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Vol. 14, No. 24, pp. 3093-3101, December 15, 2000
inhibits p70 S6 kinase via protein phosphatase 2A to induce G1 arrest
1 IMP, Research Institute for Molecular Pathology, A-1030 Vienna, Austria; 2 University of California San Francisco Cancer Center, San Francisco, California 94143, USA, and Onyx Pharmaceuticals, Richmond, California 94801, USA
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ABSTRACT |
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 Top
 Abstract
 Introduction
Results
Discussion
Material and methods
References
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On TGF-
binding, the TGF- receptor directly phosphorylates
and activates the transcription factors Smad2/3, leading to
G1 arrest. Here, we present evidence for a second, parallel,
TGF--dependent pathway for cell cycle arrest, achieved via
inhibition of p70s6k. TGF- induces association of its
receptor with protein phosphatase-2A (PP2A)-B
. Concomitantly, three
PP2A-subunits, B, A, and C, associate with p70s6k,
leading to its dephosphorylation and inactivation. Although either
pathway is sufficient to induce G1 arrest, abrogation of both, the inhibition of p70s6k, and transcription through
Smad proteins is required for release of epithelial cells from
TGF--induced G1 arrest. TGF- thereby modulates the
translational and posttranscriptional control of cell cycle progression.
[Key Words:
TGF- p70s6k; PP2A; G1
arrest; cell cycle]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Material and methods
References
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TGF- inhibits G1/S progression in a variety
of eukaryotic cell types. Among these, untransformed
epithelial cells are particularly sensitive to the growth inhibition by
TGF-. TGF- binds the TGF- receptor type I and type II
(TRI/TRII). TRI has been shown to transduce all known
signals induced by TGF- TRI binds and phosphorylates the
transcription factor Smad2 or, alternatively, its close homolog Smad3.
Smad2 and 3 associate with Smad4 and modulate transcription of
TGF- responsive genes (Massague and Chen 2000
; ten Dijke et al.
2000; Wrana and Attisano 2000).
It has been noted, however, that several other molecules interact with
TRI, indicating additional downstream effectors. FKBP12, the
cellular target of the immunosuppressant Rapamycin, for example, binds
the nonactive TRI (Wang et al. 1994, 1996; Chen et al. 1997; Huse
et al. 1999). In addition, the regulatory subunit B of protein
phosphatase 2A (PP2A) has been shown to specifically interact with the
activated TRI (Griswold-Prenner et al. 1998). Among many other
targets PP2A dephosphorylates and inactivates p70s6k (Ballou
et al. 1988; Schonthal 1998; Goldberg 1999; Millward et al. 1999), a
serine/threonine kinase that induces translation of mRNAs containing
5'TOP sequences (Jefferies et al. 1994; Pearson and Thomas 1995)
and is essential for G1/S progression (Lane et al. 1993).
In this study we first observe that inhibition of Smad signaling is not
sufficient to release epithelial cells from TGF--induced G1 arrest, indicating that there might be additional pathways for growth inhibition by TGF-. We then show that PP2A and
p70s6k are components of a TGF--induced signal
transduction pathway to control protein translation and G1/S
progression. Dependent on PP2A-B, TGF- inhibits
p70s6k by establishing or stabilizing complex formation of
PP2A with p70s6k. Finally, we argue that repression of
p70s6k functions as an alternative mechanism to Smad-mediated
transcriptional control of the cell cycle. Once activated, either
pathway is sufficient to induce TGF--dependent cell cycle arrest
in G1.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Material and methods
References
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To test whether the Smad signal transduction pathway is necessary
and sufficient for growth control by TGF-, we generated dominant-negative and constitutively active mutants of Smad2 and Smad4.
The transcriptional activity of these mutant proteins was assessed by
their potential to regulate TGF--induced transcription in
polarized mammary epithelial cells, EpH4 (Reichmann et al. 1992).
Multiclonal populations were analyzed for the retroviral-expressed mutant Smad proteins (Fig. 1A,B), the
activity of TGF- reporter constructs, endogenous TGF- target
genes (Fig. 1C), and cell cycle progression by Fluorescence
Activated Cell Scanning (FACS; Fig. 1D,E). TRI activates Smad2 by
multiple phosphorylation of its C-terminal motif, SSMS. Mutation of
those serines in Smad2 to acidic residues (Smad2EDME)
activated Smad2 to induce transcription (Fig. 1C; Macias-Silva et al.
1996; Liu et al. 1997; Souchelnytskyi et al. 1997). Mutation of the
same serines to alanines (Smad2AAMA) or expression of the
DNA-binding domain of Smad4 (Smad4MH1) inhibited
TGF--induced transcription of a transient transfected PAI1
reporter or the endogenous p15ink4b gene (Fig. 1C). Both
molecules thereby act as dominant-negative regulators of
TGF--induced transcription.
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TGF- induces G1 arrest (Fig. 1D) and Smad2EDME
indeed mimicked this effect (Fig. 1D). Surprisingly, however, neither
of the dominant-negative Smad proteins released from TGF--induced
cell cycle arrest (Fig. 1D). Because receptor-activated Smads form a
complex with Smad4, expression of Smad4MH1 should inhibit the
TGF- activated Smad2 and the Smad2EDME mutant and
relieve Smad2EDME-mediated cell cycle arrest (Fig. 1D).
Although expression of Smad4MH1 indeed reversed the cell
cycle arrest induced by the dominant active Smad2
(Smad2EDME), it failed to release the same cells from
TGF--induced G1 arrest (Fig. 1E), indicating that
TGF- induces additional pathways to induce G1 arrest,
independent of Smad-mediated transcription.
Similar experiments performed previously in other cell systems had
identified Smad-induced transcription as the only mediator of
TGF--induced G1 arrest (Liu et al. 1997). In our
experimental settings, Smad-induced transcription was likewise
sufficient to induce G1 arrest in epithelial cells, but was
clearly not the only mechanism. We attempted therefore to identify the
additional mechanism mediating TGF--induced G1 arrest.
To identify signaling pathways essential for G1/S progression
in epithelial cells, we applied known cell cycle inhibitors. We found
that Rapamycin, an immunosuppressant and Wortmannin or LY294002, two
PI3-kinase inhibitors, each induced G1 arrest in EpH4 cells
(Fig. 2A). We asked whether Rapamycin or
PI3-kinase inhibitors and TGF- would synergize to induce
G1 arrest and tested each drug for synergism with TGF-.
To pursue this approach, dose response curves for cell cycle entry in
the presence of drugs alone or with TGF- were determined (Fig. 2A;
data not shown). Rapamycin, Wortmannin, and LY294002 synergized with
TGF- to induce G1 arrest when applied at nonsaturating
conditions of either drug or factor alone (Fig. 2A), indicating that
the drugs might modulate similar downstream targets as TGF- to
control cell cycle progression. The compounds, however, did not
complement all aspects of TGF- signaling, and neither drug
significantly induced or synergized with Smad-mediated transcription
(Fig. 2B).
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All three compounds inhibit activation of p70s6k (Cheatham et
al. 1994; Chung et al. 1994; Petritsch et al. 1995). Overexpression of
p70s6k did not influence TGF--induced and Smad mediated
transcription (data not shown). Next, we asked if regulation of
p70s6k activity is a rate limiting step in the control of
G1/S progression by TGF- p70s6k was
overexpressed in EpH4 epithelial cells or derivatives coexpressing either dominant-negative Smad2 or Smad4 (Fig. 2C) and cells released into G1/S in the absence or presence of TGF- (Fig.
2D,E). In the absence of TGF- cell cycle progression was not
altered by expressing Smads and p70s6k (Fig. 2D). Expression
of both p70s6k and the Smad2AAMA, however, released
a high proportion of epithelial cells from G1 arrest into
S-phase regardless of the presence of TGF- (Fig. 2D). A kinase
deficient p70s6k (p70s6k kin.def.) was not able to
rescue from TGF--induced cell cycle arrest. Similar results were
obtained when the dominant-negative Smad4 (Smad4MH1) and
p70s6k were coexpressed (Fig. 2E; data not shown). Thus,
p70s6k confers resistance to TGF--induced, but
Smad-independent, G1 arrest.
To further test this model, we exposed cells expressing
Smad4MH1 and p70s6k to increasing concentrations of
TGF-. Whereas 100pg/mL TGF--induced complete G1
arrest in EpH4 cells expressing either p70s6k or
Smad4MH1 (Fig. 2E), cells coexpressing p70s6k and
Smad4MH1 continued to undergo G1/S progression
efficiently even at a TGF- concentration of 5ng/mL. Thus, the
cooperation of p70s6k and Smad4MH1 rendered the cells
essentially insensitive to TGF--induced inhibition of cell cycle
progression (Fig. 2E).
In mammalian cells, p70s6k becomes activated on
G0/G1 or M/G1 transition, the activity
peaks early in G1 and declines on progression into S-phase
(Edelmann et al. 1996). We therefore were interested if TGF- would
suppress p70s6k activity in G0 and early
G1 cells. EpH4 cells were cultured at confluency to induce
early G1 arrest and subsequently treated for various times
with TGF- TGF- addition lead to a decrease of
p70s6k activity by 70% within 5 min compared with untreated
lysates, and the inhibition was sustained for more than 1 h after
factor addition (Fig. 3A,B). This rapid
onset of inhibition indicates a direct posttranslational mechanism.
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Having observed a TGF--induced inhibition of p70s6k in
arrested cells we examined its effect on p70s6k activity in
cells entering the cell cycle. Because overexpression of
p70s6k and inhibition of Smad signaling released epithelial
cells from TGF--induced G1 arrest (Fig. 2D,E), we
anticipated that TGF- would inhibit activation of p70s6k
during G1/S progression. Indeed, the rapid increase of
p70s6k kinase activity observed in the untreated population
was delayed and strongly attenuated in cells treated with TGF-
(Fig. 3B).
We next looked for additive effects of inhibition of growth factor
pathways and TGF- on p70s6k activity. Activation of the
epidermal growth factor receptor (EGFR) is essential for G1/S
progression of epithelial cells (data not shown). TGF- and an
inhibitor of the EGFR tyrosine kinase, PD 153.035, both strongly
reduced the activity of p70s6k. When TGF- and PD 153.035 were added together, however, the inhibitory effect on p70s6k
was synergistic (Fig. 3C). To further examine the interplay of growth
signals, we asked if inhibition of p70s6k by TGF- would
antagonize activating signals from epithelial growth factors such as
TGF- that promote G1/S progression. Unsynchronized proliferating EpH4 cells (50% in G1 phase) were subjected to
treatment with TGF- TGF-, or a combination of both. As
expected, TGF- stimulated p70s6k and TGF- reduced the
kinase activity of p70s6k with maximal inhibition after 60 min (Fig. 3D). When both factors were given at the same time,
p70s6k activation was intermediate in value, indicating,
again, that growth inhibition by TGF- antagonizes growth promoting
signals at the level of p70s6k activation.
We next analyzed if TGF--induced inhibition of p70s6k is
mediated by the ligand-activated TRI and TRII. Accordingly,
we immunoprecipitated overexpressed p70s6k from 293 cells,
coexpressed with either wt TRI and wt TRII (Fig. 3E,F, lanes
6,7) or kinase-inactivated versions of TRI and TRII receptor
(lanes 2,3) and stimulated the cells with TGF-. The slower
migrating form of p70s6k, corresponding to a highly
phosphorylated and enzymatically active form of p70s6k
(p70s6k pp; Fig. 3E) and p70s6k activity, was
significantly reduced only in the presence of both wt TR complex
and TGF- stimulation (Fig. 3E,F, lane 7). Thus, both the
phosphorylation level and the kinase activity of p70s6k are
significantly decreased on TGF- receptor stimulation.
TGF--activated TRI has been reported to interact directly
with the regulatory subunit B of PP2A (Griswold-Prenner et al. 1998). PP2A consist of three subunits, a structural regulatory subunit
A, a variable regulatory subunit B, and catalytic subunit C. PP2A-C has
been shown to dephosphorylate and inactivate p70s6k (Ballou
et al. 1988; Peterson et al. 1999). This indicated that TGF--induced inhibition of p70s6k might be mediated by
PP2A. We therefore tested whether TGF- might regulate the
interaction of PP2A with p70s6k. HA-tagged p70s6k
was subjected to anti-HA-mediated immunoprecipitation. The endogenous catalytic unit of PP2A (PP2A-C) formed a complex with p70s6k
only in the presence of the active TGF- receptor type I and II and
only on addition of TGF- (Fig. 4A, lane
6). No interaction was observed when
kinase-dead versions of TRI and TRII were coexpressed with
p70s6k and stimulated with TGF-. Furthermore, inhibition
of PP2A catalytic activity by addition of the phosphatase inhibitor
okadaic acid at concentrations specific for PP2A disrupted formation of
the TGF--induced PP2A-C/p70s6k complex. Taken together,
these findings support the hypothesis that TGF- inhibits
p70s6k by inducing or stabilizing the interaction of PP2A-C
with its substrate p70s6k. A similar mechanism has recently
been postulated for the inhibition of p70s6k by Rapamycin
(Peterson et al. 1999).
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In certain cell lines such as mink lung epithelial cells (Mv1Lu),
TGF- does not inhibit p70s6k, and Smad signaling is both
sufficient and necessary for TGF--mediated G1 arrest
(Like and Massague 1986; Liu et al. 1997). Moreover, in contrast to
wild-type murine embryonic fibroblasts (MEFs), MEFs lacking Smad3 do
not respond to growth inhibition by TGF- (Zhu et al. 1998; Datto
et al. 1999; Yang et al. 1999). To understand this fundamental
difference between primary embryonic fibroblasts and Mv1Lu cells on the
one hand and epithelial cells on the other, we analysed the expression
of the PP2A subunits and TGF- receptors in these cell types. Both
TRI and TRII were expressed at comparable levels in all three
cell types (data not shown). We found, however, that the regulatory
subunit B of PP2A is expressed in EpH4 cells (Fig. 4B), in primary
small airway epithelial cells, and in a variety of epithelial tumor
cell lines (data not shown), but not in Mv1Lu cells or primary MEFs
(Fig. 4B). Therefore, it was not surprising that interaction of the
endogenous PP2A-B subunit was detected with endogenous
TRI/TRII in response to TGF- in EpH4 cells, but not in
Mv1Lu cells or primary MEFs (Fig. 4C). To examine the endogenous
complexes formed by PP2A and p70s6k, EpH4 and Mv1Lu cells
were treated in early G1 phase of the cell cycle with either
Rapamycin, TGF-, or Wortmannin. On p70s6k
immunoprecipitation, complex formation with PP2A-B was only detected
in EpH4 cells in response to TGF-, indicating a TGF- specific
mechanism of p70s6k inhibition presumably absent in Mv1Lu
cells (Fig. 4D). Moreover, if PP2A-B was immunoprecipitated from
lysates of EpH4 cells in early G1, we were able to detect
significantly increased amounts of p70s6k associated with
PP2A-B in TGF- treated cells (Fig. 4E). To better characterize
p70s6k regulation, we analyzed the phosphorylation status of
the Thr 389 in the linker domain of p70s6k (Fig. 4E).
Phosphorylation of Thr 389 correlates with the kinase activity of
p70s6k (Pullen et al. 1998; Weng et al. 1998). Thr 389 of
p70s6k was found to be phosphorylated in EpH4 cells in early
G1, but significantly reduced on TGF- stimulation (Fig.
4E). These data further support our model that the inhibition of
p70s6k by TGF- is dependent on PP2A-B-mediated
association of PP2A-C with p70s6k.
We then analyzed which A subunit of PP2A would interact with
p70s6k in response to TGF- The A (PR65) regulatory
subunit has recently been genetically linked to human lung and colon
cancer (Wang et al. 1998). A was expressed at comparable levels in
both cells types, EpH4 and Mv1Lu (Fig. 4F). Interestingly, we detected
A associated with p70s6k only in EpH4 cells, but not in
Mv1Lu cells, and only in response to TGF-, but not in response to
Rapamycin or Wortmannin (Fig. 4F).
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Material and methods
References
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Taken together, our results show that TGF- interferes with two
independent pathways to induce G1 arrest in epithelial cells. The first is the pathway of transcriptional control mediated by Smad
proteins (Eppert et al. 1996; Lagna et al. 1996; Macias-Silva et al.
1996; Zhang et al. 1996; Nakao et al. 1997). The second leads to the
inhibition of p70s6k via PP2A
(Fig. 5). This represents a novel
observation with potential implication for our understanding of cell
cycle regulation, cancer biology, and cancer development.
|
We show here that repression of p70s6k coincides with its
dephosphorylation and association of p70s6k with three
subunits of PP2A: PP2A-C and the two regulatory subunits PP2A-A
and PP2A-B. PP2A-B interacts with TRI and thereby physically links TGF- to the control of PP2A and to p70s6k. On
receptor activation, PP2A-B specifically binds the activated TRI and is catalytically activated by TGF- (Griswold-Prenner et al. 1998; data not shown). PP2A-B then recruits PP2A-A and PP2A-C to bind and dephosphorylate p70s6k. We were, however,
not able to detect complexes containing at the same time PP2A, TRI
and p70s6k, indicating that on activation the phosphatase is
released from the receptor to bind to the target molecule.
Immunolocalization of the endogenous proteins supports this model (M. Oft, unpubl.). p70s6k activity controls the translational
upregulation of proteins important for G1/S progression and
is itself essential for cell cycle progression (Lane et al. 1993;
Pearson and Thomas 1995). Most of the transcripts isolated to date
represent ribosomal proteins and elongation factors of protein
synthesis (Jefferies et al. 1994). TGF--induced inactivation of
p70s6k leads to the translational regulation of a group of
cell cycle regulators for G1 progression (M. Oft, unpubl.).
It remains unclear, however, if the repression of those cell cycle
regulators result from global repression of protein translation or
represent a class of specifically translationally repressed mRNAs. It
is conceivable that the regulation of crucial components of the cell
cycle machinery is mediated at the transcriptional, translational, and
posttranslational levels.
Expression of the regulatory subunit PP2A-B itself appears to be a
prerequisite for the PP2A-mediated inhibition of p70s6k by
TGF-. Cells with nondetectable PP2A-B expression remain solely
responsive to TGF--mediated transcriptional responses (Liu et al.
1997; Zhu et al. 1998; Datto et al. 1999; Yang et al. 1999).
p70s6k is not inhibited by TGF- in these cells (Like and
Massague 1986; data not shown), which reflects the differential
sensitivity of epithelial cells and mesenchymal cells to growth
inhibitory effects of TGF-. The chromosomal localization of
PP2A-B has not been investigated; PP2A-A, however, has been
mapped to a human tumor suppressor locus on 11q22-24 and appears to be
mutated in a subset of human lung tumors (Wang et al. 1998). It is
tempting to speculate that mutations of the regulatory subunits of PP2A
in human tumors abolish the regulation of p70s6k to TGF-
and confer a selective advantage to growing tumors.
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Material and methods |
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Abstract
Introduction
Results
Discussion
Material and methods
References
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Cell culture and cell cycle analysis
PAI1-promoter transcription assays were normalized to a internal
-actin--galactosidase control, and numbers represent the average of three independent experiments.
EpH4 cells were infected with supernatants of BOSC23 virus-producing
cells (Pear et al. 1993), drug selected, tested for the transgene
expression, and immediately analyzed as a mass culture to exclude
clonal variations. For G1/S progression studies, the cells
were seeded at a confluent density, arrested in
G0/G1 by confluency for two days, EDTA released and
replated as single cells. The majority of cells enter S-phase after 18 h. Samples for all G1/S profiles shown were collected 22 h
after EDTA release. The results were, however, verified extensively
throughout a 16-h to 30-h time window.
p70s6k kinase assays
Cells were washed twice with 10 mL cold Extraction Buffer (20 mM
Tris at pH 7.5, 20 mM EDTA, 15 mM MgCl2, 40 mM 4-nitrophenyl phosphate [pNPP], 1 mM dithiothreitol [DTT], and 0.1 mM
phenylmethylsulfonyl fluoride [PMSF]). For s6 kinase assays
p70s6k was immunoprecipitated using polyclonal antibodies
against p70s6k (Edelmann et al. 1996) or the HA-epitope and G
protein-coupled sepharose beads. Precipitated beads were resuspended
in S6 kinase assay buffer (50 mM Tris at pH 7.5, 0.1 mM EGTA, 5%
ethylene glycol, 5 mM DTT, 10 mM MgCl2, 0.1% Triton X-100,
and 0.25 mg/mL BSA) and assayed for S6 kinase activity using the
ribosomal 40S subunit as a substrate as described previously (Petritsch
et al. 1995). Immunoprecipitation of p90rsk and subsequent
kinase assays revealed that s6 phosphorylation by p90rsk is
negligible in EpH4 cells. All kinase assays were performed in
duplicates and repeated twice.
Immunoprecipitations
TRI, TRII or their derivatives with inactivated kinase
domains and HA-tagged p70s6k were transiently transfected
into 293 cells. Cell lysates were collected 24 h after transfection and
20 min after stimulation with TGF- or okadaic acid and subjected
to anti-HA- or anti-p70s6k immunoprecipitation.
To precipitate endogenous TRI and TRII kinase-phosphatase
complexes, lysates for immunoprecipitation were taken after 20 min of
TGF- treatment, normalized for protein content, and subjected to
immunoprecipitation using the respective antibodies and Protein G
sepharose beads.
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Acknowledgments |
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We thank J. Massague and R. Wieser for sharing the expression
vectors for various TRI and TRII and the 3TPlux construct, as
well as Ali Fattay and especially Byron Hann for helpful discussion to
improve the manuscript. C.P. was supported by a grant of the Sonderforschungsbereich.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received September 26, 2000; revised version accepted October 27, 2000.
3 Present address: Howard Hughes Medical Institute, UCSF, San Francisco, CA 94143, USA.
4 Corresponding author.
E-MAIL moft{at}cc.ucsf.edu; FAX (415) 502-6779.
Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad.854200.
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References |
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Abstract
Introduction
Results
Discussion
Material and methods
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)
Control; (
) plus 5ng/mL TGF-
