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Full-length article Acta Pharmacologica Sinica 2005 October; 26 (10): 1253-1258 Expression of proline-rich Akt-substrate PRAS40 in cell survival pathway and carcinogenesis1 Bei HUANG2,4, Gavin PORTER3 2Department of Biological Science, Anhui University School of Life Science, Hefei, Anhui 230039, China; 3Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30032, USA
1 Project supported in part by Anhui Key Laboratory of Eco-engineering and Bio-technique Fund and National Institutes of Health Grants
GM60033-03.
Aim: To study the expression of proline-rich Akt-substrate PRAS40 in the cell survival pathway and tumor progression.
Methods: The effects of three key kinase inhibitors on PRAS40 activity in the cell survival pathway, serum withdrawal,
H2O2 and overexpression of Akt were tested. The expression of PRAS40, Akt, Raf and 14-3-3 in normal cells and cancer cell lines
was determined by Western blot. Results: The PI3K inhibitors worthmannin and Ly294002, but not rapamycin, completely
inhibited the phosphorylation of Akt and PRAS40. The phosphorylation level of Akt decreased after serum withdrawal and
treatment with the MEK inhibitor Uo126, but increased after treatment with
H2O2 at low concentration, whereas none of these
treatments changed PRAS40 activity. 14-3-3 is a PRAS40 binding protein, and the expression of 14-3-3, like that of PRAS40,
was higher in HeLa cells than in HEK293 cells; PRAS40 had a stronger phosphorylation activity in A549 and HeLa cancer
cells than in HEK293 normal cells. In the breast cancer model (MCF10A/MCF7) and lung cancer model (BEAS/H1198/H1170)
we also found the same result: PRAS40 was constitutively active in H1198/H1170 and MCF7 pre-malignant and malignant
cancer cells, but weakly expressed in MCF10A and BEAS normal cell. We also discussed PRAS40 activity in other NSCLC
cell lines. Conclusion: The PI3K-Akt survival pathway is the main pathway that PRAS40 is involved in; PRAS40 is a
substrate for Akt, but can also be activated by an Akt-independent mechanisms. PRAS40 activation is an early event during
breast and lung carcinogenesis. Key words PRAS40; PI3K-Akt pathway; kinase inhibitors; 14-3-3 protein; Note: Please read the complete
full text with Figures and Tables at
Introduction
Chemoprevention is a logical and obvious strategy to help
alleviate the effects of cancer[1,2]. Much effort has been focused
on the discovery and development of new chemopreventive
agents, especially agents targeted at mechanisms known to be
involved in the process of
carcinogenesis[3,4].
The PI3K-Akt signaling pathway regulates many normal
cellular processes including cell proliferation, survival,
growth and motility, which are critical for tumorigenesis. The
role of the PI3K-Akt signaling pathway in oncogenesis has
been extensively investigated and altered expression or
mutation of many components of this pathway have been
implicated in human cancer[5] . Akt functions as a major
downstream target of phosphatilylinositol-3-kinase (PI3K),
carrying out functions including stimulation of glucose uptake
and cell growth as well as inhibition of
apoptosis[6,7].
Kovacina et al used a combination of the 14-3-3 protein
and anti-pAkt substrate antibodies to screen and isolate a
substrate of Akt, the major 14-3-3 binding protein observed
in cells after insulin treatment (a 40 kDa
molecule)[8]. This protein contains a consensus Akt phosphorylation site
(Thr-246) but not other recognizable motif. It is highly
proline-rich, with 15% of its amino acids being proline (versus 5%
for a typical protein), and these proline-rich regions are
potential SH3 and/or WW domain binding partners. The
protein has therefore been named PRAS40, which stands for the
"proline-rich Akt substrate of 40 kDa". Activation of
inducible Akt alone was sufficient to stimulate PRAS40
phosphorylation, and phosphorylation of this protein was
reduced in cells lacking Akt1 and Akt2. Thus PRAS40 is a
novel substrate of Akt, the phosphorylation of which leads
to binding of this protein to 14-3-3[8].
Saito et al demonstrated that the expression of PRAS40, PRAS40/Akt and
PRAS40/14-3-3 increased in Nerve Growth Factor (NGF)
treated mice but decreased with inhibition of PI3K and the
NGF receptor after transient focal cerebral ischemia
(tFCI) [9].
However, the critical role of PRAS40 in the biological
processes of cell proliferation and carcinogenesis remains
unknown. The aim of our study was to examine the
expression of PRAS40 in the cell survival signaling pathway, tumor
progression and its relationship with Akt and 14-3-3.
Information from this study will be helpful for further functional
studies of PRAS40.
Materials and methods
Plasmids and expression The constitutively active Akt
plasmid HA-Akt-DpH and kinase mutation Akt plasmid
HA-Akt-Km were the gift of Prof Dr Haian FU (Emory university,
USA). After transfection into human embryonic kidney cell
HEK293, the supernatant was boiled for 5 min in sodium
dodecylsulfate (SDS) sample buffer and resolved using
SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 12.5%), The
proteins were transferred to polyvinylidene difluoride (PVDF)
membranes (BIO-RAD, CA, USA). The primary antibodies
were 1:2000 dilution of mouse monoclonal antibody against
HA-Prob, Western blots were performed with horseradish
peroxidase-conjugate anti-mouse IgG. Cross-reacting
materials were visualized using ECL Detection Reagents
(Amersham Biosciences, UK).
Source of antibody Rabbit polyclonal antibody against
phosphorylated PRAS40(T246), and mouse monoclonal
antibody against PRAS40 were purchased from BioSource
International (Camarillo, CA, USA); Rabbit polyclonal
antibodies against phosphorylated Akt (S473), Akt,
phosphorylated-Raf (S338) and Raf were purchased from Cell Signaling
Technology (Beverly, MA, USA); Rabbit polyclonal antibody
against 14-3-3 (Pan-14-3-3), mouse monoclonal antibody
against tubulin and HA-Prob, goat anti-mouse IgG-HRP and
goat anti-rabbit IgG-HRP were purchased from Santa Gruz
Biotechnology (CA, USA).
Reagents Ly294002/worthmannin (PI3K inhibitor) were
purchased from Cell Signaling Technology; rapamycin
(mTOR inhibitor) was purchased from Calbiochemistry
(USA); hydrogen peroxide and U0126 (MEK inhibitor ) were
obtained from Sigma-Aldrich (St Louis, MO, USA).
Cell culture and DNA transfection HEK293, A549, HeLa
and other cancer cells were provided by the Winship Cancer
Institute (Atlanta, GA, USA) and maintained in Dulbecco¡¯s
modified Eagle¡¯s medium (DMEM) or RPMI1640 (GIBCO Invitrogen, CA, USA) with 10% heat inactivated fetal
bovine serum (FBS) and 5% CO2 at 37
oC. The confluent cells were separated and trypsinized with 0.05%
trypsin-ethylenediamine tetraacetic acid (EDTA) to produce single cells.
They were then seeded at 1×105 per
cm2 and allowed to form subcultures. Cells were transfected with plasmids using the
FuGENE6 reagent (Roche Applied Science,
USA) according to the manufacturer¡¯s instructions.
Western blot After the cells were transfected, harvested
and lysed by in 1% NP-40 lysis buffer [0.15 mol/L
NaCl, 0.01 mol/L N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid
(HEPES), 1% NP-40, 5 mmol/L sodium pyrophosphate, 5
mmol/L sodium fluoride, 2 mmol/L sodium orthovanadate, 10
mg/L aprotinin, 10 mg/L leupetin, 1 mmol/L
phenylmethlsulfonyl fluoride (PMSF)] at 4
oC for 20 min, cell extracts were clarified by centrifugation, prepared in SDS sample buffer,
boiled for 5 min, and resolved using SDS-PAGE (12.5%) for
Western blot. The enzyme-linked immunoblotting procedures
were performed essentially as described
previously[10]. Corresponding secondary antibodies were used against each
primary antibody: horseradish peroxidase-conjugated goat
anti-mouse IgG was used for monoclonal antibodies and
horseradish peroxidase-conjugated goat anti-rabbit IgG was
used for polyclonal antibodies. Cross-reacting materials were
visualized using ECL detection reagents.
Saito et al suggested that PRAS40 might play a critical
role in the neuronal cell survival pathways mediated by NGF
after cerebral ischemia[9]. In order to test which cell survival
signal cascade PRAS40 is involved in, we used three key
kinase inhibitors (Worthmannin/Ly294002, Uo126, rapamycin)
and cell death or apoptosis induction factors
(serum withdrawal, H2O2) to treat normal cell line HEK293 and cancer cell line
A549 and HeLa. The expression of PRAS40, Akt, Raf and
14-3-3 were analyzed by Western blot.
Results
PRAS40 was mainly involved in the PI3K-Akt survival
pathway After treatment with the PI3K inhibitor Worthmannin
(2 µmol/L) or Ly294002 (25 µmol/L) in HEK293 for 2 h,
Worthmannin and Ly294002 almost completely blocked
phospho-PRAS40 and phospho-Akt activity, compared with
total expression of PRAS40 and Akt. However, inhibition of
Akt phosphorylation was also induced by serum withdrawal.
Overexpression of constitutively active Akt (Akt-DpH)
promoted the expression of phopho-PRAS40 in HEK293,
compared with negative control Kinase mutation Akt
(Akt-Km)(Figure 1). Therefore, the main pathway that PRAS40 is
involved in is the PI3K-Akt pathway, and Akt is the upstream
kinase of PRAS40 .
MEK-ERK pathway is not necessary for PRAS40
activity The Raf-dependent activity of the MEK-ERK pathway
promotes cell survival by targeting various death
pathways[11]. To test whether MEK-ERK is involved in the regulation of
PRAS40/Akt activity, we used the MEK antagonist U0126
to treat HEK293, A549 and HeLa cell lines. Western blot
analysis revealed that U0126 had no effect on PRAS40 activity
(Figure 1, 2), which implies that PRAS40 does not contribute
to the MEK-ERK pathway.
ROS induced phosphorylation of Raf/Akt activity but
not PRAS40 at low concentration ASK1 is known to play an
important role in the apoptotic response induced by ROS, in
particular H2O2. Goldmann
et al showed that phosphorylation of ASK1 at Ser-976 by
H2O2 stimulation was dose-dependent, and that treatment with 1 mmol/L
H2O2 for 30 min could completely dephosphorylate ASK1 at
Ser-967[12]. In our experiment, the increase in phospho-Akt and phospho-
Raf expression was induced by treatment with 1 mmol/L
H2O2 for 20 min in HEK293 and A549, but there was no change on
PRAS40 activity (Figure 1, 2).
Akt was not the only upstream kinase of PRAS40
Akt phosphorylation could be blocked by worthmannin, U0126
and serum withdrawal in A549 and HEK293 cells; however,
only worthmannin and Ly294002 inhibited phospho-PRAS40
activity (Figure 1, 2), Therefore, Akt was not the only
upstream kinase of PRAS40. PRAS40 could also be activated
by an Akt-independent mechanism. Western blot analysis
showed that the phospho-PRAS40 and phospho-Akt
activity in A549 cancer cells were stronger than in HEK293 normal
cell line (Figure 2).
Expression of PRAS40 and 14-3-3 was higher in HeLa
than in HEK293 cells The 14-3-3 protein is an anchor
protein for some Akt substrates, and it is also a PRAS40 binding
protein. Therefore, we investigated the relationship between
PRAS40 and 14-3-3 expression in normal and cancer cells.
HEK293 and HeLa cells were treated with serum withdrawal,
H2O2, rapamycin (mTOR inhibitor), worthmannin and U0126.
Western blot demonstrated that, except for worthmannin,
these treatments produced no significant difference with
regard to PRAS40 activity in HEK293 and HeLa cells. We
found that HEK293 was more sensitive: treatment with
worthmannin only decreased the phosphorylation of PRAS40 in HeLa cells, but completely blocked PRAS40
phosphorylation in HEK293 cells. We also found that
phospho-PRAS40 and 14-3-3 were expressed more strongly in HeLa
cells than in HEK293 cells, and the total expression of PRAS40
was almost the same in the two cell lines (Figure 3).
We also noticed that there was a difference in sensitivity
to worthmannin between normal cells and cancer cells
(HEK293 and HeLa) and among cancer cells (A549 and HeLa).
PRAS40 phosphorylation activity was stronger in
cancer cells than in normal cells In order to determine if PRAS40
activity is different in normal cells, different types and
different development phases of cancer cells, we used Western
blot analysis to determine the expression of
phospho-PRAS40, PRSA40, Akt and Raf in
vitro. Because Akt and Raf are the key control factors in the cell survival pathway
(PI3K-Akt pathway and Raf-MEK pathway), we used them
to find a expression relationship between PRSA40, Akt and
Raf.
For a breast cancer model, we used the MCF10A/MCF7
cell lines. MCF10A is a normal breast cell line and MCF7 is a
breast carcinoma line. For a lung cancer model, we used the
BEAS/H1190/H1170 cell lines. BEAS cells are Human
bronchial epithelial cells, immortalized with the hybrid
adenovirus/simian virus 40. H1198 and H1170 are both derived from
BEAS cells; H1198 is pre-malignant, because 1) it is
sensitive to serum in that it was growth inhibited and terminally
differentiates into squamous cells similar to
normal HBE and BEAS-2B cells; 2) in
vitro invasiveness was detected after exposure of BEAS cells to either phorbol myristate acetate
or cigarette smoke condensate (CSC). H1170 is defined as a
malignant cell line because it exhibits several features that
are typical of invasive adenocarcinomas, including increased
expression of epidermal growth factor receptor (EGFR) and
transforming growth factor-a (TGFa)[13]. We also
investigated other non-small cell lung cancer (NSCLC) cell lines:
H460, H1299, H596, H157, H552, H1944, H1792. The details
of each cancer cell lines are given in Table 1.
We found that the expression levels of phospho-PRAS40
were higher in pre-maligment and maligmant cells than in
normal cell lines (eg MCF10A); Later phase cancer cells,
such as H1198/H1170, showed stronger PRAS40 activity than
pre-cancer cell lines (eg BEAS) except H460, H1299, H1944,
in which there were no p53 mutations. We also noticed that
there was no difference in the total expression of PRAS40
and Akt, whereas the total expression of Raf was different in
different types of cancer cell lines, for example, there was no
expression in BEAS and H1944, and weak expression in H460
and H157 (Figure 4).
Discussion
The PI3K-Akt pathway has been implicated in the
deve-lopment of multiple human
cancers[14,15]. PI3K has an active role in oncogenic transformation. Akt, an important and
probably essential downstream component of PI3K-mediated
oncogenic signaling[16], provides a critical cell survival
signal for tumor progression by phosphorylating a number of
proteins involved in cell cycle regulation and proapoptotic
factors[17]. Because only a subset of the cellular processes
regulated by the PI3K-Akt pathway, are involved in
tumorigenesis, the choice of drug targets must take into
account the adverse effects resulting from the inhibition of
other PI3K-Akt-dependent cellular processes. For example,
the effects of insulin on metabolism are mediated through
the PI3K-Akt pathway, so inhibitors of PI3K or Akt are
therefore likely to perturb glucose homeostasis. It would be
desirable, therefore, to target components of branches further
downstream in the PI3K-Akt pathway[18].
Our data provided evidence that the proline-rich
Akt-substrate PRAS40 showed higher expression levels in
cancer cells (eg A549 and HeLa) than in normal cells
(eg HEK293). In our breast cancer model (MCF10A/MCF7) and lung
cancer model (BEAS/H1198/H1170) we also found the same We tested the effect of key kinase inhibitors on PRAS40
activity: PI3K inhibitors Worthmannin or Ly294002, MEK
inhibitor Uo126 and mTOR inhibitor rapamycin. Only the
PI3K inhibitors inhibited or decreased PRAS40 activity,
therefore the PI3K-Akt survival pathway is the main pathway that
PRAS40 is involved in; The Raf-MEK pathway probably
doesn¡¯t contribute to PRAS40 activity. Also, PRAS40 is the
substrate of Akt, but it can be activated by an Akt-
independent mechanism, for example, serum withdrawal decreases
Akt activity, but has no effect on PRAS40.
14-3-3 is also a PRAS40 binding protein. A striking
feature of the 14-3-3 proteins is their ability to bind a multitude
of functionally diverse signaling proteins. This plethora of
interacting proteins allows 14-3-3 to play important roles in a
wide range of vital regulatory processes, such as mitogenic
signal transduction, apoptotic cell death and cell cycle
control[19]. Our data show that the expression level of 14-3-3, like
that of PRAS40, is higher in the HeLa cell line than in HEK293,
but exactly how 14-3-3 helps PRAS40 in the PI3K-Akt
pathway is unknown and warrants further investigation.
To our knowledge, our results provide the first evidence
that PRAS40 is constitutively active in pre-malignant and
malignant breast and lung cancer cell lines, and that PRAS40
is mainly involved in the PI3K-Akt survival
pathway. However, PRAS40 can also be activated by Akt-
independent mechanisms. We suggest that PRAS40 could be chosen as an early
detection marker in carcinogenesis, but it is also a protein that can
be targeted by anti-tumor drugs, that is a "druggable" protein.
Acknowledgements
We thank Prof Hai-an FU for the generous gift of plasmids.
We also thank the members of Prof Fu¡¯s laboratory at Emory
University for helpful discussions.
References
References
1 Khuri FR, Herbst RS, Fossella FV. Emerging therapies in
non-small-cell lung cancer. Ann Oncol 2001; 12: 739-44.
2 Mc Williams A, Lam S. New approaches to lung cancer prevention.
Curr Oncol Rep 2002; 4: 489-94.
3 Goodman GE. Lung cancer 1: prevention of lung cancer.
Thorax 2002; 57: 994-9.
4 Hong WK, Sporn MB. Recent advances in chemoprevention of
cancer. Science 1997; 278: 1073-7.
5 Vivanco I, Sawyers Cl. The phosphatidylinositol 3-kinase Akt
pathway in human cancer. Nat Rev Cancer 2002; 2: 489-501.
6 Cantley LC. The phosphoinositide 3-kinase pathway. Science
2002; 296: 1655-7.
7 Manning BD, Cantley LC. United at last: the tuberous sclerosis
complex gene products connect the phosphoinositide
3-kinase/Akt pathway to mammalian target of rapmycin (mTOR)
signaling. Biochem Soc Trans 2003; 31: 573-8.
8 Kovacina KS, Park GY, Bea SK, Gruzzetta AW, Schaefer E,
Birnbaum MJ. Identification of a proline-rich Akt substrate as a
14-3-3 binding partner. J Biol Chem 2003; 12: 10189-94.
9 Saito A, Narasimhan P, Hayashi T, Okuno S, Ferrand-Drake M,
Chan PH. Neuroprotective role of a proline-rich Akt substrate
in apoptotic neuronal cell death after Stroke: relationships with
nerve growth factor. J Neurosci 2004; 24: 1584-93.
10 Zhang L, Chen J, Fu H. Suppression of apoptosis
signal-regulating kinase 1 induced cell death by 14-3-3 protein. Proc Natl
Acad Sci USA 1999; 96: 8511-5.
11 Chen J, Fujii K, Zhang L, Roberts T, Fu H. Raf-1 promotes cell
survival by antagonizing apoptosis signal-regulating kinase 1
through a MEK-ERK independent mechanism. Proc Natl Acad
Sci USA 2001; 14: 7783-8.
12 Goldman EH, Chen J, Fu H. Activation of apoptosis
signal-regulating kinase 1 by reactive oxygen species through
dephosphorylation at Ser967 and 14-3-3 dissociation. J Biol
Chem 2004; 279: 10442-9.
13 Chun KH, Kosmeder JW, Sun S, Pezzuto JM, Lotan R, Hong WK.
Effect of deguelin on the phosphatidylinositol 3-Kinase/Akt
pathway and apoptosis in premalignant human bronchial
epithelial cells. J Nat Cancer Inst 2003; 4: 292-302.
14 Chang HW, Aoki M, Fruman D, Auger KR, Bellacosa A, Tsichlis
PN, et al. Transformation of chicken cells by the gene encoding
the catalytic subunit of PI3K-Kinase. Science 1997; 276: 1848-50.
15 Klippel A, Escobedo MA, Wachowicz MS, Apell G, Brown TW,
Giedlin MA, et al. Activation of phosphatidylinositol 3-kinase is
sufficient for cell cycle entry and promotes cellular changes
characteristic of oncogenic transformation. Mol Cell Biol 1998; 18:
5699-711.
16 Toker A, Cantley L. Signaling through the lipid products of
phosphoinositide-3-OH kinase. Nature 1997; 387: 673-6.
17 Kobayashi M, Nagata S, Iwasaki T. Dedifferentiation of
adenocarcinomas by activation of phosphatidylinositol 3-kinase. Proc
Natl Acad Sci USA 1999; 96: 4874-9.
18 Luo J, Manning BD, Cantley LC. Targeting the PI3K-akt
pathway in human cancer: Rationale and promise. Cancer Cell 2003; 19 Fu H, Subramanian HRS, Masters SC. 14-3-3 Proteins: structure,
function, and regulation. Ann Rev Pharmacol Toxicol 2000;
40: 619-49.
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