Phosphorylation of the Transcription Factor Forkhead Family
Member FKHR by Protein Kinase B*
Graham
Rena
§,
Shaodong
Guo¶,
Stephen C.
Cichy¶,
Terry G.
Unterman¶, and
Philip
Cohen
From the
Department of Biochemistry, Medical Research
Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1
5EH, Scotland, United Kingdom and the ¶ University of Illinois
College of Medicine at Chicago and Chicago Area Veterans Health Care
System (West Side Division), Chicago, Illinois 60612
 |
ABSTRACT |
Protein kinase B lies "downstream" of
phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate
many of the intracellular actions of insulin and other growth factors.
Here we show that FKHR, a human homologue of the DAF16 transcription
factor in Caenorhabditis elegans, is rapidly phosphorylated
by human protein kinase B
(PKB
) at Thr-24, Ser-256, and Ser-319
in vitro and at a much faster rate than BAD, which is
thought to be a physiological substrate for PKB. The same three sites,
which all lie in the canonical PKB consensus sequences
(Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was
cotransfected with either PKB or PDK1 (an upstream activator of PKB).
All three residues became phosphorylated when 293 cells were stimulated
with insulin-like growth factor 1 (IGF-1). The IGF-1-induced
phosphorylation was abolished by the PtdIns 3-kinase inhibitor
wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated
protein kinase cascade) or by rapamycin. These results indicate that
FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known
to be a component of a signaling pathway that has been partially
dissected genetically and includes homologues of the insulin/IGF-1
receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256,
and Ser-319 and the sequences surrounding them in DAF16 therefore
suggests that DAF16 is also a direct substrate for PKB in C. elegans.
 |
INTRODUCTION |
In recent years evidence has accumulated that many of the
metabolic actions of insulin may be mediated by a protein kinase cascade that lies "downstream" of phosphatidylinositide
(PtdIns)1 3-kinase and the
second messengers PtdIns(3,4,5)P3 and
PtdIns(3,4)P2 (reviewed in Refs. 1 and 2). A central player
in this cascade is protein kinase B (PKB, also called c-Akt). This
enzyme is activated when it becomes phosphorylated at Thr-308 and
Ser-473 (3) by 3-phosphoinositide-dependent protein kinases
1 and 2 (PDK1, PDK2), respectively (4-7). The activation of PKB by
PDK1 in vitro has an absolute requirement for
PtdIns(3,4,5)P3 or PtdIns(3,4)P2 (4), and these
mediators facilitate activation by binding to the pleckstrin homology
domains of both PKB (5, 7) and PDK1 (8). Consistent with these
observations, the phosphorylation of PKB at Thr-308, induced by either
insulin or insulin-like growth factor 1 (IGF-1) is prevented by
inhibitors of PtdIns 3-kinase (3). PDK2 has not yet been characterized
although, like the phosphorylation of Thr-308, the insulin or
IGF-1-induced phosphorylation of Ser-473 is prevented by inhibitors of
PtdIns 3-kinase (3).
PKB mediates the metabolic actions of insulin by phosphorylating
regulatory proteins at serine or threonine residues that lie in
Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs (9), of which the best
characterized are the cardiac isoform of 6-phosphofructo-2-kinase (PFK2) (2, 10), the protein kinase glycogen synthase kinase 3 (GSK3)
(11, 12), and the mammalian target of rapamycin (mTOR) (13), as well as
the proapoptotic protein BAD (reviewed in Ref. 2). Phosphorylation by
PKB activates cardiac PFK2, and this is thought to underlie the
insulin-induced stimulation of glycolysis in the heart. Phosphorylation
inhibits GSK3 and is thought to contribute to the stimulation of
glycogen synthesis and global protein synthesis by insulin (1, 2).
Phosphorylation by PKB activates mTOR, allowing it to catalyze several
phosphorylation events that enhance the translation of specific
proteins. The overexpression of PKB has also been shown to mimic other
metabolic actions of insulin, such as the stimulation of glucose (14) and amino acid transport (15).
When cells are stimulated with IGF-1, PKB is initially translocated to
the plasma membrane where it becomes activated by PDK1 and PDK2, but it
subsequently accumulates in the nucleus (16). This raises the question
of whether PKB mediates some of the effects of insulin on specific gene
transcription, and several pieces of evidence would appear to support
this contention. For example, the overexpression of constitutively
active mutants of PKB mimics the effects of insulin in stimulating the
transcription of the obesity gene product leptin (17) and in inhibiting
the transcription of IGF-binding protein 1 (IGFBP-1) (18). The
insulin-induced suppression of phosphoenolpyruvate carboxykinase (19)
and IGFBP-1 (18) is prevented by inhibitors of PtdIns 3-kinase
(wortmannin, LY 294002) and unaffected by the drugs that inhibit mTOR
(rapamycin) or the classical mitogen-activated protein (MAP) kinase
cascade (18, 19). Studies with constitutively active and dominant negative forms of PKB have shown that PKB may mediate transcriptional effects of insulin through a conserved insulin response sequence present in a number of genes known to be inhibited by insulin in the
liver, such as IGFBP-1 and PEPCK (18). This
suggests that PKB may indeed play an important role in mediating the
effects of insulin on hepatic gene expression
The insulin/IGF-1-stimulated PKB cascade has also been identified in
Caenorhabditis elegans, where it is known to stimulate metabolism, to inhibit dauer arrest and to shorten the life span of
this nematode (reviewed in Ref. 20). In this pathway, which has been
partially dissected by genetic techniques, the DAF2 gene encodes a homologue of the IGF-1 receptor and lies "upstream" of
the AGE1 gene that encodes a PtdIns 3-kinase homologue and the AKT1 and AKT2 genes that encode homologues of
PKB. Downstream of PKB is the transcription factor DAF16; mutations in
DAF16 return life span to normal that has been lengthened by
inactivating mutations in AGE1 or AKT1/2 (21,
22). Whether DAF16 is phosphorylated directly by AKT1/AKT2 is unknown,
but we noticed that it possesses three consensus sequences for
phosphorylation by PKB, all of which are highly conserved in several
mammalian DAF16 homologues, namely the "Forkhead" family members
FKHR, FKHRL1, and AFX (23). Two of the three sites are conserved in a
further DAF16 homologue encoded by the AF6q21 gene (24).
Here we establish that FKHR is phosphorylated at these three sites by
PKB in vitro and in cotransfection experiments and that the
same sites become phosphorylated in response to IGF-1 in 293 cells via
a PtdIns 3-kinase-dependent pathway that is independent of
mTOR or the classical MAP kinase cascade. The accompanying paper (25)
demonstrates that FKHR-stimulated reporter gene expression is dependent
on an intact insulin response sequence (IRS) and that transactivation
by FKHR is inhibited by insulin via the phosphorylation of Ser-256
(25). The conservation of Thr-24, Ser-256, and Ser-319 and the
sequences surrounding them in DAF16 suggests that DAF16 is likely to be
a direct substrate for PKB in C. elegans. Taken together,
these results indicate that PKB regulates the ability of FKHR to
stimulate transcription.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture reagents and IGF-1 were purchased
from Life Technologies, Inc. Laboratories (Paisley, Scotland) and
protein G-Sepharose from Amersham Pharmacia Biotech (Milton Keynes,
United Kingdom). PKI, the specific peptide inhibitor of
cAMP-dependent protein kinase (TTYADFIASGRTGRRNAIHD), was
synthesized by F. B. Caudwell in the MRC Protein Phosphorylation
Unit and other peptides by Dr. G. Bloomberg (University of Bristol).
Wortmannin was from Sigma (Poole, UK), PD 98059 and rapamycin from
Calbiochem (Nottingham, UK), and the PCR cloning vectors pCR2.1 TOPO
and pCR2.1 from Invitrogen (NV Leek, Holland). Restriction enzymes were
purchased from NEB (UK) Ltd. (Hitchin, UK) and MBI (Vilnius,
Lithuania). GST-BAD expressed in Escherichia coli was
provided by Dr. Takayasu Kobayashi of this Unit. Vectors expressing
Myc-tagged PDK1 (5) and GST-PKB[T308D/S473D] (3) were provided by Dr.
Maria Deak in the MRC Unit at Dundee.
Cell Culture, Transient Transfections, and Cell Lysis--
293
cells were cultured at 37 °C in an atmosphere of 5% CO2
in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum. Transfection of cells was carried out using the
calcium chloride precipitation method, using 10 µg of DNA per 10-cm
diameter dish. Prior to lysis, cells were serum-starved for 12 h.
Cells were lysed in 1 ml ice-cold Buffer A (50 mM Tris acetate (pH 7.5), 1 mM EGTA, 1% (w/v) Triton X-100, 1 mM EDTA, 50 mM NaF, 10 mM sodium
-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl
fluoride, and 0.1% (v/v)
-mercaptoethanol). The lysates were
centrifuged at 13,000 × g, and the supernatants were
removed, frozen immediately in liquid nitrogen, and stored at
80 °C until use.
Alignment, Cloning, Expression, and Purification of
FKHR--
Inspection of the public EST data bases identified 41 clones
from 14 different tissues that encoded fragments of FKHR. Inspection of
these sequences showed 100% identity with the nucleotide sequence of
the second FKHR sequence to be reported (GenBankTM
accession number AF032885 (26)). An epitope-tagged, full-length human
FKHR construct was generated as follows. A fusion was generated in
which the oligonucleotide 5'-GCGGG GATCC CGCCA CCATG GAGTT CATGC CCATG
GAGCT GGTGC TGCCA T-3' encoding the epitope Glu-Phe-Met-Pro-Met-Glu (termed EE-tag) preceded the first 300 base pairs of FKHR (EST 1061191). This 350-base pair oligonucleotide produced the EE-FKHR fusion in a PCR reaction with a second oligonucleotide: 5'-GGCCG CGGCG
GCCGC CGCCG CCACC GCCGC CGCCA CGGAG CC-3' corresponding to bases
259-300 of the FKHR coding sequence. This PCR product was cloned into
the TA cloning vector pCR2.1 TOPO. The remaining 1665 base pairs of
FKHR (EST 166558) were incorporated into pCR2.1 by a non-PCR TA cloning
method using the NotI and BspMI restriction sites
(27). A vector containing a construct of full-length FKHR was then
generated by ligating the two halves of FKHR in pCR2.1. This construct
was then subcloned into pCMV-5 and pEBG2T to allow expression of
recombinant FKHR in mammalian cells. Finally, the FKHR construct was
subcloned into a pGEX4T-3 vector for expression of a GST-FKHR fusion
protein in E. coli. Expressed GST-FKHR was purified by
glutathione-Sepharose affinity chromatography and stored at
80 °C
in 50 mM Tris-HCl, pH 7.5 (20 °C), 0.2 mM
glutathione at a concentration of 2 mg/ml (4).
Generation of Phosphospecific Antibodies for
FKHR--
Phosphopeptides and dephosphopeptides were synthesized
corresponding to residues 19-31 (RPRSCTpWPLPRPE), 248-262
(KSPRRRAASpMDNNSK), and 311-324 (TTFRPRSSSpNASVS) of FKHR, where p
indicates the site of phosphorylation (Thr-24, Ser-256, and Ser-319).
The peptides were conjugated to both keyhole limpet hemocyanin and
bovine serum albumin and injected into sheep at the Scottish Antibody
Production Unit (Carluke, UK). Six weeks later, antisera were passed
through a CH-Sepharose column to which a dephosphopeptide had been
coupled, followed by affinity chromatography on CH-Sepharose to which
the corresponding phosphopeptide antigen had been attached covalently. Phospho-specific antibodies were eluted with 0.1 M glycine,
pH 2.4, immediately adjusted to pH 8 with Tris base and stored at 4 °C. A further antibody was generated toward residues 636-651 of
FKHR (LPNQSFPHSVKTTTHS) that recognizes the dephosphorylated as well as
the phosphorylated form of the protein.
Activation of Protein Kinase B and Phosphorylation of FKHR and
BAD--
PKB
(0.15-5.0 units/ml) that had been expressed in
Sf9 cells and activated by phosphorylation with PDK1 (4) was
incubated for 30 min at 30 °C with FKHR (0.1 mg/ml) or BAD (0.05 mg/ml) in 50 mM Tris-HCl, pH 7.5), 0.1 mM EGTA,
2.5 µM PKI, 10 mM magnesium acetate, 0.1 mM ATP (106 cpm/nmol). One unit of PKB activity
was that amount that catalyzed the phosphorylation of 1 nmol of the
peptide GRPRTSSFAEG in 1 min (11).
To determine the stoichiometry of phosphorylation of FKHR and BAD,
aliquots of the reaction were denatured in SDS and subjected to
polyacrylamide gel electrophoresis. After staining with Coomassie Blue
and destaining, the gels were dried and the concentrations of GST-FKHR
and GST-BAD were determined by densitometry using a Fujifilm
LAS-1000 luminescent image analyzer calibrated with different
concentrations of bovine serum albumin run in parallel on the same gel.
The molecular masses of GST-FKHR and GST-BAD were taken as 97 kDa and
56 kDa, respectively.
 |
RESULTS |
Phosphorylation of FKHR by PKB in Vitro--
The observation that
FKHR contains three residues, located at Thr-24, Ser-256, and Ser-319,
that lie in consensus sequences for phosphorylation by PKB (see
introduction) led us to study the phosphorylation of this protein by
PKB in vitro. FKHR was phosphorylated by PKB to a
stoichiometry of >1 mol of phosphate/mol of protein (Fig.
1A). The initial rate of
phosphorylation was much faster than that of the pro-apoptotic protein
BAD (Fig. 1B), which is thought to be an in vivo
substrate for PKB (29).

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Fig. 1.
Relative rates of phosphorylation of FKHR and
BAD by PKB in vitro. GST-FKHR and GST-BAD (each
at 1 µM) were incubated with activated GST-PKB and
Mg[ -32P]ATP. At the times indicated aliquots of the
reaction were denatured in SDS and electrophoresed on an 8%
polyacrylamide gel. After staining with Coomassie Blue and destaining,
the gels were autoradiographed (A), and the bands
corresponding to GST-FKHR and GST-BAD were excised and counted and
phosphorylation stoichiometries (B) were calculated for FKHR
(open circles) and BAD (closed
circles) as described under "Experimental
Procedures."
|
|
To identify the sites of phosphorylation, we raised phospho-specific
antibodies that only recognize FKHR if it is phosphorylated at Thr-24,
Ser-256, or Ser-319. The specificity of these antibodies is
demonstrated in Fig. 2. The antibody
raised against a phosphopeptide corresponding to the sequence
surrounding Thr-24 recognized FKHR only after phosphorylation by PKB,
and its recognition of phospho-FKHR was prevented if the antibody was
first preincubated with the phosphopeptide used to raise it but not by
preincubation with either of the other two phosphopeptides. The
specificities of the antibodies raised against phosphopeptides
corresponding to the sequences surrounding Ser-256 and Ser-319 were
established in an analogous manner (Fig. 2). These results demonstrate
that PKB phosphorylates FKHR at Thr-24, Ser-256, and Ser-319. The
phosphorylation of all three sites approached a plateau after 5-10 min
when high concentrations of PKB were present in the assays (5 units/ml) (Fig. 3A). However, when the
concentration of PKB was reduced to 0.15 unit/ml to measure initial
rates of phosphorylation, Ser-256 was found to be phosphorylated more
rapidly than the other two sites (Fig. 3B).

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Fig. 2.
Generation of phosphospecific antibodies
against FKHR. Bacterially expressed GST-FKHR was maximally
phosphorylated with PKB (columns 2-5) or left
unphosphorylated (column 1), and aliquots (1 µg) were
spotted onto a nitrocellulose membrane. They were then immunoblotted
with affinity-purified antibodies raised against phosphopeptides
corresponding to the sequences surrounding Thr-24, Ser-256, and
Ser-319, as well as a dephosphopeptide corresponding to residues
636-651 of FKHR. In columns 3, 4, and 5, antibodies were preincubated
with 50 mM Thr-24, Ser-256, and Ser-319 phosphopeptide
antigens prior to immunoblotting as indicated below. Columns
1 and 2, antibodies used without preincubation;
column 3, antibodies incubated with the Thr-24
phosphopeptide antigen (50 mM); column 4,
antibodies incubated with the Ser-256 phosphopeptide antigen (50 mM); column 5, antibodies incubated with the
Ser-319 phosphopeptide antigen (50 mM).
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Fig. 3.
PKB phosphorylates FKHR at three
residues. A, bacterially expressed GST-FKHR was
incubated with MgATP and PKB (5 units/ml). At the times indicated,
aliquots were denatured in SDS, electrophoresed on 8% polyacrylamide
gels, transferred to nitrocellulose, and immunoblotted with the
antibodies in Fig. 2. B, same as A, except that
the concentration of PKB was reduced to 0.15 units/ml, and after
immunoblotting, the membranes were analyzed by densitometry to measure
the extent of phosphorylation of each site.
|
|
Phosphorylation of FKHR in 293 Cells--
To investigate whether
FKHR could be phosphorylated by PKB in a cellular context, we
co-transfected into 293 cells DNA encoding FKHR with that encoding a
constitutively active PKB and then examined FKHR phosphorylation by
immunoblotting with the phospho-specific antibodies. These experiments
demonstrated that Thr-24, Ser-256, and Ser-319 were phosphorylated to
low levels when FKHR was transfected alone and that phosphorylation of
each site increased greatly when FKHR was cotransfected with PKB (Fig.
4A). We also found that FKHR
became phosphorylated at all three sites when cotransfected with PDK1
(Fig. 4B), one of the upstream activators of PKB (reviewed in Refs. 1 and 2).

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Fig. 4.
Phosphorylation of FKHR in 293 cells after
cotransfection with PKB and PDK1. 293 cells were transiently
transfected with plasmids expressing GST-FKHR and/or the constitutively
active GST-PKB(T308D/S473D) mutant (A) or GST-FKHR and/or
Myc-tagged PDK1 (B). 16 h post-transfection, the cells
were serum-starved for a further 12 h and then lysed. Aliquots
were then denatured in SDS, electrophoresed on 8% polyacrylamide gels,
and after transfer to nitrocellulose, immunoblotted with the four
antibodies used in Fig. 2.
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In 293 cells PKB is maximally activated (50-fold) 5 min after
stimulation with IGF-1 with a half-time for activation of 1 min (3).
IGF-1 also stimulated the phosphorylation of FKHR at Thr-24, Ser-256,
and Thr-319 (Fig. 5), maximal
phosphorylation occurring within 10 min (data not shown). Consistent
with the IGF-1-induced phosphorylation of FKHR being mediated by PKB,
phosphorylation of Thr-24, Ser-256, and Ser-319 was prevented if the
cells were incubated with the PtdIns 3-kinase inhibitor wortmannin (100 nM) prior to stimulation with IGF-1 (Fig. 5). The basal
level of phosphorylation was also abolished by wortmannin.

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Fig. 5.
Phosphorylation of FKHR in IGF-1-treated 293 cells. 293 cells were transiently transfected with a plasmid
expressing GST-FKHR. 16 h post-transfection, the cells were
serum-starved for a further 12 h and then stimulated for 10 min
with or without 100 ng/ml IGF-1. Aliquots (10 µg of lysate protein)
were then denatured in SDS, electrophoresed on 8% polyacrylamide gels,
and after transfer to nitrocellulose, immunoblotted with the four
antibodies used in Fig. 2. Where indicated, the cells were incubated
for 10 min with 100 nM wortmannin or for 60 min with 100 nM rapamycin plus 50 µM PD 98059 prior to
stimulation with IGF-1.
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|
Residues 24, 256, and 319 all lie in Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)
sequences that are not only consensus sequences for phosphorylation by
PKB but also for phosphorylation by MAP kinase-activated protein kinase
1 (MAPKAP-K1, also called p90rsk) and p70 S6 kinase (9, 30).
MAPKAP-K1 (which lies immediately downstream of MAP kinase) and p70 S6
kinase (which lies downstream of mTOR) are both activated in response
to insulin or IGF-1, and their activation is also inhibited by
wortmannin (31, 32). To investigate which of these protein kinases
mediates the IGF-1-induced phosphorylation of FKHR in vivo,
we therefore carried out additional experiments in which 293 cells were
incubated with either rapamycin (which prevents the activation of p70
S6 kinase by inhibiting mTOR (33)) or PD 98059 (which prevents the
activation of MAP kinase kinase-1 and hence the activation of
MAPKAP-K1) (34). Neither of these drugs affected the basal or
IGF-1-induced phosphorylation of FKHR at Thr-24, Ser-256, and Ser-332
(Fig. 5) although, at the same concentrations, they prevented the basal
and IGF-1-induced phosphorylation of p70 S6 kinase and MAPKAP-K1 (data
not shown).
 |
DISCUSSION |
In this study we have demonstrated a rapid phosphorylation of FKHR
by PKB at Thr-24, Ser-256, and Ser-319. This occurs both in
vitro (Figs. 2 and 3) and in cotransfection experiments using either PKB (Fig. 4A) or PDK1 (Fig. 4B). PDK1 is
an upstream activator of PKB (reviewed in Refs. 1 and 2), and the
phosphorylation of FKHR when cotransfected with PDK1 is presumably
catalyzed by the endogenous PKB that becomes activated by the
transfected PDK1. Similar observations were made with GSK3, another
physiological substrate of PKB (35).
We have also shown that all three sites on FKHR become rapidly
phosphorylated when 293 cells are stimulated with IGF-1 (Fig. 5). The
IGF-1-induced phosphorylation is prevented by inhibitors of PtdIns
3-kinase but not by inhibitors of the activation of the MAP kinase
cascade or p70 S6 kinase. These experiments, together with the in
vitro studies and cotransfection experiments, indicate that the
IGF-1-induced phosphorylation of FKHR is mediated by PKB or a closely
related enzyme.
A pathway in C. elegans has been partially dissected by
genetic techniques in which DAF16 (an FKHR homologue) is a downstream component of a signaling cascade that includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase, and PKB (see introduction). PDK1 has not yet been identified as a component of this pathway, perhaps because its inactivation has other (lethal) consequences stemming from its role in activating additional protein kinases that
lie in distinct protein kinase cascades. This may explain why
disruption of the two PDK1 homologues in Saccharomyces
cerevisiae is also lethal (28). We have noticed that, as expected,
the genome of C. elegans does indeed encode a PDK1 homologue
located on the X chromosome (genomic clone number H33H01).
The accompanying paper (25) shows that FKHR stimulates the
transcription of a reporter gene, that this stimulation is dependent on
an intact insulin response sequence (IRS), and that transcription is
suppressed by 50% when HepG2 cells are exposed to insulin or transfected with constitutively active PKB. In this system,
phosphorylation of Ser-256 appears to be required and sufficient to
mediate the action of insulin because its mutation to alanine abolishes
the effect, whereas mutation to aspartic acid mimics it. Interestingly Ser-256 is the residue in FKHR that is phosphorylated most rapidly by
PKB in vitro (Fig. 3B). In contrast, mutation of
Thr-24 or Ser-319 to alanine has no effect on the ability of insulin or constitutively active PKB to disrupt activation by FKHR, and the functional consequences of these other phosphorylation events remain to
be determined. Nevertheless, the conservation of the sequences that
surround Thr-24 and Ser-319 in two other mammalian FKHR homologues (23)
as well as DAF16 of C. elegans (21, 22) suggests that their
phosphorylation is likely to play a role(s) in vivo.
In conclusion this study, together with that presented in the
accompanying paper (25), provides the first direct biochemical evidence
that the kinase cascade leading to the regulation of DAF16 is present
in mammalian cells. The finding that phosphorylation of FKHR inhibits
its ability to stimulate transcription is consistent with genetic
evidence in C. elegans that has shown that mutations in the
DAF16 gene have the opposite phenotype to mutations in either the AGE1 (PtdIns 3-kinase) or AKT1/2 (PKB)
genes (21-23). These findings raise the possibility that
phosphorylation of FKHR (or its homologues) underlies the regulation of
at least some of the genes whose transcription is inhibited by insulin.
 |
ACKNOWLEDGEMENTS |
We thank our colleagues Dr. Andrew Paterson
(expression and activation of PKB), Dr. Maria Deak (construction of
vectors expressing PDK1 and constitutively active PKB), and Dr.
Takayasu Kobayashi (purified BAD protein) for reagents.
 |
Note Added in Proof |
While this paper was under review,
similar results to those presented here and in the following paper (25)
have been found by three other laboratories. In analysis of Forkhead
family members namely FKHRL1 (36), AFX (37), and FKHR1 (38),
phosphorylation was accompanied by nuclear export (36, 37) as a result
of interaction of the phosphorylated transcription factor with 14-3-3 proteins (36).
 |
FOOTNOTES |
*
This work was supported by the Medical Research Council
London, the Royal Society, and the Louis Jeantet Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 44-1382-344726;
Fax: 44-1382-223778; E-mail: grena{at}bad.dundee.ac.uk.
 |
ABBREVIATIONS |
The abbreviations used are:
PtdIns, phosphatidylinositide;
PtdIns(3,4,5)P3, phosphatidylinositide 3,4,5-trisphosphate;
PtdIns(3,4)P2, phosphatidylinositide 3,4-diphosphate;
PKB, protein kinase B;
PDK, 3-phosphoinositide-dependent protein kinase;
IGF-1, insulin-like growth factor 1;
IGFBP-1, IGF-binding protein 1;
MAP, mitogen-activated protein;
MAPKAP-K1, MAP kinase-activated protein
kinase 1;
PFK2, 6-phosphofructo-2-kinase;
GSK3, glycogen synthase
kinase 3;
mTOR, mammalian target of rapamycin;
PEPCK, phosphoenolpyruvate carboxykinase;
PKI, specific peptide inhibitor of
cAMP-dependent protein kinase;
IRS, insulin response
sequence;
DAF, dauer arrest phenotype;
GST, glutathione
S-transferase;
EST, expressed sequence tag.
 |
REFERENCES |
-
Cohen, P.,
Alessi, D. R.,
and Cross, D. A. E.
(1997)
FEBS Lett.
410,
3-10[CrossRef][Medline]
[Order article via Infotrieve]
-
Alessi, D. R.,
and Cohen, P.
(1998)
Curr. Opin. Genet. Dev.
8,
55-62[CrossRef][Medline]
[Order article via Infotrieve]
-
Alessi, D. R.,
Andjelkovic, M.,
Caudwell, F. B.,
Cron, P.,
Morrice, N.,
Cohen, P.,
and Hemmings, B. A.
(1996)
EMBO J.
15,
6541-6551[Abstract]
-
Alessi, D. R.,
James, S. R.,
Downes, C. P.,
Holmes, A. B.,
Gaffney, P. R. J.,
Reese, C. B.,
and Cohen, P.
(1997)
Curr. Biol.
7,
261-269[Medline]
[Order article via Infotrieve]
-
Alessi, D. R.,
Deak, M.,
Casamayor, A.,
Caudwell, F. B.,
Morrice, N.,
Norman, D. G.,
Gaffney, P.,
Reese, C. B.,
MacDougall, C. N.,
Harbison, D.,
Ashworth, A.,
and Bownes, M.
(1997)
Curr. Biol.
7,
776-789[Medline]
[Order article via Infotrieve]
-
Stokoe, D.,
Stephens, L. R.,
Copeland, T.,
Gaffney, P. R. J.,
Reese, C. B.,
and Painter, G. F.
(1997)
Science
277,
567-570[Abstract/Free Full Text]
-
Stephens, L.,
Anderson, K.,
Stokoe, D.,
Erdjument-Bromage, H.,
Painter, G. F.,
Holmes, A. B.,
Gaffney, P. R. J.,
Reese, C. B.,
McCormick, F.,
Tempst, P.,
Coadwell, J.,
and Hawkins, P. T.
(1998)
Science
279,
710-714[Abstract/Free Full Text]
-
Currie, R. A.,
Walker, K. S.,
Gray, A.,
Deak, M.,
Casamayor, A.,
Downes, C. P.,
Cohen, P.,
Alessi, D. R.,
and Lucocq, J.
(1999)
Biochem. J.
337,
575-583[CrossRef][Medline]
[Order article via Infotrieve]
-
Alessi, D. R.,
Caudwell, F. B.,
Andjelkovic, M.,
Hemmings, B. A.,
and Cohen, P.
(1996)
FEBS Lett.
399,
333-338[CrossRef][Medline]
[Order article via Infotrieve]
-
Deprez, J.,
Vertommen, D.,
Alessi, D. R.,
Hue, L.,
and Rider, M. H.
(1997)
J. Biol. Chem.
272,
17269-17275[Abstract/Free Full Text]
-
Cross, D. A. E.,
Alessi, D. R.,
Cohen, P.,
Andjelkovic, M.,
and Hemmings, B. A.
(1995)
Nature
378,
785-789[CrossRef][Medline]
[Order article via Infotrieve]
-
van Weeren, P. C.,
de Bruyn, K. M. T.,
de Vries-Smits, A. M. M.,
van Lint, J.,
and Burgering, B. M. Th.
(1998)
J. Biol. Chem.
273,
13150-13156[Abstract/Free Full Text]
-
Scott, P. H.,
Brunn, G. J.,
Kohn, A. D.,
Roth, R. A.,
and Lawrence, J. C.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7772-7777[Abstract/Free Full Text]
-
Kohn, A. D.,
Summers, S. A.,
Birnbaum, M. J.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
31372-31378[Abstract/Free Full Text]
-
Hajduch, E.,
Alessi, D. R.,
Hemmings, B. A.,
and Hundal, H.
(1998)
Diabetes
47,
1006-1013[Abstract]
-
Andjelkovic, M.,
Alessi, D. R.,
Meier, R.,
Fernandez, A.,
Lamb, N. J. C.,
Frech, M.,
Cron, P.,
Cohen, P.,
Lucocq, J. M.,
and Hemming, B. A.
(1997)
J. Biol. Chem.
272,
31515-31524[Abstract/Free Full Text]
-
Barthel, A.,
Kohn, A. D.,
Luo, Y. N.,
and Roth, R. A.
(1997)
Endocrinology
183,
3559-3562
-
Cichy, S. B.,
Uddin, S.,
Danilkovich, A.,
Guo, S.,
Klippel, A.,
and Unterman, T. G.
(1998)
J. Biol. Chem.
273,
6482-6487[Abstract/Free Full Text]
-
Sutherland, C.,
Waltner-Law, M.,
Gnudi, L.,
Kahn, B.,
and Granner, D. K.
(1998)
J. Biol. Chem.
273,
3198-3204[Abstract/Free Full Text]
-
Wood, W. B.
(1998)
Cell
95,
147-150[Medline]
[Order article via Infotrieve]
-
Lin, K.,
Dorman, J. B.,
Rodan, A.,
and Kenyon, C.
(1997)
Science
278,
1319-1322[Abstract/Free Full Text]
-
Ogg, S.,
Paradis, S.,
Gottlieb, S.,
Patterson, G. I.,
Lee, L.,
Tissenbaum, H. A.,
and Ruvkun, G.
(1997)
Nature
389,
994-999[CrossRef][Medline]
[Order article via Infotrieve]
-
Paradis, S.,
and Ruvkun, G.
(1998)
Genes Dev.
12,
2488-2498[Abstract/Free Full Text]
-
Hillion, J.,
LeConiat, M.,
Jonveaux, P.,
Berger, R.,
and Bernard, O. A.
(1997)
Blood
90,
3714-3719[Abstract/Free Full Text]
-
Guo, S.,
Rena, G.,
Cichy, S.,
He, X.,
Cohen, P.,
and Unterman, T.
(1999)
J. Biol. Chem.
274,
17184-17192[Abstract/Free Full Text]
-
Anderson, M. J.,
Viars, C. S.,
Czekay, S.,
Cavenee, W. K.,
and Arden, K. C.
(1998)
Genomics
47,
187-199[CrossRef][Medline]
[Order article via Infotrieve]
-
Rena, G.,
and Houslay, M. D.
(1998)
Nucleic Acids Res.
26,
3867-3868[Abstract/Free Full Text]
-
Casamayor, A.,
Torrance, P. D.,
Kobayashi, T.,
Thorner, J.,
and Alessi, D. R.
(1999)
Curr. Biol.
9,
186-197[CrossRef][Medline]
[Order article via Infotrieve]
-
Datta, S. R.,
Dudek, H.,
Tao, X.,
Masters, S.,
Fu, H. A.,
Gotoh, Y.,
and Greenberg, M. E.
(1997)
Cell
91,
231-241[Medline]
[Order article via Infotrieve]
-
Leighton, I. A.,
Dalby, K. N.,
Caudwell, F. B.,
Cohen, P. T. W.,
and Cohen, P.
(1995)
FEBS Lett.
375,
289-295[CrossRef][Medline]
[Order article via Infotrieve]
-
Cross, D. A. E.,
Alessi, D. R.,
Vandenheede, J. R.,
McDowell, H. E.,
Hundal, H. S.,
and Cohen, P.
(1994)
Biochem. J.
303,
21-26[Medline]
[Order article via Infotrieve]
-
Petritch, C.,
Woscholski, R.,
Edelmann, H. M. L.,
Parker, P. J.,
and Ballou, L. M.
(1995)
Eur. J. Biochem.
230,
431-438[Abstract]
-
Thomas, G.,
and Hall, M. N.
(1997)
Curr. Opin. Cell Biol.
9,
782-787[CrossRef][Medline]
[Order article via Infotrieve]
-
Alessi, D. R.,
Cuenda, A.,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494[Abstract/Free Full Text]
-
Shaw, M.,
Cohen, P.,
and Alessi, D. R.
(1997)
FEBS Lett.
416,
307-311[CrossRef][Medline]
[Order article via Infotrieve]
-
Brunet, A.,
Bonni, A.,
Zigmond, M. J.,
Lin, M. Z.,
Juo, P.,
Hu, L. S.,
Anderson, M. J.,
Arden, K. C.,
Blenis, J.,
and Greenberg, M. E.
(1999)
Cell
96,
857-868[Medline]
[Order article via Infotrieve]
-
Kops, G. J. P. L.,
de Ruiter, N. D.,
De Vries-Smits, A. M. M.,
Powell, D. R.,
Bos, J. L.,
and Burgering, B. M. J.
(1999)
Nature
398,
630-634[CrossRef][Medline]
[Order article via Infotrieve]
-
Biggs, W. H., Meisenhelder, J., Hunter, T., Cavenee, W. K., and Arden,
K. C. (1999) Proc. Natl. Acad. Sci. U. S. A., in press
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.