(Received for publication, November 6, 1996)
From the Parathyroid hormone-related protein (PTHrP) is an
important causal factor of hypercalcemia associated with malignancy.
PTHrP also modulates cell growth and differentiation of normal cells through mechanisms that include binding to cell surface-specific receptors as well as by possible intracellular routes. To understand the regulation of intracellular PTHrP expression, post-translational processing of PTHrP was investigated. Using cell-free translations it
was shown that PTHrP can be ligated efficiently to multiple ubiquitin
moieties. Both conjugation to ubiquitin and degradation of prepro-PTHrP
synthesized in vitro were ATP-dependent.
Translation in vitro in the presence of the proteasome
inhibitor MG-132 abolished the degradation of PTHrP. Treatment of
cells, cotransfected with hemagglutinin-tagged ubiquitin and
histidine-tagged prepro-PTHrP, with MG-132, led to the accumulation of
ubiquitinated prepro-PTHrP. Deletion mutagenesis experiments indicated
that both the prepro secretory domain and a PEST (amino acid residues
Pro (P), Glu (E), and/or Asp (D), Ser (S), and Thr (T)) motif in the
COOH-terminal region of the protein were not required as
cis-acting determinants for ubiquitination. This is the
first report of a wild-type secretory polypeptide serving as a
substrate of the ubiquitin proteolytic pathway. These results suggest
that the ubiquitin-dependent proteolytic pathway is
involved in regulating the metabolic stability of intracellular PTHrP,
and this regulation may be an important mechanism for modulating its
effects on cell growth and differentiation.
Parathyroid hormone-related protein
(PTHrP)1 is a secreted peptide that is a
major pathogenic factor in hypercalcemia associated with malignancy (1,
2). PTHrP shares limited sequence homology with parathyroid hormone
(PTH), with 8 of the first 13 amino acids in PTHrP being identical to
the corresponding NH2-terminal residues of PTH (for review,
see Ref. 3). This limited homology is sufficient for PTHrP to bind to a
common PTH/PTHrP receptor and share many of the biological properties
of PTH (4, 5). Unlike PTH, PTHrP also contains a bipartite nuclear
localization signal in the midregion of the protein (6), which has
recently been demonstrated to be capable of targeting PTHrP to the
nucleolus, and nucleolar localization of PTHrP may be required for
enhancing the survival of chondrocytes in culture under conditions that
promote apoptotic cell death (7). Although PTHrP was first demonstrated
as a tumor product, it is now known to be expressed in a wide array of
normal fetal and adult tissues (for review, see Ref. 8). Unlike PTH,
PTHrP is not normally present in the circulation, suggesting that it
acts locally in an autocrine or paracrine fashion. Studies in several
cell types in vitro (6, 9-12), in vivo
experiments in transgenic mice that overexpress PTHrP (13), and
in vivo studies of PTHrP gene inactivation in mice via
homologous recombination (14-16) have all demonstrated that PTHrP
plays a role in normal cell proliferation and differentiation including
a critical role in skeletogenesis (14-16). In addition, it may also
influence tumor cell growth (17, 18).
The regulation by a variety of agents of the expression and secretion
of PTHrP has been examined in many different cells. An increase in
PTHrP mRNA transcripts is observed rapidly and transiently
following exposure of cells to serum, growth factors, and phorbol
esters through mechanisms including increased gene transcription and
mRNA stability (for review, see Ref. 8). In addition, an increase
in constitutive expression and secretion was noted in the progression
from the established to the malignant phenotype, suggesting that the
protein is dysregulated in tumor cells (12, 19).
A common feature of many regulatory proteins, including oncoproteins,
is their short half-life. As increased levels of PTHrP are associated
with the transformed phenotype (12, 19), it might be expected that
PTHrP instability, and therefore, low steady-state levels of the
protein, could be important for properly controlled cell proliferation.
To date, no study on the intracellular degradation of PTHrP has been
documented. Here we present evidence that PTHrP is a substrate of the
ubiquitin-dependent proteolytic system.
Ubiquitin-dependent protein degradation is a nonlysosomal,
ATP-dependent proteolytic pathway (for review, see Ref.
20). The biochemical mechanism involves covalent ligation of the
76-amino acid polypeptide, ubiquitin, to ATP All PTHrP constructs were derived from
the cDNA encoding rat prepro-PTHrP (PLPm10) (25). For transfection
studies, prepro-PTHrP was expressed with a histidine tag at the COOH
terminus using the mammalian expression vector pRC/CMV (Invitrogen).
Tagged prepro-PTHrP cDNA was generated by PCR using the universal
primer as the forward primer and oligonucleotide PTHrPhistag
(5 A prepro-PTHrP construct lacking the prepro sequence was derived from
PLPm10 using PCR-based mutagenesis. To create this deletion construct,
PTHrP COS-7 cells were cultured
in Dulbecco's modified Eagle's medium (plus 4.5 g/liter glucose; Life
Technologies, Inc.) supplemented with 10% fetal calf serum and
antibiotic-antimycotic (Life Technologies, Inc.) in a humidified
atmosphere at 37 °C with 5% CO2. COS-7 cells were
transiently transfected with Lipofectin (Life Technologies, Inc.)
according to the instructions of the manufacturer. The Lipofectin concentration was 12 µg/ml, and 4 µg of CMV-based expression
plasmid and 2 µg of ubiquitin expression plasmid were added per 60-mm dish.
Stable expression of prepro-PTHrP was created in a Chinese hamster lung
fibroblast cell line, E36 (kindly provided by William Dunn). The cells
were maintained at 30 °C in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. The cells were transfected
with Lipofectin reagent and CMV PTHrP-His6. Following lipofection, cells were cultured in Dulbecco's modified Eagle's medium supplemented with G418 (1 mg/ml) at 30 °C. Clonal colonies were isolated, expanded, and assayed for PTHrP expression by Northern blotting.
Conditioned media from transfected cells were collected at various
times and kept frozen at 48 h after transfection,
cells were incubated in fresh Dulbecco's modified Eagle's medium plus
10% fetal calf serum containing either 50 µM MG-132 or
dimethyl sulfoxide (Me2SO) vehicle alone (final
concentration 0.2%) for up to 24 h. In control experiments, 50 µM E64 or 50 µM calpain inhibitor II was
used in place of MG-132.
Cell
monolayers were washed once in phosphate-buffered saline (pH 7.4) and
then lysed in lysis buffer (500 µl of 8 M urea, 0.1 M sodium phosphate buffer (pH 8.0), 0.01 M
Tris-HCl (pH 8.0)). Lysates were incubated on ice for 10 min,
centrifuged at 16,000 × g for 10 min at 4 °C, and
the supernatants were stored at To isolate histidine-tagged proteins, equal amounts of total protein
from each sample (up to 300 µg of total protein) were loaded onto
Ni2+-NTA-agarose spin columns (Qiagen) pre-equilibrated
with lysis buffer. Columns were spun at 2,000 rpm for 2 min in a
microcentrifuge. Three washes were performed with 600 µl of 8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris-HCl (pH 6.3). Proteins were eluted with 200 µl of
8 M urea, 0.2 N HCl.
Equal amounts of protein from
total cell lysates or equal volumes of material eluted from the
Ni2+-NTA-agarose columns were denatured in SDS sample
buffer and separated on 12.5% polyacrylamide-sodium dodecyl sulfate
(SDS) gels. Proteins were transferred in 25 mM Tris, 192 mM glycine, 20% Methanol onto polyvinylidene difluoride
(Trans-Blot, Bio-Rad). Blots were blocked with TBS (20 mM
Tris-HCl (pH 7.5), 137 mM NaCl) plus 0.1% Tween 20 containing 3.5% bovine serum albumin. Polyclonal antiserum against
amino acids 1-34 of human PTHrP was used at a 1:400 dilution in TBS,
3.5% bovine serum albumin. The monoclonal antibody against amino acids
38-64 of human PTHrP was used at a concentration of 1 µg/ml in TBS,
3.5% bovine serum albumin. The monoclonal antibody 12CA5, specific for
a nine-amino acid HA peptide sequence (YPYDVPDYA) from influenza HA,
was used at 2.5 µg/ml. The antigen-antibody complexes were visualized
using appropriate secondary antibodies (Bio-Rad) and the ECL detection
system as recommended by the manufacturer (Amersham Corp.).
pSK (Bluescript,
Stratagene) containing rat prepro-PTHrP cDNA (PLPm10) was
linearized with BglII. pKS-hPTH was linearized with
HindIII. A 5-µg portion was transcribed in
vitro using T7 RNA polymerase (Promega Biotec) under conditions
recommended by the supplier. A 0.5-µg portion of the resulting RNA
was translated in 12.5 µl of reticulocyte lysate (Promega Biotec)
translation mix in the presence of [3H]leucine at
30 °C for the times indicated in the figure legends. Aliquots were
removed and fractionated by electrophoresis in 12.5% polyacrylamide-SDS gels. Gels were fixed and processed for fluorography with En3Hance (DuPont NEN), dried, and exposed to X-Omat AR
(Kodak) film.
Bacterial expression plasmids
for glutathione-tagged yeast ubiquitin were from J. Huibregste (27).
Glutathione S-transferase (GST) fusion constructs were
expressed in Escherichia coli DH5 Degradation assays were performed in
rabbit reticulocyte lysate translation reactions programmed with
prepro-PTHrP mRNA and [3H]leucine. After 30 min of
translation, 25-µl aliquots were diluted to a final reaction volume
of 30 µl containing 0.3 µg/ml RNase A, 15 mM
MgCl2, and either 1 mM ATP or 1 mM
ATP When used, the protease inhibitors MG-132 (50 µM) or
calpain inhibitor II (50 µM) or vehicle
(Me2SO) were added at the onset of reticulocyte lysate
translations and incubated at 30 °C for the indicated times.
Ubiquitin aldehyde was added at 30 µM.
Prepro-PTHrP mRNA was synthesized in vitro
and translated in a rabbit reticulocyte lysate in the presence of
[3H]leucine. Following SDS-PAGE and autoradiography a
principal band of protein was observed migrating at 27 kDa (Fig.
1, lane 2). The molecular mass of
prepro-PTHrP based on its deduced amino acid sequence is 14 kDa. This
aberrant migration of prepro-PTHrP has been noted previously and
results from its highly basic net charge (28). Faster migrating bands
were also observed and are presumably due to premature termination of
translation or nonspecific degradation in the lysate. In addition to
the full-length protein, translation in reticulocyte lysate generated
higher molecular mass bands which were not visualized when the same RNA
preparation was translated in a wheat germ lysate (Fig. 1, lanes
2 and 4). These higher molecular mass bands migrated
approximately 15 and 8 kDa slower than prepro-PTHrP. On longer
exposure, several additional bands with regular 8-kDa spacing were
apparent near the top of the gel (data not shown). This ladder of
higher molecular mass bands is reminiscent of the post-translational
covalent attachment of ubiquitin to target proteins in reticulocyte
lysate, which is an established source of ubiquitinating enzymes and
proteasome complexes (29). We therefore explored the possibility that
prepro-PTHrP is indeed a substrate for ubiquitin conjugation and
degradation.
To
determine if the higher molecular mass bands obtained in these
reactions were the result of prepro-PTHrP ubiquitination, we added
ubiquitin to the reticulocyte translation extract, as a fusion protein
linked to GST. If ubiquitin conjugation to prepro-PTHrP were to occur,
then the addition of the 33-kDa GST-ubiquitin protein should increase
the molecular mass of prepro-PTHrP from 27 to 60 kDa. The addition of
GST-ubiquitin but not GST alone to the translation reaction resulted in
the appearance of a novel band that migrated with the predicted
mobility of a prepro-PTHrP-GST-ubiquitin conjugate (Fig.
2, lanes 2 and 3).
To test the specificity of this ubiquitination of prepro-PTHrP, we
performed the conjugation assay on prepro-PTH, which has limited
homology to prepro-PTHrP. Prepro-PTH translated in reticulocyte lysate
was observed not to be a substrate for GST-ubiquitin conjugation (Fig.
2, lanes 4-6). This experiment suggests that ubiquitin is conjugated specifically to prepro-PTHrP in vitro.
In time course experiments, the overall
intensity of the translated PTHrP bands in the reticulocyte lysate
decreased for up to 6 h (Fig. 3, A and
B, lanes 1-4). A key feature of
ubiquitin-dependent proteolysis is its dependence on ATP,
which is required for conjugation of ubiquitin to the substrate and for
degradation of the ubiquitinated proteins. To determine whether
degradation was ATP-dependent, we depleted ATP from the
reticulocyte lysate with 2-deoxyglucose and hexokinase. This resulted
in inhibition of PTHrP proteolysis and prevented the appearance of the
higher molecular mass bands (Fig. 3A, lanes
5-8). To test the requirement for ATP hydrolysis in the
degradation assays we added ATP
To determine whether the ATP-dependent
in vitro degradation of prepro-PTHrP requires the
proteasome, we added the proteasome inhibitor MG-132 (31) or as a
control, the vehicle Me2SO, to the reticulocyte lysate
translation mix. MG-132, but not Me2SO, inhibited the
degradation of prepro-PTHrP but only minimally inhibited the turnover
of ubiquitinated forms of PTHrP (Fig. 4, lanes
1-10). To test whether the inability of MG-132 to stabilize
ubiquitinated-PTHrP was due to the action of isopeptidases in the
lysate which can remove ubiquitin from the substrate protein, we added
both MG-132 and ubiquitin aldehyde, an inhibitor of isopeptidase
function (32). This resulted in clear inhibition of both PTHrP and
ubiquitinated PTHrP turnover (Fig. 4, lanes 11-15). To
exclude the possibility that this stabilization was due to
MG-132-mediated inhibition of calpains, parallel translations were
performed in the presence of the protease inhibitor calpain inhibitor
II. Addition of calpain inhibitor II did not impede the degradation of
PTHrP (Fig. 4, lanes 16-20).
To characterize the ubiquitination of PTHrP in intact cells,
an expression plasmid containing the prepro-PTHrP cDNA encoding six
histidines at the carboxyl terminus was constructed and transiently transfected into COS-7 cells. In addition, the cells were transfected with a vector that expresses high levels of a HA-tagged human ubiquitin
(24). 48 h after transfection, cells were cultured for 6 or
24 h in medium containing either the vehicle Me2SO
(control), MG-132, calpain inhibitor II, or a cysteine proteinase
inhibitor E64. A viability test was performed on control and
MG-132-treated cells to assess for potential toxicity. Trypan blue
exclusion demonstrated that ~90% of the cells remain viable in the
presence of Me2SO- or MG-132-treated cultures. Total cell
lysates were subjected to SDS-PAGE and immunoblotted with polyclonal
antibody against PTHrP (1-34) or monoclonal antibody against PTHrP
(38-64) (Fig. 5, A and B,
respectively). Addition of MG-132, but not Me2SO, calpain
inhibitor II, or E64, induced an accumulation of higher molecular mass
bands (Fig. 5A). The antibodies against PTHrP recognized two
main products corresponding to full-length prepro-PTHrP and the
processed form following cleavage of the signal peptide, pro-PTHrP. This is more obvious in Fig. 5B since immunoblotting with
the monoclonal antibody does not detect an unrelated but similar sized protein that cross-reacts with the polyclonal antibody (Fig.
5A, lanes 1 and 2, mock transfected).
A faster migrating band accumulating in the presence of MG-132 may have
been due to enzymatic removal of a COOH-terminal fragment. Indeed,
several post-translational cleavages resulting in
NH2-terminal fragments have been reported for PTHrP (8,
33). Interestingly, when the monoclonal antibody against PTHrP (38-64)
was used (Fig. 5B), the higher molecular mass bands that
accumulate in the presence of MG-132 were not detected. This suggests
that ubiquitin may be conjugated to PTHrP in this region thus masking
the ability of the antibody to detect it. This result highlights the
necessity of using antibodies in such studies which detect different
epitopes.
To determine whether the higher molecular mass immunoreactive bands
that accumulated in the presence of MG-132 represented ubiquitinated
PTHrP, cellular extracts from Me2SO- or MG-132-treated cells were purified by Ni2+-NTA affinity chromatography.
The material that bound to and was eluted from the column was then
examined by Western blotting using a PTHrP (1-34) antibody (Fig.
6). Eluted protein from MG-132- but not
Me2SO-treated cells retained the same high molecular mass bands that were initially recognized by the PTHrP (1-34) antibody (see
Fig. 5A). The bands in Fig. 6 are specific for PTHrP because Ni2+-NTA purification and immunoblotting of
non-His6-(mock-) transfected cell extracts did not react
with PTHrP (1-34) antibody (see Fig. 9A, lanes 1 and 2). In addition, the higher molecular mass bands, purified on the Ni2+-NTA affinity column, reacted with an
antibody against the HA epitope, suggesting that these bands contain
ubiquitin (see Fig. 9B). Furthermore, the pattern of higher
molecular mass bands detected in transfected cells was identical to
that observed from in vitro translations. Taken together,
these data strongly suggest that PTHrP is multiubiquitinated in
vivo.
To address the question of whether the stability of PTHrP is altered by
treatment with MG-132, we created stably transfected Chinese hamster
lung fibroblasts that express prepro-PTHrP. PTHrP expression levels in
these cells were significantly lower than in transiently transfected
COS-7 cells (Fig. 7, compare lanes 3 and
4 with lanes 1 and 2). Treatment of
these cells with MG-132 and analysis of total protein by Western
blotting resulted in an accumulation of PTHrP, suggesting that MG-132
inhibits the degradation of PTHrP in vivo (Fig. 7).
We next evaluated whether the addition of MG-132 altered the amount of
PTHrP secreted in the conditioned medium. The levels of secreted PTHrP
in conditioned medium of Me2SO-treated (control) cells were
the same as in MG-132-, calpain inhibitor II-, or E64-treated cells in
both transiently transfected COS and stably transfected fibroblast
cells (data not shown).
To
investigate the cis-acting requirements for PTHrP
ubiquitination, we tested two His6-tagged deletion mutants
of PTHrP in the transient cotransfection assay described above. Since
previous studies have suggested that hydrophobic sequences and
amphipathic
To determine if the higher molecular mass bands contained ubiquitin, a
duplicate filter was immunoblotted with the anti-HA monoclonal
antibody. This identified those proteins that migrated as higher
molecular mass bands and contained the HA-epitope derived from
exogenous ubiquitin (Fig. 9B). HA-cross-reactive bands
accumulated in the presence of MG-132 compared with
Me2SO-treated cells for all PTHrP substrates tested
(lanes 4, 6, 8, and 10).
Evidence that MG-132 was effective was obtained by demonstrating, in
cells transfected with the HA-ubiquitin construction alone, the
accumulation of non-PTHrP, HA-reactive bands in MG-132-treated cells
but not in Me2SO-treated cells (Fig. 9B, compare
lane 2 with lane 1). PTHrP is therefore
ubiquitinated in vivo, but neither the prepro nor the
COOH-terminal PEST region appear necessary for efficient ubiquitination.
We have demonstrated ubiquitination and
proteasome-dependent degradation of a protein,
prepro-PTHrP, which is normally a secreted entity. In vitro
translation of prepro-PTHrP in reticulocyte lysate revealed the
accumulation of higher molecular mass bands. Exogenous addition of
ubiquitin in the form of a fusion protein conjugated specifically to
prepro-PTHrP and prepro-PTHrP was observed to be degraded in an ATP-
and proteasome-dependent fashion in vitro. Expression of prepro-PTHrP and an epitope-tagged ubiquitin in the
presence of the proteasome inhibitor MG-132 led to the accumulation in vivo of ubiquitin-PTHrP conjugates. These results
indicate that degradation of PTHrP by the
ubiquitin-dependent proteolytic pathway may be a mechanism
to regulate the intracellular abundance of the protein.
We also examined several putative internal signals that might target
PTHrP for ubiquitination. We observed that deletion of the prepro
sequence resulted in higher levels of PTHrP within the cell consistent
with its impaired secretion, but did not abolish efficient
ubiquitination of PTHrP in vivo. Consequently, the prepro region is as a minimum not a sole site of ubiquitination, nor is it
essential for directing ubiquitination. We therefore examined an
additional putative cis-acting signal for ubiquitination
within the mature protein, a 14-amino acid domain of PEST-rich
sequences in the COOH-terminal region of the protein. Deletion of only
these 14 amino acids did not however abolish ubiquitination. The amino acid sequence extending from residue 109 to residue 126 reveals another
domain enriched in PEST amino acids. It is possible that additional
deletion of this sequence would have influenced the ubiquitination
process, consistent with previous results that multiple PEST-like
stretches may be additive in influencing the stability of some proteins
(38). Further studies will be required to examine this possibility.
Although we were able to detect ubiquitin-PTHrP conjugates using an
antibody directed against the 1-34 sequence, we were unable to detect
such conjugates using an antibody that recognizes PTHrP (38-64) (Fig.
5 B). This lack of reactivity could be due to blockade by
ubiquitin moieties of the PTHrP epitope recognized by the antibody and
suggests that this region may be a site for ubiquitin ligation.
The subcellular compartment in which PTHrP undergoes ubiquitination and
degradation as well as the precise role that degradation plays in the
function of PTHrP remain unknown. Since PTHrP is a secreted protein,
ubiquitination could be occurring in the secretory pathway. Although
the ubiquitin proteolytic system has generally been regarded as a
cytosolic or nuclear pathway, recent studies have identified the
presence of ubiquitin and ubiquitin-activating enzyme associated with a
post-ER/pre-Golgi compartment (39). In addition, lactacystin, a
specific inhibitor of the proteasome, was shown to block the
degradation of a mutant precursor of glycosylphosphatidyl-linked protein in a pre-Golgi compartment (40). The lumenal degradation of
mutated secretory protein The ubiquitination and degradation of PTHrP may therefore be occurring
at least in part in the cytoplasmic compartment of the cell. It has
been suggested previously that the ubiquitin system could be involved
in the degradation of secreted proteins that escape the secretory
pathway (45). Thus, a possible function of ubiquitination may be to
metabolize PTHrP that has entered the cytoplasm. This may simply
provide a clearance mechanism for a peptide normally destined for
secretion. Although overexpression of PTHrP in transiently transfected
COS cells may have facilitated aberrant localization of the peptide in
the cytoplasm, we have observed a similar effect of MG-132 on PTHrP
degradation in stably transfected Chinese hamster lung fibroblasts,
which express at least 100-fold lower levels of PTHrP than do COS cells
(Fig. 7).
Although PTHrP is a secreted peptide, currently available data indicate
potential intracellular roles for this protein. It has recently been
shown that PTHrP is localized in the nucleolus where it has been
suggested to play a role in preventing cells from undergoing apoptotic
cell death (7). The mechanism for nuclear localization of PTHrP is not
known but is dependent on a consensus nuclear localization signal in
the midregion of the protein (7). Another role for PTHrP is the
regulation of growth and differentiation. Antisense RNA-mediated
inhibition of PTHrP production has shown that PTHrP can modulate the
growth of both normal and transformed cells (6, 11, 18). One
possibility is that ubiquitination of prepro-PTHrP acts to prevent
entry of the protein into the secretory pathway and may therefore
enhance availability of PTHrP for these functions. Thus early in the
translation process, ubiquitin moieties may conjugate to prepro-PTHrP
on the cytosolic side of the nascent protein as it is docked at the ER membrane, thereby redirecting it into the cytoplasm from which it may
access the nuclear compartment. The finding that a
ubiquitin-conjugating enzyme (UBC6 in yeast) has been localized to the
ER with the catalytic domain facing the cytosol (46) may support such a
mechanism.
It is conceivable however that a carefully regulated
ubiquitin-dependent mechanism exists to modulate the
half-life of PTHrP in the cytoplasm and/or nucleus for the
intracellular functions described above. Furthermore, a recent report
has suggested that intracellular PTHrP abundance may be dependent on
the stage of the cell cycle. In this study in nontransformed
asynchronously growing cells immunoreactive PTHrP was found to
accumulate in the G2+M phase of the cell cycle in the
absence of any changes in mRNA expression, suggesting that this
accumulation occurs due to inhibition of degradation rather than
enhanced synthesis (47). In contrast to the reported finding of cell
cycle-dependent PTHrP accumulation in normal cells,
immunoreactive PTHrP abundance in the squamous carcinoma cell line SCC
and in the Rice 500 Leydig cell line does not appear to display
variation during the cell cycle (47). This discrepancy may suggest that
post-translational control of PTHrP abundance may be defective in
cancer cells. Further work will be necessary to explore a potential
link between cell cycle and PTHrP ubiquitination in both normal and
transformed cells.
We thank D. Bohmann for pCMV-HA-ubiquitin, J. Huibregste for GST and GST-ubiquitin fusion constructs, A. Ciechanover
for ubiquitin aldehyde, and W. Dunn for the E36 cells.
Calcium Research Laboratory and
¶ Polypeptide Laboratory,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-amino groups of lysine
residues in target proteins by the action of ubiquitin-activating
enzyme, ubiquitin-conjugating enzymes and ubiquitin-protein ligases.
The resulting multiubiquitinated protein is either deubiquitinated by
isopeptidases or degraded by a multicatalytic protease complex, the
26 S proteasome. Multiubiquitination has been implicated, for example,
in the rapid degradation of a number of cytosolic regulatory proteins
such as transcriptional regulators, oncoproteins, and regulators of
cell cycle progression (for review, see Ref. 21). In addition,
degradation of the cystic fibrosis transmembrane conductance regulator
was reported to involve the ubiquitin-proteolytic pathway, suggesting
that ubiquitin and the proteasome play a role in the maturation of
ER-targeted proteins (22, 23). In this study, we show that prepro-PTHrP
is ubiquitinated in vitro and in vivo in cultured
cells. In addition, we also show that in transfected cells treated with
an inhibitor of the proteasome, ubiquitinated PTHrP accumulated. To
determine whether we could identify a cis-acting sequence
that confers ubiquitination, we deleted the prepro domain of PTHrP and
a COOH-terminal PEST motif and have observed that these features are
not essential for the destabilization of PTHrP. This is a novel report
of a secreted polypeptide hormone that is a substrate for the ubiquitin
proteolytic pathway. We propose that the proteasome proteolytic pathway
is involved in regulating the intracellular level of PTHrP, which in
turn would have consequences for its role as a modulator of cell growth
and differentiation.
Materials
S, hexokinase, calpain inhibitor II, E64,
and RNase A were all purchased from Boehringer Mannheim. 2-Deoxyglucose
was obtained from Sigma. The proteasome inhibitor MG-132 was a gift from MyoGenics. Ubiquitin aldehyde was a gift from A. Ciechanover. An
expression plasmid encoding ubiquitin fused at the NH2
terminus to hemagglutinin (HA) sequences was from D. Bohmann (24). The cDNAs encoding prepro-PTHrP and prepro-PTH were kindly supplied by
G. N. Hendy. Polyclonal antisera to PTHrP (1-34) was raised in a
rabbit using synthetic peptide conjugated to methylated bovine serum
albumin. The monoclonal anti-PTHrP antibody (mAb1) was purchased from
Oncogene Science. The mouse monoclonal anti-HA antibody (clone 12CA5)
was purchased from Boehringer Mannheim.
-GCTCTAGA
GTGATGGTGATGGTGATGAAATTCCCTTGAGCTGGG-3
) as the reverse primer on PLPm10 as template. The PTHrPhistag
primer encodes the last five codons of mature PTHrP except the last two codons T and H are replaced by E and F (italics). The primer also contains six histidine residues (bold type), a termination codon (underlined), and ends with an XbaI restriction site. The
amplified product was digested with HindIII and
XbaI and ligated into the HindIII and
XbaI sites of pRC/CMV, generating plasmid CMV
PTHrP-His6 for use in transient transfections.
P, two overlapping primers were synthesized which both contain
the same deletion of nucleotides encoding amino acids
36 to
1.
These "inside" primers were used in separate PCRs together with
"outside" universal primer in the left PCR and with outside PTHrPhistag primer in the right PCR. The amplified products were gel
purified. Then a subsequent reamplification of these purified overlapping fragments with only the outside primers resulted in production of the full-length product containing the deletion. This PCR
product was ligated into pRC/CMV as above generating CMV
PTHrP
P-His6. A plasmid in which the PEST region was
deleted was generated by PCR using the universal primer as the forward primer and PESThis6 as reverse primer. PESThis6
is 5
-GCTCTAGATCAGTGATGGTGATGGTGATGAAATTCGGGCTGGGGGTC-3
, which lacks
nucleotides encoding amino acids 126-139 of the mature PTHrP, leaving
intact distally the sequence encoding EFHHHHHH and a termination codon.
The PCR-amplified product was ligated into pRC/CMV as described above
generating CMV PTHrP
PEST-His6. A construct containing
the double deletion
P and
PEST was generated by PCR using the
universal and PESThis6 primers on the purified PCR product
obtained from PTHrP
P-His6. To express human prepro-PTH, cDNA encoding this peptide was excised from plasmid pPTHm125 (26) using BamHI and HindIII and then ligated into the
expression vector pKS (Stratagene) which had been cut with the same
enzymes. All constructions were verified by sequencing.
80 °C. Levels of PTHrP in the media were
determined by an amino-terminal radioimmunoassay kit from Incstar.
80 °C. Protein concentrations in
the lysates were determined by the Bradford assay (Bio-Rad).
and affinity purified
on glutathione-Sepharose (Pharmacia Biotech Inc.) under conditions
recommended by the manufacturer. Bound proteins were eluted with 10 mM glutathione (Boehringer Mannheim). For assay of PTHrP
conjugation to GST-ubiquitin, 12.5 µl of in vitro
translated 3H-labeled PTHrP was incubated at 37 °C for
up to 3 h in a total volume of 30 µl in a reaction mixture
containing 50 mM Tris-HCl (pH 7.5), 2 mM
MgCl2, 1 mM dithiothreitol, 2 mM
ATP
S, and 5 µg of GST or GST-ubiquitin. Aliquots of the reaction
were terminated with SDS sample buffer and subjected to SDS-PAGE
(12.5%), fluorography, and autoradiography.
S. In assays involving ATP depletion of the lysate, 20 mM 2-deoxyglucose and 1.5 units hexokinase were added
instead of ATP. Reactions were incubated at 30 °C for indicated
times. Aliquots of the reactions were terminated by the addition of SDS
sample buffer and analyzed on SDS-PAGE as described above.
Characterization of Prepro-PTHrP Translated in
Vitro
Fig. 1.
In vitro translation of
prepro-PTHrP. Rabbit reticulocyte lysate (lanes 1 and
2) and wheat germ lysate (lanes 3 and 4) were programmed without (lanes 1 and
3) or with (lanes 2 and 4)
prepro-PTHrP mRNA synthesized in vitro. Translation
products were analyzed on SDS-PAGE as described under "Experimental
Procedures." The arrow points to the full-length PTHrP
protein, and the asterisks mark the higher molecular mass
bands.
[View Larger Version of this Image (33K GIF file)]
Fig. 2.
PTHrP is ubiquitinated in
vitro. Rabbit reticulocyte lysate translations were
programmed with prepro-PTHrP mRNA (lanes 1-3) or
prepro-PTH mRNA (lanes 4-6) in the presence of either GST (lanes 2 and 5) or GST-tagged ubiquitin
(lanes 3 and 6). Reaction products were analyzed
by SDS-PAGE and autoradiography. The arrows point to the
full-length translation products, the asterisks indicate to
the higher molecular mass bands, and the open arrowhead
indicates the novel GST-ubiquitin-prepro-PTHrP conjugate.
[View Larger Version of this Image (50K GIF file)]
S, a nonhydrolyzable ATP analog, to
the reticulocyte lysate. This led to a slight reduction in PTHrP
proteolysis (Fig. 3B, lanes 5-8) and
stabilization of the higher molecular mass bands. PTHrP was slightly
degraded in the presence of ATP
S presumably because of residual ATP
in the lysate. The observation that the higher molecular mass bands
appear in the presence of ATP
S supports the suggestion that these
bands are ubiquitinated forms of prepro-PTHrP because ubiquitin
conjugation to target proteins requires hydrolysis of the
-
bond
and not the
-
bond in ATP (30). Moreover, the inhibition of
proteolysis observed in the absence of ATP hydrolysis suggests that the
26 S proteasome is involved.
Fig. 3.
Degradation of prepro-PTHrP in reticulocyte
requires ATP. Panel A, ATP is required for prepro-PTHrP
degradation and higher molecular mass band formation. Degradation
assays were performed where either ATP (lanes 1-4) or an
ATP-depleting system (lanes 5-8) was added after 30 min of
translation (time 0). Aliquots of the reaction were taken at
2, 4, and 6 h of incubation at 30 °C and analyzed by SDS-PAGE
and autoradiography. Panel B, hydrolysis of ATP is required
for prepro-PTHrP degradation. Degradation assays were performed in
reticulocyte translations as in panel A where either ATP
(lanes 1-4) or ATPS (lanes 5-8) was added
after 30 min of translation (time 0).
[View Larger Version of this Image (41K GIF file)]
Fig. 4.
The 26 S proteasome is responsible for
prepro-PTHrP degradation in reticulocyte lysate. Rabbit
reticulocyte lysate translations were performed as described under
"Experimental Procedures" with the addition of either Me2SO
(lanes 1-5), 50 µM MG-132 (lanes 6-10), 50 µM MG-132 plus 30 µM
ubiquitin aldehyde (Ubal; lanes 11-15), or 50 µM calpain inhibitor II (dissolved in
N,N-dimethylformamide; lanes 16-20).
The final concentration of diluents in the reaction is 0.2%. Aliquots
of each reaction were removed at 0.5, 1, 2, 4, or 6 h after the
start of translation and terminated by the addition of SDS sample
buffer. Translation products were analyzed by SDS-PAGE and
autoradiography. The arrows point to the migration of
full-length prepro-PTHrP.
[View Larger Version of this Image (71K GIF file)]
Fig. 5.
Ubiquitination of prepro-PTHrP in
vivo. Panel A, COS-7 cells transiently transfected
with pRC/CMV vector (lanes 1 and 2) or with
prepro-PTHrP-His6 (lanes 3-10) together with
CMV-HA-ubiquitin (lanes 1-10) were treated for the
indicated times with Me2SO (DMSO, lanes
1-4), 50 µM MG-132 (lanes 5 and
6), 50 µM calpain inhibitor II (lanes
7 and 8), or 50 µM E64 (lanes
9 and 10). Cell lysates (30 µg of total protein) were
immunoblotted with PTHrP (1-34) polyclonal antibody. The closed
arrow points to the migration of full-length prepro-PTHrP, and the
open arrow points to a putative signal peptide cleavage
product, pro-PTHrP. Note that prepro-PTHrP migrates just below a
cellular protein that cross-reacts nonspecifically with the PTHrP
(1-34) antibody and is indicated by an open arrowhead. The
asterisk marks the higher molecular mass bands present only in cells treated with MG-132 and recognized by anti-PTHrP (1-34) antibody. Panel B, a blot similar to that described in
panel A except immunoblotted with a monoclonal antibody
recognizing PTHrP (38-64).
[View Larger Version of this Image (39K GIF file)]
Fig. 6.
Specific binding of ubiquitinated PTHrP to
Ni2+-NTA resin. Cell lysates from COS-7 cells
coexpressing prepro-PTHrP-His6 and HA-ubiquitin which were
treated with 50 µM MG-132 (lanes 1-3) or
Me2SO (DMSO, lanes 4-6) for 24 h were fractionated on Ni2+-NTA resin as described under
"Experimental Procedures." Samples of total lysate (load; 21 µg
of total protein), flow-through unbound material (FT), and
bound, eluted material (eluate), each representing equivalent fractions of the original cell lysate, were electrophoresed and immunoblotted with PTHrP (1-34) antibody. Closed and
open arrows point to prepro-PTHrP and pro-PTHrP,
respectively. The asterisks mark the position of
ubiquitin-PTHrP conjugates formed only in the presence of MG-132.
[View Larger Version of this Image (32K GIF file)]
Fig. 9.
Expression and ubiquitination of deletion
mutants. Panel A, Western blot of Ni2+-NTA bound
and eluted material of cell lysates from COS-7 obtained after 18 h
of treatment with either Me2SO () or 50 µM
MG-132 (+). The cells were transfected with either pRC/CMV vector
(mock; lanes 1 and 2), full-length
PTHrP-His6 (lanes 3 and 4), or
PTHrP-His6 deletion mutants (lanes 5-10) and
with HA-ubiquitin. Immunoblotting was with anti-PTHrP (1-34) antibody.
Panel B, Western blot of identical samples as in panel
A except immunoblotted with anti-HA antibody. In this blot, the
PTHrP-ubiquitin conjugates are detected with all constructs. In
lanes 4 and 8 the PTHrP-ubiquitin and PTHrP
PEST-ubiquitin conjugates are indicated by closed
and open arrowheads, respectively.
[View Larger Version of this Image (30K GIF file)]
Fig. 7.
MG-132 causes accumulation of PTHrP in
transfected cells. COS-7 cells transiently transfected
(lanes 1 and 2) or E36 cells stably transfected
(lanes 3 and 4) with
prepro-PTHrP-His6 were treated for 18 h in the absence
(lanes 1 and 3) or presence (lanes 2 and 4) of 50 µM MG-132. Total protein (10 µg) from cell extracts was immunoblotted with anti-PTHrP (1-34)
antibody. The migration of prepro-PTHrP and pro-PTHrP is indicated by
closed and open arrows, respectively. The
open arrowhead indicates a nonspecific protein that
cross-reacts with the antibody.
[View Larger Version of this Image (19K GIF file)]
-helices may target proteins for ubiquitin-mediated
degradation (34), we determined whether the hydrophobic prepro sequence may target PTHrP for degradation. A truncated molecule lacking the
NH2-terminal 36-amino acid prepro sequence was therefore
created (PTHrP
P-His6, Fig. 8). PTHrP is
also rich in the amino acid residues Pro, Glu, Asp, Ser, and Thr (PEST)
in the carboxyl portion of the protein (Fig. 8). PEST-rich sequences
have been suggested to function as degradation signals (35) and have
recently been implicated to be required for recognition by the
ubiquitination process (36-38). To test whether the PEST site in PTHrP
serves as a degradation signal, amino acids 126-139 from the mature
protein were deleted (PTHrP
PEST-His6, Fig. 8). In
addition, a double mutant was created which lacked both the prepro and
the PEST motifs (PTHrP
P
PEST-His6). COS-7 cells were
cotransfected with full-length PTHrP or the deletion mutants and with
HA-tagged ubiquitin. Transfectants were exposed to Me2SO or
MG-132 for 18 h, and cell lysates were purified on
Ni2+-NTA affinity columns. Eluted material was analyzed by
immunoblotting with antibody recognizing PTHrP (1-34) (Fig.
9A). As demonstrated by this blot,
PTHrP-His6 and deletion mutants were efficiently expressed
and recovered from the transfected cells by NTA chromatography. PTHrP
cross-reactive material was present in the eluates with accumulation of
material in the MG-132-treated cultures (Fig. 9A). In
addition, overall levels of protein were higher for the leaderless
constructs because translated PTHrP was not secreted and therefore
accumulated in the cytosol. Several higher molecular mass bands with
mobilities appropriate for ubiquitin-PTHrP conjugates accumulated in
the presence of MG-132 with all PTHrP constructs tested. Strikingly,
these bands were observed in the absence of MG-132 for the two
leaderless mutants.
Fig. 8.
Schematic description of the examined
deletion mutants. The proteins used as substrates for
ubiquitination in transient transfection assays are represented as
rectangular boxes. The prepro sequence is highlighted as a
light gray box, a PEST domain is highlighted as a dark
gray box, and the six histidines tagged at the COOH terminus are
illustrated as black boxes. The sequence of amino acids
126-139 is indicated above the PEST box. Deletions of the prepro
sequence (P) and PEST (
PEST) domains are illustrated by the
dotted lines.
[View Larger Version of this Image (12K GIF file)]
1-antitrypsin is suggested to
be proteasome-dependent via association with the
transmembrane chaperone protein calnexin (41). Recent studies have
provided evidence that ER-targeted aberrant proteins can be degraded by
the 26 S proteasome in the cytoplasm. Although
ubiquitin-dependent degradation of the integral cystic
fibrosis transmembrane conductance regulator protein most likely occurs
on the cytoplasmic side of the ER membrane (22, 23), the
proteasome-dependent degradation of some aberrant lumenal
polypeptides (42-44) is proposed to be due to retrograde transport of
these proteins at least partially out of the ER into the cytosol, where
they are subsequently degraded by the proteasome. Taken together, these
studies suggest that ER-associated degradation of secretory proteins
can involve the proteasome and are consistent with our observations
that prepro-PTHrP can be a substrate for the ubiquitin-proteolytic
system.
*
This work was supported by a Medical Research Council of
Canada (MRC) fellowship (to K. M.), MRC Clinician Scientist Award and
MRC Grant MT-12121 (to S. W.), and MRC Grant MT-5775 and National Cancer Institute Grant 7620 (to D. G.).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: Calcium Research
Laboratory, Rm. H4.67, Royal Victoria Hospital, 687 Pine Ave. West,
Montreal, P.Q. Canada H3A 1A1. Fax: 514-843-1712.
1
The abbreviations used are: PTHrP, parathyroid
hormone-related protein; PTH, parathyroid hormone; ER, endoplasmic
reticulum; PEST, amino acid residues Pro (P), Glu (E) and/or Asp (D),
Ser (S), and Thr (T); ATPS, adenosine
5
-O-(thiotriphosphate); HA, hemagglutinin; CMV,
cytomegalovirus; PCR, polymerase chain reaction; Me2SO,
dimethyl sulfoxide; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.