From the Medical Research Council Protein
Phosphorylation Unit, School of Life Sciences,
University of Dundee, Dow Street, Dundee DD1 5EH, Scotland and the
¶ Cancer Research Campaign Cell Transformation Group,
Ninewells Hospital, Dundee DD1 9SY, Scotland
Received for publication, November 1, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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Peutz-Jeghers syndrome is an inherited cancer
syndrome that results in a greatly increased risk of developing tumors
in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which
LKB1 is regulated in cells is not known. Here, we demonstrate that
stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2
or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser431. We present
pharmacological and genetic evidence that p90RSK mediated
this phosphorylation in response to agonists that activate ERK1/2 and
that cAMP-dependent protein kinase mediated this
phosphorylation in response to agonists that activate adenylate
cyclase. Ser431 of LKB1 lies adjacent to a putative
prenylation motif, and we demonstrate that full-length LKB1 expressed
in 293 cells was prenylated by addition of a farnesyl group to
Cys433. Our data suggest that phosphorylation of LKB1 at
Ser431 does not affect farnesylation and that farnesylation
does not affect phosphorylation at Ser431. Phosphorylation
of LKB1 at Ser431 did not alter the activity of LKB1 to
phosphorylate itself or the tumor suppressor protein p53 or alter the
amount of LKB1 associated with cell membranes. The reintroduction of
wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed
growth, but mutants of LKB1 in which Ser431 was mutated to
Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting
growth. In contrast, a mutant of LKB1 that cannot be prenylated was
still able to suppress the growth of cells.
Peutz-Jeghers syndrome is an autosomal dominantly inherited
disorder that predisposes to a wide spectrum of benign and malignant tumors (1, 2). It is caused by mutation of a widely expressed protein
kinase of unknown function termed LKB1 (also known as STK11) (3, 4). To
date, over 60 different mutations have been mapped to LKB1, many of
which would be expected to impair LKB1 activity. These discoveries have
aroused great interest because they indicate that LKB1 is likely to
function in cells as a tumor suppressor, and consistent with this,
overexpression of LKB1 in a number of tumor cell lines has been shown
to suppress cell growth by inducing a G1 cell cycle block
(5). However, little is known regarding the mechanism by which LKB1
activity is regulated in cells, and no substrates for LKB1 have thus
far been identified.
LKB1 is a 436-amino acid protein possessing a kinase domain (residues
50-337) that is only distantly related to other mammalian kinases. The
N-terminal non-catalytic domain comprises both a nuclear localization
signal (6) and a putative cytoplasmic retention signal (7). There are
no yeast homologs of LKB1, but there are putative homologs in
Xenopus (termed XEEK1, with 84% overall identity to LKB1)
(8) and Caenorhabditis elegans (termed PAR-4, with 26%
overall identity to LKB1 and 41% identity in the kinase domain) (9).
In Drosophila, an uncharacterized protein kinase listed in
the NCBI Protein Database (NCBI accession number AAF54972) possesses
44% overall identity to LKB1.
Recently, a C-terminal fragment of LKB1 was shown to be phosphorylated
at Ser431 by the cAMP-dependent protein kinase
(10). Ser431 of LKB1 lies in the sequence
Lys-Xaa-Arg-Arg-Xaa-Ser (where Xaa is any amino acid), which is
conserved in all known mammalian LKB1 sequences and in
Xenopus XEEK1. This study did not establish whether
full-length or endogenously expressed LKB1 was phosphorylated at
Ser431 in response to stimuli that activated
cAMP-dependent protein kinase
(PKA)1 or the role that this
phosphorylation played in enabling LKB1 to suppress cell growth.
Ser431 lies in the consensus sequence for phosphorylation
by a group of kinases related to PKA, viz. p90 ribosomal S6
kinase (p90RSK), mitogen- and stress-stimulated protein
kinase (MSK1), and p70 ribosomal S6 kinase (S6K1) (11-13), that
collectively belong to the AGC kinase subfamily.
p90RSK is activated in cells by growth factors and phorbol
esters and by ERK1/2 MAPK family members (14), whereas MSK1 is
activated in vivo by two different types of MAPK family
members, viz. ERK1/2 and the stress- and cytokine-activated
p38 MAPK (13). S6K1 is activated in vivo by growth factors
through a phosphatidylinositol 3-kinase-dependent pathway
and by phorbol esters and certain cellular stresses through a
phosphatidylinositol 3-kinase-independent pathway (15).
Ser431 is located 6 amino acids from the C terminus of
LKB1, and the residues that follow Ser431 in human LKB1 are
Ala-Cys-Lys-Gln-Gln. The cysteine 2 residues C-terminal from
Ser431 (Cys433) thus lies 4 residues from the
end of the protein in a consensus sequence known as the CAAX
motif, which mediates the prenylation of many proteins (16, 17). This
sequence including the Cys residue is conserved in C-terminal sequences
of mammalian, Xenopus, and Drosophila LKB1. Uhler
and co-workers (10) have recently demonstrated that a C-terminal
fragment of LKB1, when overexpressed in 293 cells, was prenylated at
Cys433. However, these authors did not establish whether
full-length LKB1 was prenylated by addition of a geranylgeranyl
(C20) or a farnesyl (C15) moiety to
Cys433 or if prenylation of LKB1 was required for LKB1 to
suppress the growth of cells.
Here, we report that p90RSK, MSK1, S6K1, and PKA
phosphorylate full-length LKB1 specifically at Ser431
in vitro. We show that agonists that activate these kinases
in Rat-2 cells and embryonic stem (ES) cells induce the phosphorylation of endogenous LKB1 at Ser431. We use signal transduction
inhibitors and ES cells lacking p90RSK activity and ES
cells deficient in MSK1 to demonstrate that phosphorylation of LKB1
induced by EGF and TPA is likely to be mediated by p90RSK
rather than by MSK1 or S6K1 and that phosphorylation of
LKB1 induced by forskolin is mediated by PKA. We show that
full-length LKB1 expressed in 293 cells is prenylated by addition of a
farnesyl moiety at Cys433, and we provide evidence that
phosphorylation of LKB1 at Ser431, but not its
farnesylation at Cys433, is likely to be important in
mediating the ability of LKB1 to suppress cell growth.
Materials--
Protease inhibitor mixture tablets, histone 2B,
Fugene-6 transfection reagent, and G418 were from Roche Molecular
Biochemicals. PD 184352 was from Upstate Biotechnology. U0126 was from
Promega. Rapamycin, H-89, Ro 318220, PD 98059, and zwittergent 3-16
were from Calbiochem. EGF, insulin-like growth factor-1,
microcystin-LR, dialyzed fetal bovine serum, and other tissue culture
reagents were from Life Technologies, Inc.
(R)-[2-14C]Mevalonic acid lactone and
32P-labeled inorganic phosphate was from Amersham Pharmacia
Biotech. Forskolin, TPA, mevastatin, and dimethyl pimelimidate were
from Sigma. The pre-cast BisTris/SDS-4-12% gradient polyacrylamide gels were from Invitrogen. All peptides used in this study were synthesized by Dr. G. Blomberg (University of Bristol, Bristol, United
Kingdom). CREB (13) and BAD (18) were expressed as GST fusion proteins
in Escherichia coli as described previously. Mouse p53
expressed in bacteria was prepared as described previously (19).
Antibodies--
Antibodies recognizing LKB1 were raised in sheep
against peptide GELMSVGMDTFIHRID (corresponding to residues 15-30 of
mouse LKB1), and the GST-LKB1 protein expressed in E. coli.
The antibodies were affinity-purified on CH-Sepharose covalently
coupled to the antigens used to raise the antibodies. The antibody
raised against GST-LKB1 was also passed through a column of
CH-Sepharose coupled to GST, and the antibody that did not bind was
selected. The phospho-specific antibody recognizing LKB1 phosphorylated
at Ser431 (termed antibody S431-P) was raised in sheep
against peptide SNKIRRLSACKQQ (corresponding to the
residues 424-436 of mouse LKB1; the underlined residue is
phosphoserine). The antibody was affinity-purified on CH-Sepharose
covalently coupled to the phosphorylated peptide and then passed
through a column of CH-Sepharose coupled to the non-phosphorylated
peptide. Antibody that did not bind to the latter column was selected.
The antibodies raised against GST-LKB1 and antibody S431-P are
available from Upstate Biotechnology, Inc. Antibodies that recognize
S6K1 were raised against peptide AGVFDIDLDQPEDAGSEDEL (corresponding to
residues 1-20 of human S6K1). Antibodies that recognize isoforms of
p90RSK were raised against peptide RNQSPVLEPVGRSTLAQRRGIKK
(residues 712-734 of human p90RSK2). Antibodies that
recognize MSK1 were raised against peptide FKRNAAVIDPLQFHMGVER
(corresponding to residues 384-402 of MSK1) (13). Antibodies
recognizing ERK1 and ERK2, phospho-specific antibodies recognizing the
activated forms of ERK1 and ERK2, and phospho-specific antibodies
recognizing GSK3 General Methods and Buffers--
Phosphoamino acid analysis of
32P-labeled peptides, restriction enzyme digests, DNA
ligations, site-directed mutagenesis, and other recombinant DNA
procedures were performed using standard protocols. All DNA constructs
were verified by DNA sequencing. This was performed by the Sequencing
Service at the School of Life Sciences of the University of Dundee
using DYEnamic ET terminator chemistry (Amersham Pharmacia Biotech) on
Applied Biosystems automated DNA sequencers. Buffer A contained 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.27 M sucrose, and 0.1% (by volume) 2-mercaptoethanol. Buffer
B contained 50 mM Tris-HCl (pH 7.5) and 0.1 mM
EGTA. SDS-sample buffer contained 50 mM Tris-HCl (pH 6.8),
2% (by mass) SDS, 10% (by volume) glycerol, and 1% (by volume)
2-mercaptoethanol. Buffer C contained 50 mM Tris-HCl (pH
7.5), 1 mM EGTA, 1 mM EDTA, 1% (by mass)
Triton X-100, 1 mM sodium orthovanadate, 50 mM
sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 µM microcystin-LR, 0.1% (by
volume) 2-mercaptoethanol, and Complete proteinase inhibitor mixture
(one tablet/25 ml). Buffer D contained 50 mM Tris-HCl (pH
7.5), 1 mM EGTA, 1 mM EDTA, 1 mM
sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 µM microcystin-LR, 0.1% (by volume) 2-mercaptoethanol,
and Complete proteinase inhibitor mixture (one tablet/25 ml).
Cloning of Mouse LKB1--
A polymerase chain reaction-based
strategy was used to prepare an N-terminal FLAG epitope-tagged cDNA
construct encoding mouse LKB1 using, as a template, an expressed
sequence tag encoding full-length mouse LKB1 (NCBI accession number
AA542163, IMAGE number 550355) obtained from the IMAGE
consortium (20). The construct was obtained using the 5'-primer
atgcatactagtgccaccatggactactacaaggacgacgatgacaaggacgtggcggaccccgagccgttggg and the 3'-primer gacagaactagttcactgctgcttgcaggccgaga. The resulting polymerase chain reaction fragment was cloned into the pCR-Topo2.1 vector (Invitrogen) and subsequently subcloned as an
EcoRI-EcoRI fragment into the pCMV5 vector (to
encode expression of FLAG-LKB1 in mammalian cells) (21) and as a
SpeI-SpeI fragment into the pEBG-2T vector (to
encode for expression of GST-FLAG-LKB1 in mammalian cells) (22) and as
an EcoRI-EcoRI fragment into the pGEX-4T-1 vector
(to encode for expression of GST-FLAG-LKB1 in E. coli). The
indicated site-directed mutagenesis was performed using the QuickChange kit (Stratagene). To prepare the catalytically inactive mutant of LKB1 termed LKB1(KD) (where KD is kinase-dead),
Asp194 in subdomain VII of the kinase domain was mutated to Ala.
Expression of GST-LKB1 in E. coli--
The pGEX-4T-1 constructs
encoding GST-LKB1 or the indicated mutants of LKB1 were transformed
into E. coli BL21 cells, and a 0.5-liter culture was grown
at 37 °C in Luria broth containing 100 µg/ml ampicillin until the
absorbance at 600 nm was 0.6. Isopropyl- Expression of GST-LKB1 in Human Embryonic Kidney 293 Cells--
To express GST-LKB1 or the indicated mutants of LKB1 in
human embryonic kidney 293 cells, 20 dishes (10-cm diameter) of 293 cells were cultured, and each dish was transfected with 8 µg of the
pEBG-2T construct using a modified calcium phosphate method (23).
36 h post-transfection, the cells were lysed in 1 ml of ice-cold
Buffer C; the lysates were pooled and centrifuged at 4 °C for 10 min
at 13,000 × g; and the GST fusion proteins were purified by affinity chromatography on glutathione-Sepharose and stored
as described above.
Cell Culture, Stimulation, and Cell Lysis--
The rat embryonic
fibroblast cell line Rat-2 and human G361 malignant melanoma cells were
obtained from European Tissue Culture Collection. Rat-2 cells were
cultured on 15-cm diameter dishes in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal calf serum. G361 cells were
cultured on 10-cm diameter dishes in McCoy's 5a medium supplemented
with 2 mM glutamine and 10% (by volume) fetal calf serum.
The MSK1+/+ and MSK1 Phosphorylation of LKB1 by AGC Kinases--
PKA was purified
from bovine heart by Dr. C. MacKintosh in the Medical Research Council
Unit. MSK1 and p90RSK1 expressed as GST fusion proteins
were purified from TPA-stimulated 293 cells (13). Hexahistidine-tagged
S6K1, which lacks the carboxyl-terminal 104 residues and in which
Thr412 is mutated to Glu, was expressed in insect cells and
activated in vitro by phosphorylation with PDK1 (26).
GST-LKB1(KD), GST-LKB1(S431A), GST-CREB, GST-BAD, or histone 2B (all at
1 µg) and the peptide Crosstide (GRPRTSSFAEG, 30 µM) or
Kemptide (LRRASLG, 30 µM) were incubated in a total
volume of 40 µl at 30 °C with 1 units/ml PKA, GST-MSK1,
GST-p90RSK1, and His-S6K1 in Buffer B containing 10 mM magnesium acetate, 100 µM
[ Mapping the Site on LKB1 Labeled by PKA,
p90RSK1, S6K1, and MSK1--
To map the site on LKB1
phosphorylated by PKA, p90RSK1, S6K1, and MSK1,
GST-LKB1(KD) was phosphorylated by these kinases as described above,
except that the reaction was performed for 60 min, and a 10-fold higher
specific activity of ATP was employed. The reactions were terminated by
adding 1% (by mass) SDS and 10 mM dithiothreitol and
heated at 100 °C for 1 min. After cooling, 4-vinylpyridine was added
to a concentration of 1% (by volume), and the sample was left on a
shaking platform for 30 min at room temperature to alkylate cysteine
residues. The sample was subjected to electrophoresis on a
BisTris-4-12% polyacrylamide gel electrophoresis gel, and the 82-kDa
32P-labeled band corresponding to LKB1(KD) was excised and
cut into smaller pieces. These were washed sequentially for 15 min on a vibrating platform with 1 ml of the following: water, a 1:1 mixture of
water and acetonitrile, 0.1 M ammonium bicarbonate, a 1:1
mixture of 0.2 M ammonium bicarbonate and acetonitrile, and
finally, acetonitrile. The gel pieces were dried by rotary evaporation
and incubated in 0.3 ml of 50 mM ammonium bicarbonate and
0.05% (by mass) zwittergent 3-16 containing 2 µg of alkylated
trypsin. After 16 h, the supernatant was removed; the gel pieces
were washed for 10 min in a further 0.3 ml of 50 mM
ammonium bicarbonate, 0.05% (by mass) zwittergent 3-16, and 0.1% (by
volume) trifluoroacetic acid; and the combined supernatants containing
>90% of the 32P radioactivity were chromatographed on a
Vydac 218TP54 C18 column (Separations Group, Hesperia, CA)
as described in the legend to Fig. 1.
32P Labeling of 293 Cells Transfected with
LKB1--
293 cells were transfected with a pCMV5-encoded DNA
construct expressing either wild-type FLAG-LKB1 or the indicated LKB1 mutants. 36 h post-transfection, the cells were washed with
phosphate-free DMEM, incubated for 3 h with
[32P]orthophosphate (1 mCi/ml), and then left
unstimulated or stimulated with forskolin (20 µM) for 10 min. FLAG-LKB1 was immunoprecipitated from the cleared lysate with
anti-FLAG antibodies (5 µg of antibody conjugated to 5 µl of
protein G-Sepharose). The immunoprecipitates were washed 10 times with
1 ml of Buffer C containing 0.5 M NaCl and once with Buffer
A. The immunoprecipitated protein was alkylated with 4-vinylpyridine
and subjected to SDS-polyacrylamide gel electrophoresis; and following
autoradiography, the 32P-labeled band corresponding to
FLAG-LKB1 was excised, digested with trypsin, and analyzed by
chromatography on a C18 column as described above.
Phosphopeptide Sequence Analysis--
Peptides were analyzed by
MALDI-TOF mass spectrometry on a PerSeptive Biosystems Elite-STR mass
spectrometer using Immunoprecipitation of LKB1--
The polyclonal anti-LKB1
antibody raised against GST-LKB1 (1 mg) was covalently coupled to
protein G-Sepharose (1 ml) using dimethyl pimelimidate (29). Rat-2 (0.5 mg) or ES (1 mg) cell lysates were incubated for 60 min at 4 °C with
the LKB1-protein G-Sepharose conjugate (5 µl). The immunoprecipitates
were washed twice with 1 ml of Buffer C containing 0.5 M
NaCl and twice either with Buffer B for immunoblot analysis or with
Buffer A for the p53 kinase assays. For immunoblot analysis, the beads
were resuspended in SDS sample buffer that did not contain
2-mercaptoethanol.
Immunoblotting--
For blots of total cell lysates, 20 µg of
protein was used. For blots of LKB1 immunoprecipitation, 5 µl of
beads that had been incubated with 0.5 mg of Rat-2 or 1 mg of ES cell
lysate was used. Samples were subjected to SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose. For experiments in
which LKB1 and GSK3 Immunoprecipitation and Assay of p90RSK, MSK1, and
S6K1--
The indicated amounts of Rat-2 cell lysate were used to
immunoprecipitate MSK1 (500 µg of protein), p90RSK (50 µg of protein), and S6K1 (100 µg of protein). The lysates were
incubated at 4 °C for 1 h on a shaking platform with 5 µg of
each antibody coupled to 5 µl of protein G-Sepharose. The
immunoprecipitates were washed twice with 1 ml of Buffer C containing
0.5 M NaCl and twice with 1 ml of Buffer A. The assay (50 µl) contained washed protein G-Sepharose immunoprecipitate, 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.1% (by
volume) 2-mercaptoethanol, 2.5 µM protein kinase
inhibitor (TTYADFIASGRTGRRNAIHD, peptide inhibitor of PKA), 10 mM magnesium acetate, 0.1 mM
[ LKB1 Autophosphorylation and Phosphorylation of p53--
0.5
µg of GST-LKB1, GST-LKB1(KD), GST-LKB1(S431A), or
GST-LKB1(S431D) expressed in 293 cells or LKB1 that had been
immunoprecipitated from Rat-2 cells was incubated in a 50-µl reaction
mixture containing 50 mM Tris-HCl (pH 7.5), 0.1%
Preparation of Cytosolic and Membrane Fractions--
Rat-2 cells
cultured on 10-cm diameter dishes were washed once with
phosphate-buffered saline and then scraped into 2 ml of Buffer D. After
incubation on ice for 5 min, the cells were lysed by passing them six
times through a chamber containing a ball bearing, in which the space
between the chamber wall and the ball bearing was 0.014 mm. The
homogenate was centrifuged twice at 1500 × g for 10 min to remove unbroken cells and nuclei, and the mitochondrial fraction
was pelleted by centrifugation at 10,000 × g for 10 min. The supernatant was then centrifuged at 100,000 × g for 1 h to pellet the membrane fraction, and the
supernatant was used as the cytosolic fraction. The membrane fraction
was washed by resuspension in 2 ml of Buffer D containing 0.5 M NaCl, and the membranes were pelleted again by
centrifugation at 100,000 × g for 1 h. The
resulting membrane pellet was then solubilized in 0.5 ml of Buffer C
containing Triton X-100 and centrifuged at 100,000 × g
for 1 h to remove any insoluble debris. The supernatant was taken
as the membrane fraction.
Labeling of 293 Cells with [14C]Mevalonic
Acid--
293 cells were transfected with the indicated expression
constructs expressing wild-type and mutant forms of FLAG-LKB1. 16 h post-transfection, the cells were washed twice in DMEM containing 10% (v/v) dialyzed fetal bovine serum and 25 µM
mevastatin and incubated for 90 min at 37 °C. During this period,
(R)-[2-14C]mevalonic acid lactone was
evaporated to dryness under a constant stream of nitrogen at 50 °C
and converted to the sodium salt of mevalonic acid by incubation in 1 ml of 0.1 M NaOH for 1 h at 37 °C, and the mixture
was then neutralized with 2 M HCl. The cells were washed
twice with DMEM containing 10% (v/v) dialyzed fetal bovine serum and
25 µM mevastatin and then incubated in 5 ml of DMEM
containing 10% (v/v) dialyzed fetal bovine serum, 25 µM
mevastatin, and 2 µCi/ml
(R)-[2-14C]mevalonic acid lactone. After
20 h at 37 °C, the cells were lysed in Buffer C, and FLAG-LKB1
and Ras were immunoprecipitated from the cleared lysate with anti-FLAG
or anti-Ras antibodies (5 µg of antibody conjugated to 5 µl of
protein G-Sepharose). The immunoprecipitates were washed 10 times with
1 ml of Buffer C containing 0.5 M NaCl and once with Buffer
A and then resuspended in SDS sample buffer. The samples were subjected
to SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose, and the 14C-labeled proteins were detected
using standard PhosphorImager analysis with a screen that detects
14C radioactivity.
Growth Suppression of G361 Cells--
G361 cells were cultured
to 50% confluence on 10-cm diameter dishes and transfected with 2.5 µg of the indicated wild-type and mutant LKB1 in the pCMV5 vector
together with 2.5 µg of the pCI-neo vector (Promega) using Fugene-6
transfection reagent following the manufacturer's protocol. A
triplicate set of dishes was used for each condition. After 48 h,
G418 was added to the medium to a final concentration of 3 mg/ml, and
the medium was changed every 48 h, maintaining G418. After 16 days
the cells were Giemsa-stained, and the average number of colonies
present per cm2 on each dish was counted.
Phosphorylation of LKB1 at Ser431 by Different AGC
Kinases--
To compare the phosphorylation of LKB1 by different AGC
kinase members, we expressed a catalytically inactive point mutant of
LKB1 in E. coli as a fusion protein with GST (hereafter
termed GST-LKB1(KD)). PKA, p90RSK, MSK1, and S6K1, but not
PKB (all at ~1 unit/ml), phosphorylated GST-LKB1(KD) (Fig.
1A). Control experiments
showed that, under the same conditions, the pro-apoptotic protein BAD
was phosphorylated with similar efficiency by PKA, p90RSK,
PKB, and MSK1 (18), whereas as expected, the transcription factor CREB
was phosphorylated to a similar extent by MSK1 and PKA, but at a vastly
lower rate by p90RSK (Fig. 1A) (13).
p90RSK, S6K1, MSK1, and PKA (all at ~1 unit/ml)
phosphorylated GST-LKB1(KD) to 0.5-0.8 mol of phosphate/mol of protein
after 60 min. Digestion of labeled GST-LKB1(KD) with trypsin, followed
by chromatography on a C18 column, revealed that these
kinases had phosphorylated GST-LKB1(KD) at one major tryptic
phosphopeptide termed P1, eluting at 12.5% acetonitrile (Fig.
1B). Phosphoamino acid analysis revealed that peptide P1
contained only phosphoserine. After solid-phase sequencing,
32P radioactivity was released after the third cycle of
Edman degradation (data not shown). The molecular mass of P1 determined
by MALDI-TOF mass spectrometry (862.400 Da) was identical to
that expected for the tryptic phosphopeptide comprising residues
429-434 that is phosphorylated at Ser431 and in which
Cys433 is pyridylethylated due to alkylation of LKB1 with
4-vinylpyridine prior to digestion with trypsin. This was confirmed by
gas-phase Edman sequencing of this peptide (data not shown). Moreover,
when Ser431 on GST-LKB1 was mutated to Ala, the resulting
mutant was no longer phosphorylated significantly by
p90RSK, S6K1, MSK1, or PKA (Fig. 1C).
Generation of a Phospho-specific Antibody That Recognizes LKB1
Phosphorylated at Ser431--
We prepared a
phospho-specific antibody that recognized only LKB1 phosphorylated at
Ser431, termed antibody S431-P. Its specificity was
established by the finding that it only recognized GST-LKB1 after
phosphorylation in vitro by p90RSK1 and did not
recognize GST-LKB1(S431A) (Fig. 2).
Furthermore, the recognition of phosphorylated LKB1 was abolished when
antibody S431-P was incubated with the phosphopeptide used to raise it, but not the non-phosphorylated form of this peptide (Fig. 2).
To identify cell lines that express significant levels of endogenous
LKB1, we immunoblotted lysates derived from nine different cell lines
with a polyclonal anti-LKB1 antibody raised against bacterially
expressed GST-LKB1. This antibody recognized a single immunoreactive
band migrating with slightly lower apparent molecular mass than FLAG
epitope-tagged LKB1 (55 kDa) in most cell lines tested. These included
NIH3T3 cells, which have previously been reported to express LKB1, but
not HeLa cells, which have previously been reported not to express LKB1
(5, 10). We also failed to detect any expression of LKB1 in KB cells
(Fig. 3). A similar pattern of expression
of LKB1 was also observed using a different polyclonal anti-LKB1
antibody raised against an N-terminal region of LKB1 (data not shown).
Rat-2 embryonic fibroblasts expressed the highest levels of LKB1 (Fig.
3) and were therefore used in the experiments described below.
Forskolin and a Cell-permeable Analog of cAMP Induce
Phosphorylation of Endogenous LKB1 at Ser431--
Rat-2
cells were stimulated with the adenylate cyclase activator forskolin;
the cells were lysed; and endogenous LKB1 was immunoprecipitated. The
immunoprecipitates were immunoblotted with antibody S431-P as well as
with an antibody recognizing the LKB1 protein to quantitate the amount
of LKB1 immunoprecipitated. In unstimulated cells, the level of
phosphorylation of LKB1 was low, but increased strikingly in response
to forskolin, reaching a plateau within 2 min, which was maintained for
40 min (Fig. 4A). This
phosphorylation is likely to be mediated by PKA, as the isoquinoline
derivative H-89, which is a potent inhibitor of PKA (31, 32), largely
prevented the forskolin-induced phosphorylation of LKB1 (Fig.
4B). Other signal transduction inhibitors, including three
structurally unrelated inhibitors of MAPK kinase-1 activation (PD 98059 (33), PD 184352 (34), and U0126 (35)), an inhibitor of
phosphatidylinositol 3-kinase (wortmannin (36)), and an inhibitor of
S6K1 activation (rapamycin (37)), that would not be expected to affect
PKA activation had no effect on phosphorylation of LKB1 induced by
forskolin (Fig. 4B). Forskolin also induced phosphorylation of CREB at Ser133, a known substrate of PKA, and this
phosphorylation was also inhibited by H-89, but not by other signal
transduction inhibitors (Fig. 4B). Stimulation of Rat-2
cells with the cell-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP,
which activates PKA, also induced phosphorylation of LKB1 at
Ser431, and this was inhibited by H-89 (Fig.
4C).
EGF Induces Phosphorylation of Endogenous LKB1 at
Ser431--
EGF induced a substantial activation of
p90RSK (Fig.
5A), MSK1 (Fig.
5B), and S6K1 (Fig. 5C) in Rat-2 cells, as
expected. The activation of p90RSK and MSK1 was rapid and
reached near-maximum levels within 5 min. However, whereas the activity
of p90RSK was only moderately reduced by 40 min, the
activation of MSK1 was more transient and had decreased to near-basal
levels by 40 min. As expected, the activation of p90RSK and
MSK1 was completely inhibited by incubating cells with PD 184352 prior
to stimulation with EGF (Fig. 5, A and B). The
activation of S6K1 by EGF was slower, reaching a plateau after 10 min.
As expected, the activation of S6K1 was prevented by the
immunosuppressant drug rapamycin (Fig. 5C) and the
phosphatidylinositol 3-kinase inhibitor wortmannin (data not
shown).
EGF stimulation of Rat-2 cells induced a significant phosphorylation of
LKB1 at Ser431 within 2 min, which reached a maximum within
10 min before declining to lower levels by 40 min (Fig.
6A). PD 184352 (Fig.
6A) and PD 98059 and U0126 (Fig. 6B) completely
inhibited EGF-induced phosphorylation of LKB1. In contrast, rapamycin
and wortmannin had no effect on the EGF-mediated phosphorylation of
LKB1 (Fig. 6B).
Ro 318220 is a bisindolylmaleimide that was originally developed as an
inhibitor of protein kinase C, but that also inhibits p90RSK (38) and MSK1 (39) with similar potency in
vitro. In contrast, PKA is inhibited by Ro 318220 only at far
higher concentrations (40, 41). Ro 318220 did not affect the
forskolin-induced phosphorylation of LKB1 (Fig. 4B), but
almost completely inhibited the EGF-stimulated phosphorylation of LKB1
(Fig. 6B).
Pharmacological Evidence That p90RSK Mediates LKB1
Phosphorylation--
The results presented above are consistent for a
role for either p90RSK or MSK1 in mediating the EGF-induced
phosphorylation of LKB1 at Ser431. Recent studies indicated
that H-89 inhibits MSK1 (IC50 = 0.12 µM) with
a similar potency to PKA (IC50 = 0.13 µM),
but inhibition of p90RSK is much weaker (IC50 = 2.6 µM) (40, 42). This indicates that cellular responses
mediated by MSK1, but not those mediated by p90RSK, should
be sensitive to H-89 (42, 43). H-89 at a concentration of 5 µM had no detectable effect on the phosphorylation of
LKB1 induced by EGF; and even at concentrations as high as 10 and 20 µM, H-89 had only a small effect (Fig.
7A). In contrast, the
phosphorylation of CREB at Ser133 in response to EGF, which
is thought to be mediated by MSK1 rather than p90RSK (13,
24, 43), was virtually abolished even at 5 µM H-89 (Fig.
7A). Consistent with previous studies in other cells, the EGF-induced phosphorylation of CREB in Rat-2 cells was inhibited by PD
184352 or Ro 318220 (Fig. 6B). Similarly, 20 µM H-89 had no effect on the activation of
p90RSK (Fig. 7B) and MSK1 (Fig. 7C)
induced by EGF after these kinases were immunoprecipitated from cells
and assayed in the absence of H-89 (Fig. 7B).
Genetic Evidence That p90RSK Rather than MSK1 Mediates
LKB1 Phosphorylation in Vivo--
p90RSK, in addition to
requiring phosphorylation by ERK1/2, also needs to be phosphorylated at
Ser222 (a site phosphorylated by PDK1) (14, 45) to become
activated (44). We have recently prepared mouse ES cells deficient in the expression of PDK1 (termed PDK1
We therefore decided to investigate whether TPA induced the
phosphorylation of LKB1 in PDK1 Evidence That Phosphorylation of LKB1 at Ser431 Does
Not Affect Its Activity--
No substrates for LKB1 have been
identified thus far, and the only assay that has been used to gauge
LKB1 activity has been to measure its autophosphorylation. We have
confirmed that recombinant wild-type GST-LKB1, but not GST-LKB1(KD),
expressed in 293 cells autophosphorylated in the presence of MnATP
(Fig. 9A), but not MgATP (data
not shown), as reported by others (5, 48). We also demonstrated that
the extent of autophosphorylation of wild-type GST-LKB1 was comparable
to that of GST-LKB1(S431D) and GST-LKB1(S431A). We have also tested 25 peptides and 50 proteins routinely used to assay protein kinases and
found just one, viz. the p53 tumor suppressor protein, that
was phosphorylated in vitro by wild-type LKB1, but not by a
catalytically inactive mutant (Fig. 9A). The extent to which
p53 was phosphorylated by wild-type GST-LKB1 was similar to that to
which it was phosphorylated by GST-LKB1(S431D) and GST-LKB1(S431A)
(Fig. 9A). As many protein kinases phosphorylate p53
in vitro, but not in vivo, further work is
required to establish whether p53 is a physiological substrate for
LKB1. However, this finding was useful for the development of an assay
for LKB1 activity. In Fig. 9B, we demonstrate that
stimulation of Rat-2 cells with forskolin and EGF did not
affect the extent to which the endogenous LKB1 immunoprecipitated from
these cells autophosphorylated or the degree to which it phosphorylated
p53.
Evidence That a Small Pool of Endogenous LKB1 Associates with
Membranes--
Although one previous study has indicated that the
C-terminal fragment of LKB1, when transfected into cells, is prenylated and localized at cell membranes (10), other localization studies of
full-length LKB1 expressed in various cell lines have indicated that
LKB1 is expressed in both the nucleus and cytoplasm rather than at the
plasma membrane (5, 7). To investigate whether endogenously expressed
LKB1 is associated with cell membranes, we prepared cytosolic and
membrane fractions of unstimulated Rat-2 cells or Rat-2 cells
stimulated with EGF or forskolin. An equal amount of cytosolic protein
and membrane protein was immunoblotted with antibodies recognizing
LKB1, LKB1 phosphorylated at Ser431, Ras (a prenylated
membrane protein), and glyceraldehyde-3-phosphate dehydrogenase (a
cytosolic protein). As expected, Ras was localized exclusively in the
membrane fraction, whereas glyceraldehyde-3-phosphate dehydrogenase was
localized only in the cytoplasmic fraction. Although LKB1 was mainly
localized in the cytosol, there was a small but significant amount of
LKB1 associated with the membrane fraction (Fig.
10). Stimulation of Rat-2 cells with
EGF and forskolin did not significantly alter the amount of LKB1
localized at the membrane; but interestingly, phosphorylation of LKB1
at Ser431 was detected only in the membrane fraction, and
not in the cytosolic fraction (Fig. 10A).
Evidence That Phosphorylation of Ser431 Does Not Affect
Prenylation of LKB1--
To establish whether full-length LKB1
expressed in cells was prenylated, we transfected 293 cells with
wild-type FLAG-LKB1. The cells were metabolically labeled with
[14C]mevalonic acid (a precursor in isoprenoid
biosynthesis) for 24 h and lysed. FLAG-LKB1 was immunoprecipitated
with an anti-FLAG antibody, and Ras was also immunoprecipitated as a
control. Wild-type FLAG-LKB1 and Ras were significantly
14C-labeled, indicating that they were prenylated (Fig.
10B). FLAG-LKB1(S431A) and FLAG-LKB1(S431D) expressed in 293 cells were 14C-labeled to the same degree as wild-type
LKB1, suggesting that phosphorylation of LKB1 at Ser431
does not affect prenylation of LKB1. A mutant of LKB1 in which the
conserved Cys residue predicted to be a prenyl acceptor residue of LKB1
was mutated to Ala (FLAG-LKB1(C433A)) was not 14C-labeled
(Fig. 10B), confirming that Cys433 is likely to
be the site of prenylation.
Evidence That Phosphorylation of LKB1 at Ser431 Is
Required for Its Ability to Inhibit Cell Growth--
Makela and
co-workers (5) have demonstrated that expression of wild-type LKB1, but
not of a catalytically inactive mutant of LKB1, in G361 melanoma cells,
which do not express LKB1, potently suppresses the ability of these
cells to grow. We have confirmed that G361 cells do not express LKB1
(Fig. 11A). To determine
whether mutation of Ser431 of LKB1 to either Ala or Asp
affected the ability of LKB1 to suppress growth of G361 cells, we
transfected these cells with an expression vector encoding either
wild-type LKB1 or catalytically inactive LKB1 (LKB1(S431A) or
LKB1(S431D)) together with a plasmid encoding a neomycin/G418
resistance gene using the same protocol as Makela and co-workers (5).
After 16 days of selection with G418, as expected from the previous
study (5), a 10-fold lower number of colonies were recovered when the
G361 cells were transfected with a plasmid encoding wild-type LKB1
compared with catalytically inactive LKB1 (Fig. 11). However, when the
G361 cells were transfected with LKB1(S431A) or LKB1(S431D), a 7-fold
greater number of colonies were obtained compared with transfections
with wild-type LKB1 (Fig. 11). This indicates that phosphorylation of
LKB1 at Ser431 is likely to play a role in enabling LKB1 to
inhibit cell growth.
Role of Prenylation in Regulating LKB1 Function--
To
investigate whether prenylation of LKB1 was required for its ability to
suppress cell growth, we compared the ability of wild-type LKB1 and the
mutant of LKB1 that cannot be prenylated (LKB1(C433A)) to prevent the
growth of G361 cells. In Fig. 12
(A and B), we demonstrate that LKB1(C433A) was
equally efficient at suppressing growth of G361 cells as wild-type
LKB1, indicating that the prenylation of LKB1 is not essential for it
to suppress the growth of these cells. To establish whether prenylation
was required for LKB1 to be phosphorylated at Ser431, we
transfected 293 cells with wild-type and mutant LKB1. Stimulation of
these cells with either EGF to activate p90RSK or forskolin
to activate PKA induced significant phosphorylation of wild-type LKB1
and the non-prenylated mutant, LKB1(C433A) (Fig. 12B). As
controls, we show that EGF and forskolin induced phosphorylation of
LKB1(KD), but not of LKB1(S431A) (Fig. 12B). Purified
GST-LKB1(C433A) phosphorylated itself and p53 in vitro to
the same extent as wild-type GST-LKB1 (Fig. 12C), indicating
that prenylation of LKB1 is not required for the activity of the enzyme
in vitro.
Cys433 Is Modified by Farnesylation--
We decided to
isolate the LKB1 tryptic peptide containing Cys433 and to
determine its mass to establish whether it was modified by
farnesylation or geranylgeranylation. As the tryptic peptide containing
Cys433 will be in the same peptide as Ser431,
we decided to 32P label 293 cells expressing wild-type
LKB1, LKB1(S431A), and LKB1(C433A); stimulate them with forskolin;
immunoprecipitate LKB1; and perform standard tryptic peptide map
analysis to purify the tryptic peptide containing Ser431
and Cys433. These experiments demonstrated that forskolin
stimulated phosphorylation of both wild-type LKB1 and mutant
LKB1(C433A), but not mutant LKB1(S431A) (Fig.
13A).
32P-Labeled LKB1 from these experiments was digested with
trypsin, and the resulting peptides were separated by chromatography on a C18 column. A number of minor 32P-labeled
peptides were recovered from wild-type and mutant LKB1 derived from
unstimulated cells (Fig. 13, B-D). Forskolin stimulated the
phosphorylation of two peptides of wild-type LKB1: one termed peptide
PA, eluting at 12.5% acetonitrile (the same position as peptide P1 in Fig. 1B), and the other termed peptide
PB, eluting at 48% acetonitrile (Fig. 13B).
Peptide PA corresponds to the tryptic phosphopeptide
comprising residues 429-434 that is phosphorylated at
Ser431 and in which Cys433 is pyridylethylated
because of the alkylation of free Cys residues of LKB1 with
4-vinylpyridine prior to digestion with trypsin. The mass of this
peptide determined by MALDI-TOF mass spectrometry is 862.400 (the
predicted mass for this peptide is 862.401), and it contains
phosphoserine. 32P radioactivity was released at the third
cycle of solid-phase Edman sequencing (data not shown). As expected,
peptide PA was absent from the tryptic peptide map derived
from forskolin-stimulated LKB1(S431A), whereas this peptide eluted
slightly earlier on the C18 column from the
forskolin-stimulated LKB1(C433A) sample, as it was not pyridylethylated
(the mass of this peptide was determined as 725.3721 Da, coinciding
with the predicted mass of 725.3711 Da for the tryptic phosphopeptide
comprising residues 429-434 that is phosphorylated at
Ser431 and in which the residue equivalent to
Cys433 is an Ala). Peptide PB was observed in
the tryptic peptide map derived from forskolin-stimulated wild-type
LKB1, but was not observed in the maps from forskolin-stimulated
LKB1(S431A) and LKB1(C433A) (Fig. 13, B-D). The mass of
peptide PB is 961.5319 Da (Fig. 13E), identical
to that of the peptide comprising residues 429-434 of LKB1
(Arg-Leu-Ser-Ala-Cys-Lys) that is phosphorylated at Ser431
and farnesylated at Cys433 (predicted mass of 961.5310 Da).
Had this peptide been geranylgeranylated, its mass would have been
68.070 Da higher. Consistent with this analysis, peptide PB
contains phosphoserine, and solid-phase sequencing indicated that
32P radioactivity was released after the third cycle of
Edman sequencing (data not shown). This also confirms the mass
spectrometry analysis result that peptide PB is not
carboxymethylated; otherwise, it would not be possible to couple it
through its C-terminal carboxyl residue to the arylamine residue for
solid-phase sequencing, and it would have an observed mass of 16.0 Da
greater. In the fraction adjacent to peptide PB in
forskolin-stimulated wild-type LKB1, the
non-Ser431-phosphorylated peptide comprising residues
429-434 of LKB1 that is farnesylated at Cys433 with a mass
of 881.576 Da (predicted mass of 881.5647 Da) was also observed (Fig.
13F).
One of the major findings of this study is that we identified
Ser431 as an in vivo phosphorylation site in
LKB1. In support of a role of PKA in mediating the phosphorylation of
LKB1, we found that stimulation of Rat-2 cells (Fig. 4) and embryonic
stem cells (Fig. 8B) with forskolin, an activator of PKA,
induced the phosphorylation of endogenous LKB1 at Ser431
and that this was inhibited by H-89 (Fig. 8). EGF also induced the
phosphorylation of Ser431, and this is likely to be
mediated by p90RSK. This event is prevented by inhibitors
of MAPK kinase-1 activation and an inhibitor of p90RSK and
MSK1 (Ro 318220), but not by concentrations of H-89 that selectively
inhibit MSK1. We supported this finding by demonstrating that, in
control ES cell lines and in an MSK1-deficient ES cell line (24), TPA
potently activated p90RSK in these cells and still induced
the phosphorylation of LKB1 at Ser431 and that this
phosphorylation was inhibited by PD 184352 and Ro 318220, but not by
H-89 (Fig. 8). In contrast, in a PDK1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phosphorylated at Ser21 and GSK3
phosphorylated at Ser9 were from New England Biolabs Inc.
Antibodies recognizing CREB, phospho-specific antibodies recognizing
CREB phosphorylated at Ser133, and the antibodies used for
immunoblotting Ras were from Upstate Biotechnology, Inc. The antibodies
used for immunoprecipitating Ras were a generous gift from R. Marais
(Institute for Cancer Research). Secondary antibodies coupled to
horseradish peroxidase used for immunoblotting were from Pierce, and
monoclonal antibodies recognizing GST and FLAG epitope tags were from Sigma.
-D-galactosidase (250 µM) was added, and the cells were cultured for a
further 16 h at 26 °C. The cells were resuspended in 25 ml of
ice-cold Buffer C and lysed by one round of freeze/thawing, and the
lysates were sonicated to fragment the DNA. The lysates were
centrifuged at 4 °C for 30 min at 20,000 × g, and
the supernatant was filtered through a 0.44-µm filter and incubated
for 60 min on a rotating platform with 1 ml of glutathione-Sepharose
previously equilibrated in Buffer C. The suspension was centrifuged for
1 min at 3000 × g, and the beads washed three times
with 15 ml of Buffer C containing 0.5 M NaCl and then a
further 10 times with 15 ml of Buffer A. The protein was eluted from
the resin at ambient temperature by incubation with 2 ml of Buffer A
containing 20 mM glutathione, and the beads were removed by
filtration through a 0.44-µm filter. The eluate was divided into
aliquots, snap-frozen in liquid nitrogen, and stored at
80 °C.
/
ES cells
(24) and the PDK1+/+ and PDK1
/
ES cells (25) were cultured on gelatinized 15-cm diameter dishes in
KnockOutTM DMEM supplemented with 10%
KnockOutTM serum replacement, 0.1 mM
nonessential amino acids, antibiotics (100 units of penicillin G and
100 mg/ml streptomycin), 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 1000 units/ml ESGROTM
(murine leukemia inhibitory factor) to prevent differentiation of the
cells. Prior to stimulation, Rat-2 cells were cultured in the absence
of serum overnight, whereas the ES cell lines were deprived of serum
for 4 h. Inhibitors were dissolved in Me2SO at a
1000-fold higher concentration than they were used. These inhibitors or
the equivalent volume of Me2SO as a control was added to
the tissue culture medium 30 min prior to stimulation unless indicated
otherwise. The cells were stimulated with the indicated agonists, lysed
in 1 ml of ice-cold Buffer C, and centrifuged at 4 °C for 5 min at
16,000 × g. The supernatants were frozen in liquid
nitrogen and stored at
80 °C. Protein concentrations were
determined using the Bradford method (58), and bovine serum albumin was employed as the standard.
-32P]ATP (1000 cpm/pmol), and 1 µM
microcystin-LR. After incubation for 15 min, incorporation of phosphate
into peptides was determined using phosphocellulose P-81 paper (27),
and the incorporation of phosphate into LKB1, CREB, BAD, and histone 2B
was determined following the electrophoresis of samples on
BisTris-4-12% polyacrylamide gel electrophoresis gels and
autoradiography of the gels.
-cyanocinnamic acid as the matrix. Spectra were
obtained in both the linear and reflector modes. The sequence identity
of each peptide was also confirmed by Edman sequencing on an Applied
Biosystems 476A sequencer, and the site of phosphorylation was
determined by solid-phase Edman degradation of the peptide coupled to
Sequelon-AA membrane (Milligen) as described previously (28).
isoforms were being immunoblotted, the membranes
were incubated in 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.5% (by volume) Tween, and 10% (by mass) skimmed
milk for 7 h at 4 °C in the presence of 1 µg/ml antibody.
Immunoblotting with antibody S431-P (1 µg/ml) was carried out as
described above, except that non-phosphorylated peptide (10 µg/ml)
corresponding to the antigen used to raise the antibody was included.
For experiments using all other commercial antibodies, we used a
1000-fold dilution of the stock antibody and 5% (by mass) bovine serum
albumin in place of skimmed milk. Detection was performed using
horseradish peroxidase-conjugated secondary antibodies and the enhanced
chemiluminescence reagent (Amersham Pharmacia Biotech).
-32P]ATP (~1000 cpm/pmol), and Crosstide
(GRPRTSSFAEG, 30 µM) (30). The assays were carried out
for 15 min at 30 °C, with the assay tubes being agitated
continuously to keep the immunoprecipitate in suspension, and then
terminated and analyzed as described previously (27). 1 milliunit of
activity is the amount of enzyme that catalyzes the phosphorylation of
1 pmol of Crosstide in 1 min.
-mercaptoethanol, 0.1 mM EGTA, 10 mM
manganese chloride, 0.5 µM microcystin-LR, and 100 µM [
-32P]ATP (1000 cpm/pmol) in the
presence or absence of 2 µg of bacterially expressed mouse p53. After
incubation for 60 min at 30 °C, the reactions were terminated by
addition of SDS sample buffer, and the samples were electrophoresed on
BisTris-4-12% polyacrylamide gel electrophoresis gels and analyzed by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phosphorylation of LKB1 at Ser431
by AGC kinase. A, GST-LKB1(KD), GST-CREB, GST-BAD, or
histone 2B and the peptide Crosstide or Kemptide were incubated with
the indicated AGC kinase members in the presence of magnesium and
[ -32P]ATP as described under "Experimental
Procedures." Phosphorylation of protein substrates was determined
following electrophoresis on a 4-12% gradient polyacrylamide gel, and
the Coomassie Blue-stained bands corresponding to each substrate was
autoradiographed. Phosphorylation of Crosstide and Kemptide was
determined following adsorption of these peptides to phosphocellulose
P-81 paper. ND, not determined. Similar results were
obtained in three separate experiments. B, GST-LKB1(KD) that
had been phosphorylated with the indicated kinases was digested with
trypsin and chromatographed on a Vydac 218TP54 C18 column
equilibrated in 0.1% (by volume) trifluoroacetic acid in water. The
column was developed with a linear acetonitrile gradient
(diagonal lines) at a flow rate of 0.8 ml/min, and fractions
of 0.4 ml were collected. 80% of the radioactivity applied to the
column was recovered from the major 32P-containing peptide
(peptide P1) at 12.5% acetonitrile. C, GST-LKB1 or
GST-LKB1(S431A) expressed in E. coli was phosphorylated with
the indicated AGC kinases as described for A. Similar
results were obtained in two separate experiments.
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Fig. 2.
Generation of phospho-specific antibodies
against LKB1. Bacterially expressed GST-LKB1 or GST-LKB1(S431A)
was incubated for 60 min with MgATP in the presence or absence of 1 unit/ml p90RSK1. Aliquots containing 10 ng of GST-LKB1 were
electrophoresed on a 4-12% gradient polyacrylamide gel, transferred
to nitrocellulose, and immunoblotted with antibody S431-P in the
presence of either the phosphopeptide antigen used to raise this
antibody (phospho S431 peptide) or the dephosphorylated form
of this peptide (dephospho S431 peptide). The samples were
also immunoblotted with the anti-LKB1 antibody raised against GST-LKB1.
Similar results were obtained in at least three separate
experiments.
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Fig. 3.
Expression of endogenous LKB1 in different
cell lines. Cell lysates from the indicated cell lines (20 µg)
were electrophoresed on a 4-12% gradient polyacrylamide gel,
transferred to nitrocellulose, and immunoblotted with the anti-LKB1
antibody raised against the GST-LKB1 protein. As a control, a 293 cell
lysate (0.3 µg) overexpressing FLAG epitope-tagged LKB1 was also
immunoblotted. No LKB1 immunoreactive band was observed when the
equivalent amount of 293 cell lysate not expressing FLAG-LKB1 was
immunoblotted (data not shown). PC12 refers to
pheochromocytoma cells; HEK 293 refers to human embryonic
kidney cells; and KB refers to human oropharyngeal
epidermoid carcinoma cells.
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Fig. 4.
Phosphorylation of endogenous LKB1 at
Ser431 is stimulated by forskolin. A, Rat-2
cells were stimulated for the times indicated with 20 µM
forskolin. The cells were lysed, and LKB1 was immunoprecipitated,
subjected to electrophoresis on a 4-12% gradient polyacrylamide gel,
transferred to nitrocellulose, and immunoblotted with either antibody
S431-P or the anti-LKB1 antibody raised against the GST-LKB1 protein.
Similar results were obtained in three separate experiments.
B, same as described for A, except that prior to
stimulation with forskolin for 10 min, the cells were pretreated for 30 min with 0.1 µM rapamycin, 10 µM H-89, 5 µM Ro 318220, 2 µM PD 184352, 50 µM PD 98059, 1 µM U0126, or 0.1 µM wortmannin, except that this was added to the cells 10 min prior to stimulation. Cell lysates were also immunoblotted with a
phospho-specific antibody that recognizes CREB phosphorylated at
Ser133 (P-CREB) and with an antibody that
recognizes the CREB protein (Total CREB). C, same
as described for A, except that Rat-2 cells were
stimulated for 10 min with 20 µM forskolin or 100 µM 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) in
the presence or absence of 10 µM H-89.
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Fig. 5.
Activation of p90RSK, MSK1, and
S6K1 in EGF-stimulated Rat-2 cells. Rat-2 cells were pretreated
for 30 min in the presence ( ) or absence (
) of 2 µM
PD 184352 (A and B) or 100 nM
rapamycin (C) prior to stimulation with 100 ng/ml EGF for
the times indicated. The cells were lysed, and p90RSK
(A), MSK1 (B), and S6K1 (C) were
immunoprecipitated from the same lysate and assayed. The data are
presented as the means ± S.E. for two separate experiments, with
each determination carried out in triplicate.
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Fig. 6.
Effect of signal transduction inhibitors on
phosphorylation of LKB1 induced by EGF. A, Rat-2 cells
were pretreated for 30 min in the presence or absence of 2 µM PD 184352 prior to stimulation with 100 ng/ml EGF for
the times indicated. The cells were lysed, and LKB1 was
immunoprecipitated, subjected to electrophoresis on a 4-12% gradient
polyacrylamide gel, transferred to nitrocellulose, and immunoblotted
with either antibody S431-P or the anti-LKB1 antibody raised against
the GST-LKB1 protein. Cell lysates (20 µg of protein) from these
stimulations were also immunoblotted with a phospho-specific antibody
that recognizes the activated forms of ERK1 and ERK2
(Phospho-ERK1/2) as well as with an antibody that recognizes
ERK1 and ERK2 proteins (Total ERK1/2). Similar results were
obtained in three separate experiments. B, same as described
for A, except prior to stimulation of Rat-2 cells with 100 ng/ml EGF for 10 min, the cells were pretreated for 30 min with 0.1 µM rapamycin, 5 µM Ro 318220, 2 µM PD 184352, 50 µM PD 98059, 1 µM U0126, or 0.1 µM wortmannin, except that
this was added to the cells 10 min prior to stimulation. Cell lysates
were also immunoblotted with a phospho-specific antibody that
recognizes CREB phosphorylated at Ser133
(Phospho-CREB) and with an antibody that recognizes the CREB
protein (Total-CREB).
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Fig. 7.
Effect of H-89 on phosphorylation of LKB1 and
CREB induced by EGF. Prior to stimulation of Rat-2 cells with 100 ng/ml EGF for 10 min, the cells were pretreated for 30 min with the
indicated concentrations of H-89. A, LKB1 was
immunoprecipitated, subjected to electrophoresis on a 4-12% gradient
polyacrylamide gel, transferred to nitrocellulose, and immunoblotted
with either antibody S431-P or the anti-LKB1 antibody raised against
the GST-LKB1 protein. Cell lysates (20 µg of protein) from these
stimulations were also immunoblotted with a phospho-specific antibody
that recognizes CREB phosphorylated at Ser133
(Phospho-CREB) and with an antibody that recognizes the CREB
protein (Total-CREB). The lysates were also immunoblotted
with an antibody that recognizes the activated forms of ERK1 and ERK2
(Phospho-ERK1/2) as well as with an antibody that recognizes
ERK1 and ERK2 proteins (Total-ERK1/2). Similar results were
obtained in three separate experiments. B and C,
p90RSK and MSK1, respectively, were immunoprecipitated and
assayed. The data are presented as the means ± S.E. for two
separate experiments, with each determination carried out in
triplicate.
/
cells); and as expected, these cells possessed no detectable p90RSK activity even after TPA stimulation, which activates
ERK1/2 in these cells (25). Importantly, in
PDK1
/
ES cells, TPA still induced
activation of MSK1 to the same extent as observed in control ES cells
(25). Recently, MSK1-deficient ES cells have also been generated; and
in these cells, TPA failed to stimulate the phosphorylation of CREB,
despite p90RSK being activated normally (24).
/
and
MSK1
/
ES cells. LKB1 was expressed in
control mouse ES cells (Fig. 3), and we demonstrate in Fig.
8A that TPA induced the
phosphorylation of LKB1 at Ser431 in both the control and
MSK1
/
ES cell lines, but not in the
PDK1
/
ES cell line. The phosphorylation of
LKB1 in these cells, like that observed in response to EGF in Rat-2
cells, was inhibited by either PD 184352 or Ro 318220 (Fig.
8B). ERK1 and ERK2 were activated by TPA in both the
PDK1
/
and MSK1
/
ES cells (Fig. 8A) in a PD 184352-sensitive, but Ro 318220- and H-89-insensitive manner (Fig. 8B). p90RSK is
thought to mediate the TPA-stimulated phosphorylation of GSK3
at
Ser21 and of GSK3
at Ser9 (46, 47). We
demonstrate in Fig. 8A that the phosphorylation of GSK3
and GSK3
was stimulated by TPA in MSK1
/
ES cells. In contrast, no detectable phosphorylation of GSK3
or
GSK3
was observed in either unstimulated or TPA-stimulated PDK1
/
ES cells (Fig. 8A), as
reported previously (25). Stimulation of the control,
PDK1
/
, and
MSK1
/
ES cell lines with forskolin induced
a potent phosphorylation of LKB1 at Ser431. Consistent with
this being mediated by PKA, it was inhibited by H-89, but not by PD
184352 or Ro 318220 (Fig. 8C).
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Fig. 8.
Phosphorylation of endogenous LKB1 in
MSK1 /
and
PDK1
/
ES
cells. A, the indicated ES cell lines were stimulated
for 20 min with 400 ng/ml TPA. The cells were lysed, and LKB1 was
immunoprecipitated, subjected to electrophoresis on a 4-12% gradient
polyacrylamide gel, transferred to nitrocellulose, and immunoblotted
with either antibody S431-P or the anti-LKB1 antibody raised against
the GST-LKB1 protein. The lysates (20 µg of protein) were also
immunoblotted with an antibody that recognizes the activated forms of
ERK1 and ERK2 (Phospho-ERK1/2) as well as with an antibody
that recognizes ERK1 and ERK2 proteins (Total ERK1/2). The
lysates were also immunoblotted with an antibody that recognizes
GSK3
phosphorylated at Ser21 and GSK3
phosphorylated
at Ser9 (Phospho-GSK3
/
) as well
as with an antibody that recognizes GSK3
(GSK3
).
B, same as described for A, except that the cells
were incubated in the presence or absence of PD 184352 (2 µM), or Ro 318220 (5 µM), or dimethyl
sulfoxide (DMSO) as a control for 30 min prior to stimulation with TPA.
C, same as described for A, except that the cells
were incubated in the presence or absence of H-89 (10 µM)
and then stimulated with 20 µM forskolin for 10 min.
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Fig. 9.
Evidence that phosphorylation of LKB1 at
Ser431 does not affect its activity. A,
wild-type (WT) GST-LKB1 or the indicated mutants of GST-LKB1
expressed in 293 cells were incubated for 30 min with
manganese/[ -32P]ATP in the presence or absence of
mouse p53 (2 µg) and electrophoresed on a 4-12% gradient
polyacrylamide gel, which was autoradiographed. The samples were also
immunoblotted with antibodies recognizing the GST tag to ensure that
comparable amounts of wild-type and mutant GST-LKB1 were used.
B, Rat-2 cells were stimulated for 10 min with 100 ng/ml EGF
or 20 µM forskolin or were left unstimulated. The cells
were lysed, and LKB1 was immunoprecipitated and assayed by incubation
for 30 min with manganese/[
-32P]ATP in the presence or
absence of p53 to measure autophosphorylation activity.
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Fig. 10.
Evidence that phosphorylation of
Ser431 does not affect membrane association or prenylation
of LKB1. A, Rat-2 cells were left unstimulated or
stimulated for 30 min with 100 ng/ml EGF or 20 µM
forskolin. The cells were lysed in a lysis buffer without Triton X-100
(Buffer D), and cytosolic and membrane fractions were prepared as
described under "Experimental Procedures." The membrane fraction
was washed with Buffer D containing 0.5 M NaCl and then
resuspended in lysis buffer containing Triton X-100 (Buffer C). The
cytosol and membrane were immunoblotted with the anti-LKB1 antibody
raised against the GST-LKB1 protein (40 µg of protein), antibody
S431-P (20 µg of protein), an antibody that recognizes all Ras
isoforms (10 µg of protein), or an antibody that recognizes
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5 µg of
protein). Similar results were obtained in two separate experiments.
B, 293 cells were transfected with wild-type (WT)
FLAG-LKB1, the indicated mutant forms of FLAG-LKB1, or empty pCMV5
vector. The cells were labeled with [14C]mevalonic acid
for 24 h and lysed in Buffer C, and LKB1 and Ras were
immunoprecipitated with the anti-FLAG or anti-Ras antibody. 90% of the
immunoprecipitate was electrophoresed on 4-12% polyacrylamide gel,
transferred to nitrocellulose, and autoradiographed for 14C
radioactivity. The remaining 10% of the immunoprecipitate was
immunoblotted with the anti-FLAG antibody to monitor the amount of
wild-type and mutant FLAG-LKB1 in each immunoprecipitate. The results
of two separate experiments are shown.
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Fig. 11.
Evidence that phosphorylation of LKB1 at
Ser431 is necessary for its ability to inhibit cell
growth. G361 cells were transfected with the indicated
wild-type (wt) and mutant forms of FLAG-LKB1 in the presence
or absence of the pCI-neo expression vector, which encodes for G418
resistance. After 4 days, the samples were immunoblotted with the
anti-LKB1 antibody to ensure that comparable amounts of wild-type and
mutant forms of LKB1 were expressed (A). After 16 days of
G418 selection, Giemsa-stained colonies were counted (B) and
photographed (C). The S.D. values and photographs are from
three independent dishes. Similar results were obtained in two separate
experiments, with each condition carried out in triplicate.
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Fig. 12.
Role of prenylation of LKB1 in regulating
phosphorylation of Ser431 and activity and ability of LKB1
to suppress cell growth. G361 cells were transfected with the
indicated wild-type (WT) and mutant forms of FLAG-LKB1 in
the presence or absence of the pCI-neo expression vector, which encodes
for G418 resistance. A, after 4 days, the samples were
immunoblotted with the anti-LKB1 antibody to ensure that comparable
amounts of wild-type and mutant forms of GST-LKB1 were expressed.
B, after 16 days of G418 selection, Giemsa-stained colonies
were photographed. C, 293 cells were transfected with the
indicated N-terminal FLAG epitope-tagged wild-type and mutant forms of
LKB1 or the empty pCMV5 vector ( ) as a control. After 24 h, the
cells were deprived of serum overnight and left unstimulated or were
stimulated for 10 min with 100 ng/ml EGF or 20 µM
forskolin. The cells were lysed, and 1 µg of cell lysate was
immunoblotted with antibody S431-P to measure phosphorylation of LKB1
at Ser431 or with the anti-FLAG antibody to assess the
level of expression of LKB1 in the lysate. D, wild-type
GST-LKB1 or the indicated mutants of GST-LKB1 expressed in 293 cells
were incubated for 30 min with manganese/[
-32P]ATP in
the presence or absence of mouse p53 (2 µg) and electrophoresed on a
4-12% gradient polyacrylamide gel, which was autoradiographed. The
samples were also immunoblotted with antibodies recognizing the GST tag
to ensure that comparable amounts of wild-type and mutant forms of
GST-LKB1 were used. Similar results were obtained in two separate
experiments for all data presented, with each condition in B
carried out in triplicate.
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Fig. 13.
LKB1 is farnesylated at
Cys433. 293 cells were transiently transfected
with DNA constructs encoding wild-type (WT) FLAG-LKB1 or
mutant LKB1(S431A) or LKB1(C433A). After 24 h, the cells were
deprived of serum overnight and then washed in phosphate-free medium
and incubated for 3 h with 32P-labeled inorganic
phosphate. The cells were either left unstimulated or stimulated for 10 min with 20 µM forskolin. The cells were lysed; the
32P-labeled LKB1 protein was immunoprecipitated from the
lysates using an anti-FLAG antibody, treated with 4-vinylpyridine, and
electrophoresed on a 4-12% polyacrylamide gel; and
32P-labeled LKB1 was visualized by autoradiography
(A). 32P-Labeled LKB1 was excised from the gel,
digested with trypsin, and chromatographed on a Vydac 218TP54
C18 column equilibrated in 0.1% (by volume)
trifluoroacetic acid, and the columns were developed with a linear
acetonitrile gradient (diagonal lines). The flow rate was
0.8 ml/min, and fractions of 0.4 ml were collected (B-D).
The two major forskolin-stimulated 32P-labeled peptides
eluting at 12.5% (peptide PA) and 48% (peptide
PB) acetonitrile are indicated. The MALDI-TOF mass spectrum
profile of fraction 215, in which peptide PB derived from
forskolin-stimulated wild-type LKB1, corresponds to residues 429-434
of LKB1 (Arg-Leu-Ser-Ala-Cys-Lys) that is phosphorylated at
Ser431 and farnesylated at Cys433
(E). The MALDI-TOF mass spectrum profile of fraction 214 is
also shown, which contains the peptide comprising residues 429-434 of
LKB1 that is farnesylated at Cys433, but that is not
phosphorylated at Ser431 (F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
ES
cell line (25), in which TPA activated ERK1/2 and MSK1, but not
p90RSK, this agonist failed to induce phosphorylation of
LKB1 at Ser431 (Fig. 8). Therefore, the combined
pharmacological and genetic data that we have obtained in Rat-2 and ES
cells, which are summarized in Fig. 14,
strongly support a role for p90RSK, rather than MSK1 or
S6K1, in mediating the phosphorylation of LKB1 at Ser431 in
response to agonists that activate ERK1/2.
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Fig. 14.
LKB1 is phosphorylated at Ser431
in response to agonists that activate p90RSK and PKA.
The signal transduction pathways and the sites of action of the kinase
inhibitors used in this study are indicated. IGF-1,
insulin-like growth factor-1; PI3K, phosphatidylinositol
3-kinase.
Interestingly, MSK1 phosphorylated LKB1 in vitro at an ~2-fold higher initial rate than p90RSK or S6K1; but under identical conditions, MSK1 phosphorylated CREB at an ~100-fold higher rate than p90RSK (Fig. 1A). Previous work has shown that there is typically a 20-fold higher level of p90RSK activity in cells compared with MSK1 activity (13, 43). This is also the case in Rat-2 cells, in which, after 5 min of EGF stimulation, MSK1 activity reached ~6 milliunits/mg (Fig. 5A) and p90RSK activity reached ~100 milliunits/mg (Fig. 5B). The larger amount of p90RSK activity in cells may explain why p90RSK rather than MSK1 phosphorylated LKB1 in vivo. Thus, in general, substrates that are phosphorylated in vitro at a similar initial rate by MSK1 and p90RSK are perhaps more likely to be physiological substrates for p90RSK rather than for MSK1. In contrast, it could be expected that in vivo MSK1 substrates such as CREB will turn out to be vastly superior in vitro substrates for MSK1 compared with p90RSK.
To determine whether the activation of MSK1 in the absence of p90RSK activity could induce phosphorylation of LKB1, we stimulated Rat-2 cells with cellular stresses, including UV irradiation, hydrogen peroxide, and sorbitol, which activate MSK1 to the same extent as EGF, but do not activate p90RSK. None of these agonists induced a significant phosphorylation of LKB1 at Ser431 (data not shown), further indicating that MSK1 does not phosphorylate LKB1 in vivo. It should also be noted that stimulation of Rat-2 cells with insulin-like growth factor-1, which potently activates S6K1, but not p90RSK and MSK1, also did not induce a notable phosphorylation of LKB1 at Ser431 (data not shown). This is consistent with the observation that S6K1, in the absence of p90RSK and MSK1 activity, does not phosphorylate LKB1 in vivo (data not shown). Ser431 does not lie in a consensus motif required for phosphorylation by PKB (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)) (11) and would not be expected to be phosphorylated by PKB. However, another residue on LKB1 (Thr336) lies in a consensus motif for phosphorylation by PKB, and this site is conserved in the Xenopus LKB1 homolog XEEK1. The finding that bacterially expressed GST-LKB1 (Fig. 1A) or GST-LKB1(KD) prepared from serum-starved transfected 293 cells, in which PKB is inactive (data not shown), was not significantly phosphorylated by PKB in vitro suggests that Thr336 may not be a physiological PKB phosphorylation site.
Previous studies have indicated that LKB1 may have a very narrow substrate specificity, as it did not phosphorylate a number of substrates routinely used to assay protein kinases (3-5). An alternative explanation is that LKB1 requires a regulatory subunit for activity; and thus, the catalytic subunit, when expressed alone, may possess only a basal level of activity. We found that only one substrate out of nearly 80 that we tested, viz. the p53 tumor suppressor protein, was phosphorylated by LKB1 prepared from transfected 293 cells, albeit at a low rate. A catalytically inactive mutant of GST-LKB1 in which a single residue had been mutated was unable to phosphorylate p53 (Fig. 9B). p53 was also phosphorylated in vitro by endogenous LKB1 immunoprecipitated from Rat-2 cells. Furthermore, phosphorylation of p53 occurred only in the presence of MnATP, but not MgATP, consistent with other studies showing that LKB1 is only active in the presence of MnATP. These observations suggest that LKB1, rather than a contaminating kinase, was the enzyme phosphorylating p53 in these experiments. GST-LKB1 expressed in E. coli, unlike GST-LKB1 expressed in 293 cells, did not phosphorylate itself or p53 (data not shown), indicating either that the bacterially expressed enzyme is misfolded or that LKB1 expressed in mammalian cells undergoes some modification or interaction with a regulatory component that activates it. Clearly, further studies are required to establish whether p53 is a physiological substrate for LKB1.
The finding that LKB1 autophosphorylation or its activity for p53 was not altered following mutation of Ser431 to Asp or Ala in vitro or phosphorylation of Ser431 in vivo (Fig. 9) indicates that phosphorylation of Ser431 may not regulate the catalytic activity of LKB1 directly. One mechanism by which phosphorylation of Ser431 could regulate LKB1 function would be to alter its cellular location or to enable it to interact with a regulatory subunit or a substrate. Ser431 lies 2 residues N-terminal to a potential prenylation site (Cys433); and therefore, phosphorylation of Ser431 could potentially regulate prenylation of LKB1 in vivo. A C-terminal fragment of LKB1 expressed as an N-terminal green fluorescent protein fusion was shown to be prenylated in vivo, but it was not demonstrated in this study whether full-length LKB1 was prenylated (10). We have demonstrated here that full-length LKB1, when overexpressed in 293 cells, is farnesylated, but that a mutant form of LKB1 in which the predicted farnesyl acceptor Cys residue is mutated to Ala (LKB1(C433A)) is not prenylated (Figs. 10B and 13). As LKB1(S431A) and LKB1(S431D) are prenylated to a similar extent as wild-type LKB1 (Fig. 10B), this indicates that phosphorylation of LKB1 at Ser431 may not affect prenylation of this enzyme. Unlike Ras, which is prenylated and exists solely at the membranes of cells, subcellular fractionation experiments indicated that only a small fraction of endogenous cellular LKB1 in Rat-2 cells was associated with the membrane fraction (Fig. 10A). Furthermore, stimulation of cells with EGF or forskolin to induce phosphorylation of Ser431 did not significantly alter the amount of LKB1 present in the membrane fractions of these cells. Although most of the LKB1 was located in the cytosolic fraction of cells, no phosphorylation of cytosolic LKB1 was observed following stimulation of cells with forskolin and EGF. In contrast, these stimuli induced potent Ser431 phosphorylation of LKB1 associated with cell membranes (Fig. 10). This could be explained if membrane-associated LKB1 was a better substrate for p90RSK and PKA than cytosolic LKB1. It is also possible that both membrane and cytosolic LKB1 are phosphorylated, but cytosolic LKB1 may be more efficiently dephosphorylated by a protein phosphatase than the membrane-associated form of LKB1. Alternatively, cytosolic LKB1 may be associated with another protein that prevents it from becoming phosphorylated at Ser431.
There is considerable evidence that PKA (49, 50) as well as p90RSK (14, 51, 52) could play important roles in regulating proliferation and cell survival. One of the cellular targets that p90RSK and PKA may phosphorylate to protect cells from apoptosis is BAD. This protein, in its dephosphorylated form, interacts with the Bcl family member Bcl-xL and induces apoptosis of some cells. However, after BAD is phosphorylated at Ser112 by p90RSK (53, 54) or at Ser155 by PKA (18, 55-57), it dissociates from Bcl-xL and interacts with 14-3-3 instead, and apoptosis is prevented. However, BAD has a very restricted tissue distribution, suggesting that p90RSK and PKA may arrest the apoptotic pathway or regulate proliferation by phosphorylating additional targets. LKB1 could represent one of these targets, as there is strong evidence that it functions as a tumor suppressor and thus could play a role in regulating cellular transformation. This is based on the findings that many of the mutations identified in LKB1 in patients with Peutz-Jeghers syndrome would be expected to impair its activity (1, 3, 4) and that overexpression of LKB1 in several cancer cells inhibited the proliferation of these cells (5). We have confirmed these findings and demonstrated that mutation of Ser431 to either Ala or Asp greatly reduced the ability of LKB1 to suppress the growth of G361 melanoma cells (Fig. 11). As LKB1(S431A) is similar to LKB1(S431D) in this assay, it is possible that mutation of Ser431 to Asp is not sufficient to mimic phosphorylation of LKB1 at this residue. It also cannot be discounted that both the phosphorylation and dephosphorylation of Ser431 are required for LKB1 to suppress growth. The discovery that LKB1 is a physiological target of p90RSK and PKA is intriguing, and it is tempting to speculate that some of the effects of p90RSK and PKA on cell survival and proliferation could be mediated through phosphorylation of LKB1.
The finding that prenylation of LKB1 is not essential for its ability
to suppress the growth of G361 cells (Fig. 12B) indicates that farnesylation of LKB1 instead of activation of LKB1 may play a
role in inhibiting the function of LKB1 in vivo. A mutant of LKB1 that is not prenylated still can phosphorylate itself and p53 to a
similar extent as wild-type LKB1 (Fig. 12B) and is still phosphorylated at Ser431 in cells in response to agonists
that activate PKA and p90RSK (Fig. 12C).
However, as only ~10% of LKB1 that is expressed in 293 cells is
prenylated (Fig. 13), it is not possible to conclude whether or not
prenylation of LKB1 negatively regulates LKB1 activity, as it would be
necessary to obtain a population of LKB1 that was farnesylated to a
high stoichiometry to address this question. It is possible that
prenylation may regulate the cellular location of LKB1, the stability
of LKB1, or its interaction with a regulatory subunit or substrate. In
the future, it will be important not only to identify the function of
phosphorylation of LKB1 at Ser431, but also to discover the
role that farnesylation and/or defarnesylation of Cys433
plays in enabling LKB1 to inhibit cell transformation.
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ACKNOWLEDGEMENTS |
---|
We thank R. Marais for generously donating the immunoprecipitating anti-Ras antibodies and J. Leitch for preparation of sheep antibodies. We thank the Sequencing Service (School of Life Sciences, University of Dundee) for DNA sequencing.
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FOOTNOTES |
---|
* 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 a Diabetes UK studentship.
Supported by Diabetes UK and the United Kingdom Medical
Research Council. To whom correspondence should be addressed. Tel.: 44-1382-344241; Fax: 44-1382-223778; E-mail:
d.r.alessi@dundee.ac.uk.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009953200
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ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; p90RSK, p90 ribosomal S6 kinase; MSK1, mitogen- and stress-activated protein kinase-1; S6K1, p70 ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; ES, embryonic stem; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CREB, cAMP response element-binding protein; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PDK1, 3-phosphoinositide-dependent protein kinase; GSK3, glycogen synthase kinase-3; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PKB, protein kinase B; AGC, protein kinases similar to PKA, PKG, and PKC.
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