecipitates were washed twice with the same
buffer and twice with 10-fold diluted PBS. Immune complexes were
analyzed by polyacrylamide gel electrophoresis in the presence of SDS
together with total cellular lysate and transferred to polyvinylidene
difluoride paper. The blots were immunostained using either Mab 4G10
(anti-PY), anti-GHR, or commercial JAK2 antibody. After incubating the
blots with rabbit anti-mouse IgG (RAMPO) or protein A conjugated to
horseradish peroxidase, antigens were visualized using the ECL system
(Roche Molecular Biochemicals).
and
supplemented with 20 mM Hepes and 8 nM hGH. The
cells were then washed once to remove unbound GH and incubated at
30 °C for different periods of time. Cells were put on ice after which the cell surface-labeled GH was removed by two times 30 s
with acidic solution of 50 mM glycine, 150 mM
NaCl, pH 2.5. The cells were washed with PBS and lysed in 0.1% Triton
X-100, 1 mM EDTA, 0.5% bovine serum albumin, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 1 mM Na3VO4, and 50 mM NaF in PBS. Immunoprecipitations were performed in the
same buffer with GH antiserum for 2 h on ice. The immune complexes were treated as above. The blots were immunostained using Mab 4G10 or
JAK2 antibody. To control for GH rebinding during and after cell lysis,
lysis of cells expressing the (full-length) wtGHR was performed in the
presence of increasing amounts of cell extracts from unstimulated
(truncated) GHR 1-369 cells. Detection was performed with anti-GHR Mab5.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of MG132 on GHR-phosphorylation.
Cells were incubated for 2 h at 30 °C without
(Control) or with 20 µM MG132 and supplemented
with 8 nM hGH for the time periods indicated. Cell lysates
were prepared and subjected to immunoprecipitation (IP) of
the GHR by using anti-GHR(T) and subjected to immunoblot analysis with
an antibody against phosphotyrosine residues (anti-PY). A,
ts20 cells, expressing the wtGHR. C, ts20 cells expressing
the GHR F327A. B and D, the PY blots were
reblotted with anti-GHR(T). Arrows indicate mature (mGHR)
and precursor (pGHR) forms of the GHR.
receptors after ligand stimulation, showing sustained
tyrosine phosphorylation of both the receptors and JAK1/JAK3 (17). To determine whether the effect of MG132 on GHR phosphorylation is caused
by sustained activation of JAK2 kinase, anti-JAK2 immunoprecipitates were prepared from cell lysates and analyzed by immunoblotting with an
antibody to phosphotyrosine. As shown in Fig.
2A, GH induced a transient
phosphorylation of JAK2 with a maximum at 15 min in the absence of
proteasomal inhibitor, declining to nearly basal levels after 2 h.
However, treatment of the cells with MG132 prevented the
dephosphorylation of JAK2, correlating well with the sustained GHR
activity (compare lanes 4 and 8). Reblotting with
an anti-JAK2 antibody showed similar amounts of immunoreactive protein
in all samples indicating that MG132 had little effect on the stability of the protein (Fig. 2B). Shorter preincubation periods with
MG132 were as effective in stabilizing the tyrosine phosphorylation of
JAK2, suggesting that its mechanism of action is specific and not
caused by general cell toxic effects. The same was observed for the GHR
F327A mutant (Fig. 2, C and D). JAK2 activation
was transient in this mutant, but as for the wtGHR, MG132 treatment prolonged JAK2 phosphorylation in a similar way. Taken together, these
results demonstrate that MG132 prolongs the GH-induced activity of both
GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine
phosphorylation. Thus, the proteasome plays a role in decreasing GHR
signal transduction.
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Fig. 2.
Effect of MG132 on JAK2 phosphorylation.
The procedure was as described in the legend to Fig. 1, except that
anti-JAK2 immunoprecipitations were subjected to an anti-PY immunoblot
analysis. A, ts20 cells expressing the wtGHR. C,
ts20 cells expressing the GHR F327A. In B and D,
the resulting immunoprecipitates were reblotted with anti-JAK2.
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Fig. 3.
Effect of MG132 on JAK2 phosphorylation in
truncated GHR-expressing cells. Ts20 cells expressing GHR-(1-399)
(A) or GHR-(1-369) (C) were incubated at
30 °C without (Control) or with 20 µM MG132
and supplemented with 8 nM hGH for the time periods
indicated. The truncated forms of the GHR were immunoprecipitated with
anti-GHR(T) and immunoblotted with anti-PY. Reblots with anti-GHR(T)
are shown in B and D.
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Fig. 4.
Acid-resistant GH·GHR complexes are found
in the cell. A, cells expressing full-length wtGHR,
B, GHR-(1-369), and D, GHR F327A were incubated
with 8 nM hGH on ice for 1 h, followed by incubation
at 30 °C for the time periods indicated. The cells were then
subjected or not to an acid-wash procedure. The cell lysates were
immunoprecipitated with anti-GH and blotted with anti-GHR (Mab5,
B), or with anti-GHR (C) for cells
expressing wtGHR.
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Fig. 5.
Acid-treatment control.
A, cells expressing wtGHR were treated with GH for 15 min
and acid washed as previously, but lysis was performed in the presence
of cell extracts from unstimulated GHR-(1-369) cells at 1, 2, or
3-fold excess concentrations of wtGHRs. The cell lysates were
immunoprecipitated with anti-GH and blotted with anti-GHR (Mab5).
B, total lysates were blotted with anti-GHR (Mab5).
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Fig. 6.
Acid-resistant GH·GHR complexes contain
JAK2. A, immunoprecipitates of GH·GHR complexes were
blotted for anti-JAK2 for wtGHR, GHR-(1-369), GHR F327A and ts20
untransfected cell lines. In B, the direct lysates were
blotted for anti-JAK2 in the different cell lines as indicated.
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Fig. 7.
Phosphorylation of proteins attached via the
GH·GHR complex after internalization. The same procedure was
used as described in the legend to Fig. 4. Immunoprecipitates of
GH·GHR complexes from wtGHR, and GHR-(1-369) cells were blotted with
anti-PY and Mab5, as shown in A and B,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (35). This would explain why in the
presence of MG132, phosphatase inhibitors prevent dephosphorylation of
the JAK2 by SHP-1, and thereby prolonging phosphorylation of both JAK2
and the GHR. In support of this model, SHP-1 degradation has been shown
to be ubiquitin-dependent in mast cells (36), suggesting
that the proteasome is involved in SHP-1 regulation. SHP-2, however,
has been shown to interact directly with the tail of the GHR (residues
484-620) and associate with JAK2 and SIRP
1, a member of a family of
transmembrane glycoproteins identified by its association to SH2
domain-containing SHP-2. In response to GH, JAK2 associates with
SIRP
1 and rapidly stimulates tyrosine phosphorylation of both
SIRP
1 and SHP-2, and enhances association of these two molecules
(37). Recently, it was shown that SIRP
1 is acting as a negative
regulator of GH signaling by its ability to bind SHP-2 (38). The
proteasome could therefore play a role in SHP-2/SIRP
1 association
and binding to JAK2. As SHP-2 is known to associate to other signaling
molecules as IRS-1 (39) and p85-PI3K (40), future studies will indicate
whether the MG132 effect on these molecules is directly related to
SHP-2 activity. However, it cannot be excluded that activated JAK
kinases themselves are subject to proteasome-mediated degradation.
Support for this comes from a recent identified negative regulatory
pathway of the GHR signaling involving the SOCS proteins. GH
preferentially induces the rapid, transient expression of
SOCS-3, a member of the SOCS family that is known to
inhibit cytokine receptor signaling. Expression of other SOCS genes,
SOCS-1, SOCS-2, and CIS, was also up-regulated by GH, although to a lesser extent than SOCS-3
and with different kinetics (14). Recently, it was shown that the highly conserved C-terminal homology domain of the SOCS proteins, termed the SOCS box, mediates interactions with elongins B and C, which
in turn may couple SOCS proteins and their substrates to the
proteasomal protein degradation pathway (41). How SOCS proteins inhibit
JAK kinase activity is still not clear, but analogous to the family of
F-box-containing proteins, SOCS box interaction with elongins B and C
potentiates interaction with the proteasome complex. This would explain
why, in the presence of MG132, degradation of SOCS proteins and its
associated proteins like JAK2 would be prevented, and therefore induce
sustained activation of JAK2 and, consequently the GHR. Evidence for a
role of the ubiquitin-proteasome system in signal transduction came
from the experiments of Verdier et al. (19) who showed that
a Cis member of the SOCS family was ubiquitinated upon erythropoietin
receptor activation. Thus, at least two mechanisms for the termination
of the GHR phosphorylation might depend on proteolysis: the regulation
of phosphatases and of the SOCS proteins.
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ACKNOWLEDGEMENTS |
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We thank Jurgen Gent, Julia Schantl, Toine ten Broeke, and Martin Sachse for creative discussions.
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FOOTNOTES |
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* This work was supported by Grant NWO-902-68-244 from The Netherlands Organization for Scientific Research and by European Union Network Grant ERBFMRXCT96-0026.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.
To whom correspondence should be addressed. Tel.: 31 30-2506476;
Fax: 31 30-2541797; E-mail: strous@med.uu.nl.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M003635200
2 C. Alves dos Santos, and G. Strous, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: GHR, growth hormone receptor; GH, growth hormone; JAK, Janus kinase; STAT, signal transducer and activator of transcription; SHP, SH2-containing phosphatase; IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3-kinase; SOCS, suppressor of cytokine signaling; SIRP, signal regulatory protein; E1, ubiquitin-activating enzyme; MEM, minimal essential medium; Mab, monoclonal antibody; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase.
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