From the Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Received for publication, November 30, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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Ectopic expression of Jab1/CSN5 induces specific
down-regulation of the cyclin-dependent kinase (Cdk)
inhibitor p27 (p27Kip1) in a manner dependent upon
transportation from the nucleus to the cytoplasm. Here we show that
Grb2 and Grb3-3, the molecules functioning as an adaptor in the signal
transduction pathway, specifically and directly bind to p27 in the
cytoplasm and participate in the regulation of p27. The interaction
requires the C-terminal SH3-domain of Grb2/3-3 and the proline-rich
sequence contained in p27 immediately downstream of the Cdk binding
domain. In living cells, enforcement of the cytoplasmic localization of
p27, either by artificial manipulation of the nuclear/cytoplasmic
transport signal sequence or by coexpression of ectopic Jab1/CSN5,
markedly enhances the stable interaction between p27 and Grb2.
Overexpression of Grb2 accelerates Jab1/CSN5-mediated degradation of
p27, while Grb3-3 expression suppresses it. A p27 mutant unable to bind
to Grb2 is transported into the cytoplasm in cells ectopically
expressing Jab1/CSN5 but is refractory to the subsequent degradation.
These findings indicate that Grb2 participates in a negative regulation of p27 and may directly link the signal transduction pathway with the
cell cycle regulatory machinery.
The proliferation of mammalian cells is strictly regulated by
extracellular signals, which largely exert their effects during the
G1 phase of the cell cycle. Among G1 regulatory
factors, the Cdk inhibitor p27 (p27Kip1), which negatively
regulates cell proliferation, is the downstream target of
mitogen-stimulated signal transduction, and modulation of p27 activity
is one of the most important steps not only in the control of mammalian
cell proliferation but also in the regulation of normal tissue
development and in the suppression of malignant transformation (1, 2).
The expression level of p27 is regulated in several ways, among which
cell cycle-dependent and substrate-specific proteolysis
seems to be the most important (1, 2). Down-regulation of p27 has been
reported to involve (i) phosphorylation of the Thr187
residue by the cyclin E-Cdk2 complex (3, 4), (ii) transport from
the nucleus to the cytoplasm (5), (iii) ubiquitination mediated by the
ubiquitin ligase SCFSKP2 complex (6-8), and (iv)
proteolysis by the 26 S proteasome. However, the precise biochemical
link between these events and the biochemical reaction that initiates
p27 degradation remains to be clarified.
Recently, we have isolated a new regulator of p27, Jab1/CSN5 (5).
Jab1/CSN5 was originally identified as a coactivator of c-Jun
transcription factor (9), and recent findings indicate that Jab1/CSN5
is the fifth component of the COP9 signalosome complex (10) (the
nomenclature of the eight subunits is now unified as CSN1-8 (11)).
Although the COP9 signalosome was originally identified in
Arabidopsis as a negative regulator of photomorphogenesis, purification of the COP9 signalosome complex from mammalian cells has
revealed that its function is not necessarily restricted to light/dark-mediated signal transduction in plants. Genetic analysis has
demonstrated that the complex plays a pivotal role in the regulation of
early development (12), but the specific biochemical functions of the
complex are not fully clarified yet. Jab1/CSN5 directly interacts with
p27 in vitro as well as in vivo. In cells expressing ectopic Jab1/CSN5, p27 is exported from the nucleus to the
cytoplasm and is induced to be degraded in a manner sensitive to
chemical inhibitors of CRM1-dependent nuclear export and
the 26 S proteasome. Jab1/CSN5 overexpression enables mouse fibroblasts to progress from G0 to S phase in low serum, indicating
that Jab1/CSN5 plays an important role in G1 progression
and cell proliferation (5).
Many growth factor receptors exhibit tyrosine kinase activity triggered
by oligomerization upon binding to their specific ligands, leading to
autophosphorylation (13). Phosphotyrosine residues contained in the
activated receptor serve as docking sites for a variety of downstream
effector molecules such as Grb2. Grb2 functions as an adaptor and
contains two types of interaction domains, SH2 and SH3 (14). SH2
mediates binding to phosphotyrosine residues contained in the receptor,
and SH3 directs Grb2 to the proline-rich motif, through which Grb2
associates with SOS, resulting in activation of Ras protein. The
mitogen-activated protein kinase cascade consisting of Raf kinase,
mitogen-activated protein kinase kinase, and mitogen-activated
protein kinase (Erk1 and Erk2) as well as another Ras target,
phosphatidylinositol 3-kinase, acts downstream of Ras and facilitates
cell cycle progression through G1. Introduction of a
dominant negative form of Ras into proliferating cells up-regulates p27
protein expression (15, 16), while activation of Ras induces p27
down-regulation (17). Moreover, pharmacological inhibition of
mitogen-activated protein kinase kinase or phosphatidylinositol
3-kinase counteracts the action of Ras to down-regulate p27 (16, 17).
These findings strongly suggest that the
receptor-Ras-(mitogen-activated protein kinase, phosphatidylinositol
3-kinase) signaling pathway plays an important role in p27 regulation.
In the present study, we show that Grb2 and its alternatively spliced
form, Grb3-3, bind to p27 in the cytoplasm and that the interaction
between Grb2 and p27 is required for efficient degradation of p27. Our
findings may, at least in part, explain why nuclear p27 is translocated
to the cytoplasm before degradation and provide a novel pathway from
the signal transduction machinery directly to the cell cycle regulator.
Recombinant Proteins and in Vitro Protein Binding Assay--
To
construct the p27(PA) mutant, overlapping fragments of p27, introducing
the relevant nucleotide changes, were made. The 3'-fragment was made by
PCR1 using wild-type p27
cDNA as a template and the PA mutation primer (5'-C TAC TAC AGG GCC
GCG CGC GCC GCC AAG AGC GCC TGC-3') and 3'-p27 primer (5'-AAGCTT CGT
CTG GCG TCG AAG G-3'). The 5'-fragment was made using the same template
but with the reverse PA mutation primer (5'-GCA GGC GCT CTT GGC GGC GCG
CGC GGC CCT GTA GTA G-3') and 5'-p27 primer (5'-GGATCC ATG TCA AAC GTG
AGA GTG T-3'). The overlapping PCR fragments were purified, mixed, and
used as the template for the second PCR. The full-length p27(PA) mutant
fragment was amplified by PCR using the 5'- and 3'-p27 primers
introducing a 5' BamHI and a 3' HindIII site,
respectively. cDNA fragments encoding p27 variants, Grb2, Grb3-3,
and a variety of SH3-containing proteins were amplified by PCR using a
pair of primers specific to each of them. The resulting PCR fragments
were cloned, sequenced to confirm sequence integrity, and inserted into
pGEX (Amersham Pharmacia Biotech) in frame with glutathione
S-transferase (GST) and pBluescript. GST fusion proteins
were expressed in bacteria and purified as described (18). Crude cell
extracts containing recombinant mammalian cyclin D1-Cdk4 complex
expressed in Sf9 cells by infection with baculovirus expression
vectors were prepared as previously described (19). pBluescript
plasmids containing cDNA were transcribed and translated in
vitro in the presence of [35S]methionine using the
TNT T7/T3 Coupled Reticulocyte Lysate Systems kit (Promega) according
to the manufacturer's instructions. Binding was performed as described
(5) in buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40, and
the protein complexes were washed in the same buffer.
Cell Culture and High Efficiency Transfection--
NIH3T3 and
COS7 cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Grb2/Grb3-3 cDNA was
inserted into the pFLAG-CMV-2 expression vector (Eastman Kodak Co.) in
frame with a FLAG epitope. The expression vectors for p27 variants and
Jab1 were previously described (5). Cells were transfected with vectors
by a modified calcium phosphate-DNA precipitation method (20). The
highest efficiency was obtained as described (5). Consistently,
50-80% of the transfected cells expressed exogenous proteins coded in
the plasmid. Antisense oligonucleotides and controls directed to Grb2
have been designed and manufactured by Biognostik, Germany, and were
directly added to the medium (final concentration 2 µM).
Cells were cultured in the same medium for 3 days and harvested. Cell
lysates were analyzed by immunoblotting using antibodies specific to
Grb2, p27, and p21.
Protein Analyses--
Cells were harvested 24 h (for
detection of the complex) and 48-72 h (for detection of p27
down-regulation) after transfection. Metabolic labeling, cell lysis,
immunoprecipitation, gel electrophoresis, and immunoblotting were
performed as described (5, 18, 19). Polyclonal antibodies to GST and
p27 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Mouse monoclonal antibodies to HA and FLAG peptide epitopes were
obtained from Roche Molecular Biochemicals and Kodak, respectively.
Immunofluorescent Staining--
Cells were fixed in 3%
paraformaldehyde, permeabilized in 0.5% Triton X-100, stained with
anti-HA mouse monoclonal antibody (Roche Molecular Biochemicals), and
incubated with Texas Red-linked anti-mouse IgG (Amersham Pharmacia
Biotech). For determination of BrdU incorporation, cells stained with
anti-HA rabbit polyclonal antibody (Babco) followed by Texas Red-linked
antirabbit IgG (Amersham Pharmacia Biotech) were treated with 1.5 M HCl and stained with anti-bromodeoxyuridine mouse
monoclonal antibody (Amersham Pharmacia Biotech) and fluorescein-linked
anti-mouse IgG (Amersham Pharmacia Biotech). The cell samples were
viewed by phase-contrast and/or fluorescence microscopy. More than 500 cells were examined for each transfectant.
Identification of Grb2 and Grb3-3 as New Interactors Specific to
p27--
Using a yeast two-hybrid screen, we identified three kinds of
cDNAs (5), all of which encode polypeptides capable of specifically interacting with the C-terminal domain of p27Kip1. We
previously showed that one of them encodes Jab1/CSN5, which negatively
regulates p27 by translocating it from the nucleus to the cytoplasm and
subsequently inducing its degradation (5). The second class of cDNA
contained the C-terminal SH3 domain of Grb2, which is well known to
function as an adaptor molecule in the signal transduction pathway
(14). To determine the specificity of the interaction between p27 and
Grb2, we analyzed the capability of p27 to bind to other SH3-containing
proteins. We selected from the data base several genes that encode
proteins containing a SH3 domain closely related to the C-terminal SH3
domain of Grb2 (more than 40% identity), including STAM, SH3P8,
SH3P13, and mNck-
Among a variety of GST fusion proteins containing a portion of the p27
molecule (Fig. 1B), all that contained amino acids 89-96
bound to 35S-labeled, in
vitro-transcribed/translated Grb2 and Grb3-3 proteins in a test
tube (Fig. 1C). The N terminus deletion mutant,
p27-(89-197), associated with Grb2/3-3 slightly weaker than
other mutants, suggesting that amino acids adjacent to 89 are required
for efficient interaction. The region of amino acids 89-96 is rich in
proline residues and contains two overlapping SH3-binding motifs
(PXXP) (14). Alteration of all of these 4 proline
residues to alanine (RPPRPPK to RAARAAK) completely abolished the
binding activity for Grb2/3-3, while this mutant, designated as
p27(PA), retained the capability of interacting with cyclin D1-Cdk4
complexes (Fig. 1D), inhibiting the kinase activity of
cyclin-Cdk complexes and inducing G1 arrest when
overexpressed in mouse fibroblasts (data not shown, but see Fig.
4C). This proline-rich motif is unique to p27 and not found in sequences of other similar Cdk inhibitors, p21 and p57. In fact, we
did not detect interaction between p21 and Grb2/Grb3-3 under these
conditions in vitro (negative data not shown).
Grb3-3 consisting of two functional SH3 domains but lacking half of the
SH2 domain (21) is able to bind to p27, indicating that the SH3 but not
SH2 domain is involved in interaction with p27. To determine more
precisely the binding domain within Grb proteins, we generated
truncated mutants of Grb3-3, one containing the N terminus SH3 domain
(SH3N) and the other the C terminus SH3 (SH3C) (Fig. 1E),
and then tested them for activity to bind to 35S-labeled
full-length p27 proteins in vitro (Fig. 1F). We
found that the GST fusion protein containing the C terminus SH3 but not
the N terminus SH3 interacted with p27. Thus, these results clearly
indicate that p27 and Grb2/3-3 specifically interact with each other at
least in vitro through the proline-rich motif and a specific
SH3 domain.
Grb2 and Grb3-3 Bind to p27 in the Cytoplasm in Vivo--
In mouse
fibroblasts, which express p27 and Grb2 but not Grb3-3 (data not
shown), we were unable to detect a complex between endogenous p27 and
Grb2; nor did we see any interaction between the two in COS cells
overexpressing exogenous HA-tagged full-length p27 and FLAG-tagged Grb2
proteins (data not shown). This could be because the intracellular
localization of the two proteins is different (p27 is in the nucleus
and Grb2 in the cytoplasm) and because the intermediate p27-Grb2
complex exists only for a very limited time. We examined these
possibilities by using p27 mutants. When we ectopically expressed
FLAG-tagged Grb proteins together with the p27 mutant (p27(NES)) that
localizes mainly in the cytoplasm because of the artificially fused NES
sequence (5), we found that p27(NES) efficiently formed a complex with Grb2 and Grb3-3 in vivo (Fig.
2A). In addition, we detected
very stable interaction between Grb2/Grb3-3 and another p27 mutant, p27-(1-151), which lacks the nuclear localization signal and
remains in the cytoplasm (5) (data not shown). In another experiment, we observed a p27/Grb association when we used degradation-resistant p27 mutants (e.g. p27(T187A), p27-(1-186) (3-5)) (data not
shown, but see Fig. 2C and below). With wild-type p27,
Grb3-3, but not Grb2, formed a detectable amount of complex (Fig.
2B, seventh lane from the
left). Thus, we conclude that (i) p27 binds to Grb proteins
when it localizes in the cytoplasm, (ii) the p27/Grb2 interaction is
very transient, and (iii) inhibition of Thr187
phosphorylation in p27 or disruption of SH2 function in Grb2 increases
the stability of the complex (see below). In control experiments, we
tested for p21 in lieu of p27 in normal NIH3T3 mouse fibroblasts and
transfected COS cells and found that the interaction with Grb2/Grb3-3
was specific to p27 (negative data not shown).
As for the factor that causes the cytoplasmic localization of p27, we
focused on Jab1/CSN5, which directly binds to p27 and down-regulates
the protein by translocating it from the nucleus to the cytoplasm (5).
Since ectopic coexpression of HA-Jab1 (CSN5) induces down-regulation of
wild-type p27 and its effect is prominent 48-72 h after transfection,
we assayed for the complex formation 24 h after transfection, at
which time p27 is not markedly down-regulated. Fig. 2B shows
that coexpression of HA-Jab1 significantly increased the amount of
complex formed between ectopic p27 and Grb3-3 proteins
(seventh and eighth lanes from the
left). Interestingly, upon immunoprecipitation with antibody
specifically recognizing the C-terminal half of the p27 protein, Grb3-3
proteins were not coprecipitated. Since we did not detect Grb3-3 in
anti-HA-immunoprecipitates from cells expressing HA-Jab1 and
FLAG-Grb3-3, it seems likely that this anti-p27 antibody interfered
with the formation of complex between p27 and Grb proteins, or,
alternatively, HA-Jab1 may assist in increasing the stability of the
p27-Grb protein complex. We detected interaction between wild-type p27
and Grb2 in the presence of ectopic HA-Jab1, but not in its absence
(Fig. 2C). Mutation of the C terminus phosphorylation site
(T187A), which renders p27 more stable (3-5), enhanced the complex
formation, while mutation in the Grb2-binding site (p27(PA)) or
deletion of the Jab1-binding domain (p27(Del 97-151)) abolished the
interaction. The p27/Grb2/Jab1 interaction was completely
disrupted in the presence of leptomycin B, a specific inhibitor of the
NES/CRM1-dependent nuclear export (22) (data not shown).
Ectopic Jab1/CSN5 did not induce complex formation between p21 and
Grb2/3-3 (negative data not shown), confirming the specificity of the
interaction. Thus, ectopic expression of Jab1 enables specific p27-Grb2
interaction by translocating p27 from the nucleus to the cytoplasm and
by increasing the stability of the complex.
Grb2 Facilitates Down-regulation of p27 in Vivo--
We next
examined the effect of Grb2 and Grb3-3 on p27 stability. Fig.
3A shows that ectopic p27 was
relatively stable (open circle), and coexpression
of Jab1/CSN5 markedly reduced its stability (closed
circle) as previously reported (5). Additional coexpression of Grb2 further reduced the stability of p27 (open
triangle), while coexpression of Grb3-3 blocked
Jab1/CSN5-mediated down-regulation of p27 (closed
triangle). Therefore, although only the SH3 domain is
required for direct binding to p27, the integrity of the SH2 domain in
Grb2 is important for induction of p27 down-regulation. The simplest
interpretation is that the recruitment of a certain cellular protein,
which most likely contains phosphorylated tyrosine residues, into the
Grb2-p27-Jab1 complex would facilitate down-regulation of p27. However,
Grb2 is well known to function in the signal transduction pathway (14),
especially upstream of the Ras/Raf/mitogen-activated protein kinase
pathway, the activation of which is reported to induce degradation of
p27 (15-17). To analyze the possible involvement of the Ras signaling
pathway in down-regulation of p27, we examined the effect of chemical
inhibitors to mitogen-activated protein kinase kinase (PD98059
(PD); see Ref. 17) and phosphatidylinositol 3-kinase
(wortmannin (WT); see Ref. 16) on the level of p27 in NIH3T3
cells transfected with HA-p27 together with Jab1/CSN5 and Grb2 (Fig.
3B). We found that Grb2 accelerated the degradation of p27
in the presence and absence of these inhibitors. These results strongly
support our interpretation that Grb2 plays an important role in the
regulation of p27 by the direct binding but not by activating the Ras
signaling pathway.
Direct Binding of Grb2 Is Required for Down-regulation of
p27--
To investigate the requirement of direct Grb binding in p27
down-regulation, we utilized the PA mutant of p27 (p27(PA)), which is
unable to bind to Grb proteins due to amino acid substitutions in the
SH3-binding motif but retains the capability of binding to other p27
binding proteins such as cyclin-Cdk complexes and Jab1 (Fig.
1D and data not shown). p27(PA) was located in the nucleus
and transported to the cytoplasm in the presence of ectopic Jab1/CSN5
(Fig. 4A), indicating that the
proline-to-alanine mutation did not affect nuclear import and
Jab1/CSN5-mediated nuclear export of p27. The intensity of the nuclear
staining was indistinguishable between the wild type and the PA mutant,
but the signal of p27(PA) was much stronger than that of wild-type p27
in cells coexpressing Jab1/CSN5, implying that p27(PA) was not
down-regulated in the presence of ectopic Jab1/CSN5. To directly
examine this, we measured the half-life of these two p27 molecules in
the presence and absence of ectopic Jab1 (Fig. 4B).
Exogenous p27 was a relatively stable protein (open
circle) and was induced to be degraded by coexpression of
Jab1/CSN5 (closed circle). The PA mutant was kept
stable whether Jab1/CSN5 was cotransfected or not (open and
closed squares, respectively). In addition,
although the ability of wild-type p27 to inhibit growth was partially
rescued by Jab1/CSN5 coexpression, the PA mutant was quite resistant to
the neutralizing effect of Jab1/CSN5 (Fig. 4C). These
results, together with the observation that most of the cell lines we
examined so far expressed Grb2 but not Grb3-3, indicate that the direct
binding of Grb2 is required for cytoplasmic degradation of p27 but not
for transportation of p27 from the nucleus to the cytoplasm.
Grb2 Is Required for Maintenance of Low Expression of p27 in
Proliferating Fibroblasts--
To investigate the physiological
importance of Grb2 in the regulation of p27 in vivo, we
manipulated the expression of the Grb2 protein by antisense technology
and examined the effect on p27 expression. The addition of the
antisense oligonucleotides to the medium did not have any apparent
effect on NIH3T3 fibroblasts during the first 24 h; however, at 3 days post-treatment, the rate of proliferation gradually slowed.
Importantly, most cells neither exhibited a round morphology nor
detached from the solid support during this period, indicating that few
cells lost their viability due to the treatment with Grb2-specific
antisense oligonucleotides. In these cells, the expression of Grb2
proteins was reduced to 36% compared with that in control cells. In
contrast, p27 expression was 5 times higher than that in cells
untreated or treated with random oligonucleotides. Importantly, Grb2
antisense oligonucleotides did not significantly alter the level of
endogenous p21 proteins, indicating that the effect of Grb2 was
specific to p27 (Fig. 5). These results
demonstrate that Grb2 specifically functions upstream of p27 and is
required for maintaining the low level of p27 protein.
Proteolytic down-regulation specifically linked to transportation
from the nucleus to the cytoplasm is occasionally observed in the
control of key regulators of cell proliferation, such as cyclin D1,
p53, p27, and p27 is subject to multiple forms of regulation (1, 2). It is
transcriptionally activated by the CBP coactivator in response to
retinoic acid treatment (27) and by the Ah receptor (28). Translational
control of p27 expression is also reported (29-31). However, because
the level of p27 protein fluctuates during the cell cycle (high in
G0/G1 and low in S, G2, and M),
while the level of p27 mRNA is constant (29, 30), the main
regulatory mechanism for p27 seems to be post-translational, mostly due
to the activation and inactivation of substrate-specific and cell cycle-dependent proteolysis. Degradation of p27 has been
reported to involve phosphorylation of Thr187 by cyclin
E-Cdk2 complex (3, 4), nuclear export induced by Jab1/CSN5 (5),
ubiquitination mediated by the ubiquitin ligase
SCFSKP2 complex (6-8), and proteolysis by the 26 S
proteasome. However, the precise biochemical link between these events
and the biochemical reaction that initiates p27 degradation remains to
be clarified. Nullification of Skp2, an F-box-containing subunit
of the SCF ubiquitin ligase, results in up-regulation of p27 (32).
Introduction of the dominant negative form of Jab1 increases levels of
p27 expression in proliferating mouse
fibroblasts.2 Therefore,
these two pathways seem to significantly participate in the regulation
of p27 in vivo. But we have not obtained any evidence that
p27 exported from the nucleus by Jab1/CSN5 is ubiquitinated by
SCFSKP2 in the cytoplasm. This may suggest that
these two pathways are independent. Although experiments using
Skp2 Cancers with low p27 protein expression are reported to be well
correlated with poor prognosis (33-35). This finding was originally made in breast and colorectal carcinomas and now is the case for a wide
variety of human tumors. Since the p27 gene is rarely altered in human
cancers, the genetic target for malignant transformation seems to be
the gene functioning upstream of p27. Overexpression of the SKP2
gene is occasionally observed in transformed cells (36) but is not
necessarily correlated with low expression of p27. So far, no
alteration of Jab1/CSN5 expression has been reported, but because other
components of the COP9 signalosome complex are capable of
down-regulating p27,2 it is necessary to investigate
whether any other CSN is involved in human cancers. In addition, our
results suggest that the possible role of Grb2 and its associated
proteins in tumorigenesis has to be reevaluated in terms not only of
activation of the signal transduction pathway leading to Ras activation
but also of direct involvement in control of the key cell cycle regulator.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(accession numbers are U43900, U58885, U58887, and
AF043259, respectively) in addition to Grb2 and its alternatively
spliced form, Grb3-3 (21). We cloned their coding sequences by PCR
using a pair of primers specific to each of them,
transcribed/translated them in vitro in the presence of
[35S]methionine, and used these 35S-labeled
proteins to assay in vitro the binding to GST and
GST-fused p27 recombinant proteins preabsorbed onto glutathione beads.
Fig. 1A shows that
35S-labeled Grb2 and Grb3-3 but not the others associated
with GST-p27 fusion proteins in a test tube. The amounts of bound Grb2
and Grb3-3 were almost the same, and neither molecule bound to GST alone, indicating that the interaction between p27 and Grb proteins is
specific and that Grb2 and Grb3-3 have equivalent binding capabilities at least in vitro.
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Fig. 1.
Specific interaction of Grb2 with p27
in vitro. A, Grb2 and its variant
Grb3-3 specifically interact with p27 in vitro. GST or
GST-p27, immobilized on glutathione beads, was incubated with the
in vitro translated 35S-labeled proteins shown
at the top of the panel. Bound 35S-labeled proteins were
detected by autoradiography. B, p27 mutants. C,
in vitro interaction of GST-fused p27 deletion mutants with
35S-labeled Grb2 and Grb3-3. To confirm that equal amounts
of GST fusion proteins were used, the results of immunoblotting using
antibody to GST are also shown. D, a proline-rich sequence
contained in p27 is required for interaction with Grb2/3-3. The binding
assay was performed as in C. The results of anti-GST
immunoblotting and a control binding assay with recombinant cyclin
D1-Cdk4 complex (detected by anti-cyclin D1 immunoblot) are shown.
E, Grb2 mutants. F, the C terminus SH3 domain of
Grb2 is required for interaction with p27. The assay of the binding
between GST-fused Grb2 variants and 35S-labeled p27 was
performed as in C.
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Fig. 2.
Specific interaction of Grb proteins with
cytoplasmic p27 in vivo. COS cells were
transfected with the expression vectors shown at the top of
the panels. Lysates from cells harvested 24 h
post-transfection were directly analyzed by immunoblot with antibodies
to HA and FLAG epitopes or subjected to immunoprecipitation
(IP)/immunoblot analysis using the same antibodies.
A, Grb2/3-3 form stable complexes with a p27 mutant
containing artificial NES. B, binding of Grb3-3 with p27 was
enhanced by ectopic expression of Jab1. C, both binding
sites for Grb2 and Jab1 contained in p27 are required for p27/Grb2
interaction.
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Fig. 3.
Effect of Grb proteins on the stability of
p27. A, NIH3T3 cells were transfected with expression
vectors (open circle, p27 alone;
closed circle, p27 and Jab1; open
triangle, p27, Jab1, and Grb2; closed
triangle, p27, Jab1, and Grb3-3), pulse-labeled with
[35S]methionine for 30 min at 48 h post-transfection
and chased with excess cold methionine. At the indicated times, cells
were collected, and the relative 35S in HA-p27 was
measured. B, activation of the Ras signaling pathway is
dispensable for accelerated degradation of p27 by Grb2. NIH3T3 cells
were transfected with GFP together with p27, Jab1/CSN5, and Grb2 as
indicated at the top, incubated for 6 h in the presence
of chemical inhibitors (10 µM PD98059 (PD) and
2 µM wortmannin (WT)) at 48 h
post-transfection, and harvested. Cell lysates containing the same
amount of protein were analyzed by immunoblotting with antibodies
specifically recognizing p27, Grb2, Jab1/CSN5, and GFP, respectively.
We confirmed that PD98059 suppressed the activation of
mitogen-activated protein kinase under these conditions. Wortmannin was
used as described (16). DMSO, dimethyl sulfoxide.
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Fig. 4.
The integrity of the Grb2 binding site is
required for Jab1-mediated down-regulation of p27. A,
subcellular localization of the p27(PA) mutant in the presence and
absence of Jab1. NIH3T3 cells were transfected with an HA-tagged
p27(PA) mutant alone (top panels) or
together with a Jab1 expression vector (bottom
panels) and, at 48 h post-transfection, stained with
antibody to an HA epitope followed by the Texas Red-tagged secondary
antibody. The results obtained by phase-contrast (PC) and
fluorescence microscopy (aHA) are shown. B,
stability of wild-type and PA mutant p27 proteins in the presence and
absence of ectopic Jab1. NIH3T3 cells were transfected with HA-p27
wild-type (circles) and PA mutant (squares)
plasmids alone (open symbols) or with HA-Jab1
(closed symbols), and the relative
35S in HA-p27 was measured as in Fig. 3. C,
p27(PA) is resistant to Jab1-mediated neutralization of the
proliferation-inhibitory activity of p27. NIH3T3 cells were transfected
with HA-p27 wild-type or PA mutant plasmids with or without FLAG-Jab1.
After 48 h, cells were incubated with bromodeoxyuridine
(BrdU) for 24 h and simultaneously stained with anti-HA
and anti-bromodeoxyuridine antibodies. Percentages of
bromodeoxyuridine-positive cells among HA-positive cells are
shown.
View larger version (54K):
[in a new window]
Fig. 5.
Grb2 is required for down-regulation of p27
in proliferating fibroblasts. NIH3T3 cells (~10% confluence)
were incubated in medium supplemented with 2 µM antisense
oligonucleotides directed to Grb2 and control oligonucleotides and
harvested after 3 days. Cell lysates containing the same amount of
protein were analyzed by immunoblotting with antibodies specifically
recognizing Grb2, p27, and p21.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin. Nuclear-cytoplasmic transportation and
subsequent degradation of cyclin D1 is regulated by phosphorylation of
a specific threonine residue (Thr286) by GSK-3
kinase
(23), which is a component of the signal transduction pathway.
Ubiquitination and cytoplasmic transportation of p53 is mediated by
MDM2 (24, 25), one of the p53-responsive gene products, thereby
creating a negative feedback loop to make the effect of p53 temporal.
Nuclear export of p27 is induced by Jab1/CSN5, which facilitates
degradation of p27 in the cytoplasm. In this case, the cytoplasmic
location of p27 alone is not sufficient for induction of p27
degradation (5), suggesting that Jab1/CSN5 has some role other than
cytoplasmic shuttling of p27. Subcellular localization and turnover of
-catenin is regulated by nuclear-cytoplasmic shuttling of APC (26).
The question then arises as to why the protein needs to go to the
cytoplasm before degradation. The simplest interpretation is that a
certain factor compartmentalized to a specific area in the cytoplasm is
required for degradation of these proteins, although no such factor has
been identified yet. In the present study, we have found that Grb
proteins specifically bind to p27 in the cytoplasm, which is required
for efficient down-regulation of p27. Because Grb proteins function as
an adaptor in the signal transduction pathway, one can easily speculate
that Grb proteins mediate interaction between p27 and some unknown factor that may contain activity for ubiquitination or proteolysis. Grb2 accelerates degradation of p27 and Grb3-3 exhibits an opposite effect, suggesting that the SH2 domain is important presumably for
recruitment of such a factor. Although we have no clues as to the
molecular identity of the factor, it is feasible that tyrosine phosphorylation of the factor by the growth factor receptor triggers association with the p27-Grb2 complex.
/
cells need to be performed to
clarify this issue, it is feasible that the down-regulation of p27
occurs in several steps. Jab1/CSN5 and Grb2 may be involved in the
down-regulation during early to mid-G1, and the cyclin
E-Cdk2-SCFSkp2 pathway may govern the late G1
to S phase event.
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ACKNOWLEDGEMENTS |
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We thank Drs. C. J. Sherr and J. Fujisawa for the plasmids and baculoviruses.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, and Culture of Japan and the Special Coordination Funds of the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese government.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.
Supported by Research Fellowships of the Japan Society for
the Promotion of Science for Young Scientists.
§ To whom correspondence should be addressed. Tel.: 81-743-72-5541; Fax: 81-743-72-5549; E-mail: jkata@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010811200
2 K. Tomoda, Y. Arata, T. Tanaka, N. Yoneda-Kato, and J. Kato, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; HA, hemagglutinin; SH2 and SH3, Src homology domain 2 and 3, respectively; GST, glutathione S-transferase.
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