Báo cáo khoa học: Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade pot

By using COS-7 cells, glutathione-S-transferase GST-tagged full-length TORCs were coexpressed with con-trasting SIK1 mutants; one was a kinase-defective mutant K56M and the other was a m

by the LKB1-SIK signaling cascade

Yoshiko Katoh1, Hiroshi Takemori2, Xing-zi Lin1, Mitsuhiro Tamura1, Masaaki Muraoka3,

Tomohiro Satoh3, Yuko Tsuchiya4, Li Min1, Junko Doi5, Akira Miyauchi3, Lee A Witters6,

Haruki Nakamura4 and Mitsuhiro Okamoto7

1 Molecular Physiological Chemistry, Osaka University Medical School, Japan

2 Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka, Japan

3 ProteinExpress Co Ltd, Chiba, Japan

4 Institute for Protein Research, Osaka University, Japan

5 Food and Nutrition, Senri Kinran University, Osaka, Japan

6 Departments of Medicine and Biochemistry, Dartmouth College, Hanover, NH, USA

7 Department of Food and Nutrition, Tezukayama University, Nara, Japan

Cyclic AMP-responsive element (CRE)-binding protein

(CREB) is a transcription factor that plays an

import-ant role in numerous physiological events, such as cell

proliferation, survival, tumorigenesis, glucose metabo-lism and memory, in a phosphorylation-dependent manner [1,2] Upstream signals arriving at CREB are

Keywords

cAMP responsive element; CRE-binding

protein; LKB1; salt-inducible kinase;

transducer of regulated CREB activity

Correspondence

H Takemori, Laboratory of Cell Signaling

and Metabolism, National Institute of

Biomedical Innovation, 7-6-8, Asagi, Saito,

Ibaraki, Osaka, 567-0085, Japan

Fax: +81 72 641 9836

Tel: +81 72 641 9834

E-mail: takemori@nibio.go.jp

(Received 16 January 2006, revised 19 April

2006, accepted 24 April 2006)

doi:10.1111/j.1742-4658.2006.05291.x

Cyclic AMP responsive element (CRE)-binding protein (CREB) is known

to activate transcription when its Ser133 is phosphorylated Two independ-ent investigations have suggested the presence of Ser133-independindepend-ent acti-vation One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner The inactivation of kinase cascades by 10 nm staurosporine in LKB1-posit-ive HEK293 cells also induced unregulated, constitutLKB1-posit-ively activated, CRE activity Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another tar-get of LKB1 Constitutive activation of CREB in LKB1-defective cells or

in staurosporine-treated cells was not accompanied by CREB phosphoryla-tion at Ser133 The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation

of TORC, and such silencing may be indispensable for the regulated activa-tion of CREB

Abbreviations

A-loop, activation loop; AMPK, AMP-activated protein kinase; bZIP, basic leucine zipper domain; CRE, cAMP-response element; CREB, CRE-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; KID, kinase-inducible domain; moi, multiplicities of infection; PKA, protein kinase A; RT, reverse transcription; SIK, salt-inducible kinase; TORC, transducer of regulated CREB activity.

conveyed to transcriptional machineries via two distinct

domains of CREB The kinase-inducible domain (KID)

is located in the N-terminal region, which contains an

activating phosphoacceptor residue, Ser133 The other

domain in the C-terminus is composed of a basic

leu-cine zipper (bZIP) that is responsible for dimerization

and binding to the CRE Phosphorylation of Ser133

alters the affinity of KID to the KIX domain of CREB

and p300, resulting in enhanced transcription of

CRE-dependent genes [3] Development of a specific

anti-body that recognized phospho-Ser133 [4,5] enabled

investigators to monitor the level of ‘activated CREB’,

which has now significantly accelerated the studies of

phosphorylation-dependent CREB activation

Possible involvement of the bZIP domain of CREB

in the regulation of CRE-dependent gene expression

has been suggested by the results of two lines of

research One was initiated by an mRNA subtraction

study to isolate a specific molecule induced in the

adre-nal gland under the stress of consuming a high-salt diet

The molecule isolated was a kinase, and, thus, we

named it salt-inducible kinase (SIK) [6] SIK is a

mem-ber of the AMP-activated protein kinase (AMPK)

fam-ily [7] A gene database search found three isoforms of

SIK, SIK1–3 [8,9] In Y1 mouse adrenocortical tumor

cells, levels of mRNA, protein and kinase activity of

SIK1 were elevated within 30 min after the initiation of

the cAMP–protein kinase A (PKA) cascade

Overex-pression of SIK1 inhibited gene exOverex-pression(s) induced

by cAMP [10] Analyses of the promoter regions of

such genes indicated that CREs in the promoters were

the sites where SIK-mediated transcriptional repression

occurred, and, thus, SIK1 was thought to repress

CREB activity [11] Although SIK seemed not to

phos-phorylate CREB directly, it repressed CREB in a

kinase-activity-dependent manner Mapping the region

where SIK1 exerted its repressive action suggested that

SIK1 repressed CREB by acting on the bZIP domain

[11] In reporter gene assays, overexpression of the

kin-ase domain of SIK1 repressed CRE-dependent

tran-scription completely, even when CREB was supposed

to be fully activated by overexpression of PKA We

therefore thought that the phosphorylation of CREB at

Ser133 was not sufficient for making ‘activated CREB’

The second line of research began in an attempt to

isolate novel factors that could modulate CREB

activ-ity by using high-throughput transformation assays

[12,13] Expression vectors containing full-length

cDNAs were cotransformed with reporter vectors in

HEK293 cells, and a new family of coactivators was

identified They were named transducer of regulated

CREB activity (TORC) 1–3 The N-terminal region

of TORCs formed a coiled-coil structure, which

interacted with the bZIP domain of CREB [12] Once the TORCs had been overexpressed in HEK293 cells, CREB-dependent transcriptions were upregulated at,

or beyond, the levels induced by cAMP Activation of CREs by an overexpression of TORCs required CREB-, but not Ser133 phosphorylation, and, thus, TORCs were thought to be coactivators that did not require CREB phosphorylation Cytochemical studies

of TORCs, however, showed that the activating signals that could phosphorylate CREB, such as cAMP and

Ca2+, also induced the nuclear import of TORCs [14,15], suggesting that combination of Ser133 phos-phorylation and the binding of TORCs to the bZIP domain produces the fully ‘activated CREB’

The above observations indicate that SIKs and TORCs share a common feature regarding the regula-tion of CREB activity, both acting on the bZIP domain

of CREB in a phospho-Ser133-independent manner Having examined this feature further, we found that SIK2 phosphorylated TORC2 at Ser171 The resulting phospho-TORC2 was exported from the nucleus to the cytoplasm, and this led to the apparent inactivation of the CREB activity [14] We also showed that PKA phosphorylated SIK1 [16] and the phospho-SIK1 could not induce the nuclear export of TORC2 [17]

The importance of the phospho⁄ dephospho regula-tion of TORC, by regulating CREB, was also shown as

a physiological impact in hepatic gluconeogenesis [18] However, it remains to be clarified as to whether the phospho⁄ dephospho regulation of TORC is one of the regulatory mechanisms for the CREB activity or the cascade indispensable for the CREB action AMPK family kinases, including SIK, have flexible activation loops (A-loops) near their substrate-binding pockets Phosphorylation in the A-loop induces a structural change in the catalytic site, which turns on the kinase activities Recently, the LKB1 tumor suppressor kinase [19] was reported to be a major upstream activator of AMPK family kinases [20] LKB1 phosphorylates Thr residues in the A-loops of SIKs By using LKB1-defect-ive HeLa cells and a compound inhibiting TORC-kinases, including SIKs, we tried to elucidate the importance of the Ser133-independent activation of CREB The results suggested that the phospho⁄ dephos-pho regulation of TORC plays an indispensable role in the regulated activation of CREB

Results

All TORCs are substrates of SIK1 One of the downstream branches of the SIK-signaling cascade leads to the regulation of CRE-dependent

transcription, and we recently succeeded in identifying

TORC2, a CREB-specific coactivator, as an

endog-enous substrate of SIK2 Because mammals have three

TORC isoforms, we decided to clarify which isoform

could act as the endogenous substrate of SIK1

Figure 1A shows that the SIK-phosphorylation motif

is highly conserved among the three isoforms SIK1

was able to phosphorylate all the TORC peptides

except for the S171A-TORC2 (Fig 1B)

The levels of SIK1-dependent phosphorylation of

TORC isoforms were also examined in cultured cells

By using COS-7 cells, glutathione-S-transferase

(GST)-tagged full-length TORCs were coexpressed with

con-trasting SIK1 mutants; one was a kinase-defective

mutant (K56M) and the other was a mutant

constitu-tively phosphorylating TORC [17] (S577A mutant) As

shown in Fig 1C, the levels of phosphorylation at

Ser171 of TORC2 and Ser163 of TORC3 were

strik-ingly elevated in the presence of SIK1 (S577A) (see the

lanes indicated by 577) However, the corresponding

residue, Ser167 of TORC1, seemed to be

phosphor-ylated even in cells expressing inactive SIK1 (the lanes indicated by 56), and its phosphorylation level was enhanced slightly in cells expressing SIK1 (S577A) In contrast to the phosphorylation at Ser167, binding of 14-3-3 to TORC1 was significantly enhanced by SIK1 (S577A), suggesting that SIK1 could phosphorylate TORC1, but some as yet unidentified kinases, other than SIK1, might phosphorylate at Ser167

To evaluate the direct action of SIK1 on the trans-activation activity of TORCs, assays were performed using Gal4-fused TORCs (Fig 1D) SIK1 was able to completely inhibit the transactivation activities derived from all TORCs Together, these results suggested that SIK1 could phosphorylate all TORCs and thereby repress their transactivation activities

SIK1 is unable to induce the nuclear export

of TORC in HeLa cells The nucleo-cytoplasmic redistribution of TORC2 is important for both the stimuli-induced CRE activation

D C

Fig 1 SIK affects the functions of all TORCs (A) Amino acid alignment of the SIK phosphorylation motif (box) of TORC1–3 SIK2 was shown to phosphorylate Ser171 of TORC2 [14] The corresponding Ser residues, Ser167 (TORC1), Ser171 (TORC2) and Ser163 (TORC3) are indicated in bold (B) GST–TORC peptides were prepared in E coli and used as substrates for an in vitro kinase assay ([ 32 P]dATP[aP]) GST-tagged SIK1(1–354) prepared in COS-7 cells was used as an enzyme (Upper) Incorporation of 32P into GST–TORC peptides (Lower) Coomassie Brilliant Blue (CBB) staining of the substrates GST–Syntide2 was used as a positive control substrate (C) COS-7 cells were co-transformed with pEBG-TORC1 (1.5 lg), pEBG-TORC2 (4 lg) or pEBG-TORC3 (6 lg) and pTarget-SIK1s (2 lg each) pEBG is a mammalian expression plasmid for the GST-fusion protein After 48 h, GST–TORCs were purified by glutathione columns (CP:) and then subjected to western blot analyses (WB:) using anti-GST (upper), anti-(phospho-Ser171 TORC2) (middle) and anti-(14-3-3) (lower) sera 577 indicates the SIK1 mutant (S577A) which represses the CRE activity constitutively 56 indicates the SIK1 mutant (K56M) with no kinase activity (D) HEK293 cells were cotransformed with the expression plasmids for Gal4-fusion TORC1–3 (0.05 lg) and a 5xGAL4-luciferase reporter plasmid (0.2 lg) with an internal reporter phRL-TK(Int–) (0.03 lg) in the presence or absence of the SIK1 (S577A) plasmid (0.1 lg) The specific trans-activation activities of TORCs were expressed as the fold-trans-activation of the empty Gal4 vector, pM Means and SD are indicated (n ¼ 4).

and the SIK-mediated CRE repression Interestingly,

Bittinger et al found that TORC2 and TORC3 never

moved out of the nucleus in HeLa cells [15] Because

HeLa cells lacked LKB1, which had been reported to

phosphorylate SIKs and activate them [20], we thought

that the impaired nuclear export of TORC2 in HeLa

cells was a result of the loss of SIK activity [20] To

test this, we compared the behaviors of TORCs in

HeLa and COS-7 cells using a green fluorescent

pro-tein (GFP)-fusion technique Because GFP–TORC1

was present in the cytoplasm of COS-7 cells (Fig 2A),

we were unable to see the SIK1-induced intracellular

redistribution of GFP–TORC1 [compare SIK1(–) with

SIK1(+)] In contrast to TORC1, GFP–TORC2

clearly showed SIK1-dependent nuclear export GFP–

TORC3 also moved out of the nucleus in a slightly

lower level As expected, overexpression of SIK1 did

not induce the intracellular redistribution of TORCs in

HeLa cells (Fig 2B)

Overexpression of LKB1 in HeLa cells restores

the nucleo-cytoplasmic shuttling of TORC2

To elucidate the mechanism underlying the impaired

nucleo-cytoplasmic shuttling of TORC in HeLa cells,

we first tested whether overexpression of LKB1 in this

cell line could restore the SIK1-induced nuclear export

of TORC2 (Fig 3A) As shown in the third panel of the ‘upper’ set, a small population of GFP–TORC2 moved to the cytoplasm in LKB1-overexpressing HeLa cells Furthermore, expression of LKB1 and SIK1 in combination could completely induce the nuclear export of GFP–TORC2 (final panel) As expected, dis-tribution of GFP–TORC2 was not influenced by the overexpression of LKB1 in LKB1-positive COS-7 cells (lower set)

The Thr182 of SIK1 is phosphorylated by LKB1, resulting in conversion from inactive SIK1 to the act-ive form The importance of phospho-Thr182 was also supported by the fact that substitution of the Thr with

a negatively charged residue produced a constitutive active enzyme [20]; hence we prepared the T182E mutant As shown in Fig 3B, however, neither SIK1 (T182E) nor SIK1 (T182A) could enhance LKB1-sup-ported nuclear export of GFP–TORC2 in HeLa cells

Differential properties of the A-loops of the individual isoforms of SIK1–3

Because the SIK1 (T182E) mutant did not induce the nuclear export of TORC2, we assayed the kinase activ-ity of this mutant The T182E mutant, prepared as a GST-fusion protein using COS-7 cells, was much less active than wild-type SIK1 (Fig 4A) As expected,

A

B

Fig 2 SIK1 alone is unable to induce the nuclear export TORC2 in

HeLa cells (A) COS-7 cells cultured on cover slips were

cotrans-formed with expression vectors for GFP-tagged TORC1–3 with (+)

or without (–) the SIK1 (S577A) plasmid After 24 h, the cells were

fixed for cytochemical analyses as described in Experimental

proce-dures Green fluorescent signals of GFP–TORC1–3 (upper) and blue

fluorescent signals of nuclear staining with DAPI (lower) are shown.

(B) The same experiments were performed using HeLa cells More

than 80% of GFP-positive cells had similar patterns as shown in

each representative panel.

A

B

Fig 3 LKB1 is essential for the SIK1-induced nuclear export of TORC2 in HeLa cells (A) LKB1-defective HeLa cells (upper) and LKB1-positive COS-7 cells (lower) were cotransformed with the GFP–TORC2 expression plasmid and SIK1 expression plasmid in the presence or absence of the LKB1 expression plasmid (pEBG-LKB1) (B) Thr182, the LKB1-dependent phosphorylation site, of SIK1 was substituted with Glu or Ala, and the resultant mutants were subjected to cytochemical analyses of GFP–TORC2 HeLa cells were transformed with plasmids as in Fig 2.

neither SIK1 (T182A) nor a negative control mutant,

K56M, showed kinase activities The discrepancy

between our T182E mutant and the mutant in previous

reports [20,21] might be caused by the different

sources, Escherichia coli or cultured cells However,

similar discrepancies have also arisen in studies of

AMPK [22]

There are three isoforms of SIK in the

AMPK-rela-ted kinase family Although the overall sequence of

A-loops in SIKs is highly conserved (Fig 4B), some

variety is found in the N-terminal side of the

LKB1-phosphorylatable Thr in SIK3 To see whether the

SIK2 and SIK3 isoforms behave similarly to SIK1

with regard to LKB1-dependent phosphorylation of

Thr, we prepared several mutants in which the

corres-ponding Thr residues were substituted Kinase assays

of GST–SIK2s (Fig 4C) produced results similar to

those of SIK1s However, GST–SIK3s (Fig 4D)

provi-ded results quite different from the others SIK3

(T163A) had a little peptide phosphorylation activity,

and SIK3 (T163E) had activity as high as that of the

wild-type enzyme

SIK kinase activity is sufficient to induce the

nuclear export of TORC2 in HeLa cells

The finding of the constitutive active SIK3 mutant,

SIK3 (T163E), prompted us to investigate whether the

kinase activity of SIK was sufficient to export TORC2

even under LKB1-defective conditions Expression plasmids for GFP–TORC2 and SIK3s were cotrans-formed into HeLa cells In LKB1-overexpressing HeLa cells, GFP–TORC2 was exported from the nucleus to the cytoplasm by either wild-type SIK3 or SIK3 (T163E) mutant (Fig 5, upper) SIK3 (T163A) mutant was unable to enhance the nuclear export of GFP– TORC2 As expected, even in LKB1-nonexpressing HeLa cells (Fig 5, lower), SIK3 (T163E) could induce the nuclear export of GFP–TORC2, although wild-type SIK3, induced little export These results suggested

A

B

D C

Fig 4 The effect of the Thr to Glu substitu-tion in the A-loop on the kinase activities of SIKs (A) GST–SIK1 (1–354) and its mutants were prepared using COS-7 cells and were subjected to an in vitro kinase assay K56M

is kinase-defective SIK1, a negative control mutant GST–Syntide2 was used as a sub-strate (B) Alignment of amino acid sequences of the A-loops of SIK1–3 and AMPKa1 Conserved residues are marked

by black boxes, and the Thr residues repor-ted to be phosphorylarepor-ted by LKB1 are indi-cated by a phosphor symbol (C) Thr175 of GST–SIK2 (full-length), corresponding to Thr182 of SIK1, was replaced with Glu or Gly, and the resultant mutants were subjec-ted to an in vitro kinase assay K49M is kin-ase-defective SIK2 (D) Thr163 of the SIK3 kinase domain (1–340) was substituted with Ala or Glu K37M is kinase-defective SIK3 These panels were one of representative sets using SIK enzymes that had been pre-pared using COS-7 cells at least three times.

Fig 5 The constitutive active SIK3 exports TORC2 without LKB1

in HeLa cells The effects of substitutions at Thr163 of SIK3 on the intracellular localization of GFP–TORC2 in HeLa cells in the pres-ence (upper, +LKB1) or abspres-ence (lower, –LKB1) of LKB1 as des-cribed in Figs 2 and 3 pEBG–SIK3 (1–340) was used for the overexpression of SIK3.

that LKB1 could regulate the intracellular distribution

of TORC2 through SIKs, and that SIK kinase activity

might be sufficient to induce the nuclear export of

TORC2

Environments of CRE-dependent transcription

in HeLa cells

Next, we examined the expression of an endogenous

target of CREB, NR4A2 (Nurr1) gene (Fig 6A) The

level of NR4A2 mRNA was significantly induced by

forskolin treatment in HEK293 cells In HeLa cells,

however, it had already been expressed moderately,

and its level was not enhanced strongly by forskolin

treatment The level of 36B4 RNA, generally used as

an internal standard, showed no change

To find out why HeLa cells expressed NR4A2 mRNA constitutively, we compared the expression level and the status of components in the TORC– CREB system between HeLa and HEK293 cells The mRNA levels of TORCs in HeLa cells did not differ substantially from those in HEK293 cells (Fig 6B) However, protein levels of TORCs seemed to be much lower in HeLa cells than in HEK293 cells (Fig 6C,D) TORC2 proteins in HEK293 cells are known to migrate as two bands on SDS⁄ PAGE (Fig 6C)

A slowly moving form was the major form in nonstim-ulated HEK293 cells and was shown to be the phos-phorylated form However, the rapidly moving form was the major form in forskolin-treated cells and was shown to be the dephosphorylated form [14,15]

In HeLa cells, however, only the rapidly moving

D C

E

Fig 6 Impaired CRE-dependent

transcrip-tion in HeLa cells (A) Quantificatranscrip-tion of the

mRNA levels for NR4A2 and 36B4 in

HEK293 and HeLa cells using real-time PCR

analyses Forskolin (20 l M ) was added to

the culture medium 4 h prior to the harvest

for total RNA extraction One unit is

equival-ent to 6 pg of the standard plasmids

con-taining respective amplicons To quantify

36B4 RNA, a reverse-transcribed mixture

was further diluted at 1:100 Means and SD

are indicated (n ¼ 3) (B) Quantification of

the mRNA levels for TORC1–3 in HEK293

and HeLa cells by real-time PCR analyses.

Forskolin (20 l M ) was added to the culture

medium 4 h prior to the harvest for total

RNA extraction (C) Analyses of the level

and modification of the TORC2 protein in

HEK293 and HeLa cells Forskolin (20 l M )

was added to the culture medium 2 h prior

to the harvest for immunoprecipitation (IP)

followed by western blot analyses (WB) (D)

Analyses of the levels and modifications of

TORC1 and TORC3 in HEK293 and the

HeLa cells (E) The phosphorylation of CREB

at Ser133 is impaired in the HeLa cells.

Forskolin (20 l M ) was added to the culture

medium 30 min prior to the harvest for

western blotting.

dephospho form was seen Because our antibody raised

against TORC1 was able to detect both TORC1 and

TORC3 with equal efficiency, analyses of these two

TORCs were carried in a single blot (Fig 6D)

Simi-larly to the case of TORC2, TORC1 and TORC3 also

formed two bands in the gel The cases of TORC1 and

TORC3 were apparently the same as TORC2, but

might be substantially different, because all of the

bands responded to forskolin treatment in HEK293

cells and shifted to lower positions However, in HeLa

cells the bands remained at the same positions

irres-pective of treatment It should be mentioned here that

Ser133 phosphorylation of CREB might not be strong

in forskolin-treated HeLa cells (Fig 6E), suggesting

that more than one step in the regulatory pathway of

CREB might be impaired in HeLa cells

LKB1 restores the accentual regulation of

CRE-dependent transcription

To examine whether the constitutive expression of

NR4A2 mRNA in HeLa cells suggested the impaired

regulation of CREs, and if so, to test whether LKB1

could restore the forskolin-induced activation of

CRE-dependent transcription, we tried to perform reporter

assays Because plasmid-based reporters could not

pro-vide a high enough level of reporter activities in HeLa

cells (not shown), we prepared an adenovirus-mediated

reporter system As shown in Fig 7A, weak

enhance-ment of CRE activity by forskolin was observed in the

control HeLa cells (LacZ) Coinfection with the

LKB1-adenovirus (LKB1) repressed basal CRE activity to

one-tenth of its original level within 24 h At this time

point, forskolin was unable to substantially induce

CRE activity At 48–72 h post infection, however, a

large induction of the CRE activity by forskolin was

observed In addition, forskolin induced NR4A2

mRNA in LKB1 expressing HeLa cells (Fig 7B)

To investigate whether the impaired regulation of

CRE-dependent transcription in HeLa cells resulted

from the dysfunction of the overall phosphorylation

cascades in the SIK–TORC system or the particular

combination of SIK- and TORC isoforms, the levels of

proteins and the phosphorylation of individual isoforms

were examined As shown in Fig 7C, the level of SIK1

protein was elevated slightly by forskolin in control

cells (LacZ) When the LKB1-adenovirus was infected,

the basal level of SIK1 decreased significantly, and the

level was elevated prominently by forskolin These

results agreed with the fact that SIK1 gene expression

depended on its own CREs [18] In vitro kinase assays

using the SIK1 protein purified by immunoprecipitation

indicated that overexpression of LKB1 restored SIK1

kinase activity in HeLa cells (see the panel indicated by

32P-ATP As expected, Thr182 was not phosphory-lated in the LKB1 nonexpressing HeLa cells Because sensitivity of the anti-(phospho-Thr182 IgG) was less than that of the anti-(SIK1 IgG), we could not dis-cuss whether Thr182 was phosphorylated in the LKB1-expressing cells when cells were not stimulated with forskolin (third lane from the left) After forskolin treatment, however, the level of SIK1 protein had risen sufficiently so that we were able to detect phospho-Thr182 (the final lane)

In the case of SIK2 (Fig 7D), the protein level was not influenced by overexpression of LKB1 The levels

of kinase activity and phospho-Thr175 were elevated

in the LKB1-expressing HeLa cells By contrast, the protein level of SIK3 increased in LKB1-expressing HeLa cells, and restoration of the kinase activity and phosphorylation at Thr163 also occurred (Fig 7E) Next, we examined the phosphorylation of TORCs

in the same way (Fig 7F) Similarly to the cases for HEK293 cells, overexpression of LKB1 restored phos-pho-⁄ dephospho regulation of TORCs in HeLa cells

We describe briefly here the results of TORC1 A small part of the TORC1 population had already been phos-phorylated in control HeLa cells (lanes indicated by LacZ), and LKB1 enhanced the phosphorylation of TORC1 (third lane from the left) Forskolin treatment stimulated the dephosphorylation of TORC1 a little (final lane) Results in Figs 7F and 1C suggest that multiple cascades might be operating differentially in the phosphorylation of TORC1, and these cascades may be classified into three categories, LKB independ-ent, SIK independent and SIK dependent

Finally, we assayed the level of CREB phosphoryla-tion (Fig 7G) Forskolin-induced phosphorylaphosphoryla-tion of CREB at Ser133 was evident in LKB1-overexpressing HeLa cells These results suggested that LKB1 could modulate the actions of all participants in the SIK– TORC–CREB cascade

SIK activity restores the regulation of CRE-dependent transcription in HeLa cells

To obtain direct evidence of SIK-mediated phosphory-lation of TORC in HeLa cells, the constitutive active SIK3 mutant was overexpressed in this cell line As shown in Fig 8A, the constitutive active SIK3 mutant, T163E, could phosphorylate TORC2 even in the absence of LKB1 Moreover, the T163E mutant could recover the forskolin-dependent induction of CRE activity without LKB1 (Fig 8B)

Finally, it should be noted that the forskolin-induced CREB phosphorylation at Ser133 was restored

by overexpression of SIK3 (T163E) (Fig 8C) These

observations suggested that restoration of SIK activity

might be sufficient for repairing impaired

CREB-dependent transcription in HeLa cells

LKB1-mediated phosphorylation of SIK1 at

Thr182 enhances phosphorylation at Ser577

We noticed a large discrepancy between the increase in

total SIK1 activity and the decrease in the level of

TORC phosphorylation in forskolin-treated LKB1-expressing HeLa cells (Fig 7C,F) In this regard, we found a similar case when CREB was activated by PKA; PKA also phosphorylated SIK1 at Ser577, which diminished SIK1-mediated cytoplasmic retention

of TORC2 [17] Interestingly, phosphorylation at Ser587 of SIK2 (corresponding to Ser577 of SIK1) was not obvious in LKB1 nontransformed HeLa cells (Fig 7D, lower) To compare the specific level of SIK1 phosphorylation at Ser577 with that at Thr182 and the

C

Fig 7 LKB1 restores forskolin-induced CRE activity in HeLa cells (A) HeLa cells were cotransfected with adenovirus reporters, Ad-CRE-fLuc

or Ad-TK-rLuc (an internal standard), at moi 3 and an LKB1-adenovirus or a lacZ-adenovirus at moi 30 After the indicated periods, cells were harvested for the luciferase assays Forskolin (20 l M ) was added to the culture medium 8 h prior to the harvest (B) HeLa cells were trans-fected with the adenovirus of LKB1 or lacZ After 72 h, total RNA was purified from the cells and the levels of mRNA for NR4A2 (Nurr1) and 36B4 were quantified using real-time PCR analysis as described in the Experimental procedures Forskolin (20 l M ) was added to the culture medium 8 h prior to the harvest n ¼ 3 (C–G) HeLa cells were transfected with the LKB1-adenovirus or the lacZ-adenovirus After 72 h, cells were treated with forskolin (20 l M ) for 1 h and then subjected to immunoprecipitation (IP:) using anti-SIK1 (C), anti-SIK2 (D), anti-SIK3 (E), anti-TORC2 or anti-TORC1 ⁄ 3 (F) sera followed by western blotting Immunopurified SIK enzymes were also subjected to in vitro kinase assays ([32P]dATP[aP]) using GST–Syntide2 as a substrate The panels represent one of duplicate experiments Anti-(phospho-Thr182 SIK1) was able to detect phospho-Thr175 of SIK2 and phospho-Thr163 of SIK3 Anti-(phospho-Ser577 SIK1) cross-reacted with phospho-Ser587 of SIK2 To detect overexpressed LKB1 and total- ⁄ phospho-CREB, cell lysate was subjected to western blotting (G).

kinase activity, GST-fusion SIK1 was overexpressed

in HeLa cells As shown in Fig 9A, Ser577 was

not phosphorylated in control HeLa cells and was

apparently less phosphorylated by forskolin treatment (indicated by LacZ) Overexpression of LKB1 induced phosphorylation at Ser577, and its level was signifi-cantly elevated after forskolin treatment (indicated by LKB1), suggesting that phospho-Ser577 might be the result of an autophosphorylation of SIK1 Other indi-cators, such as phospho-Thr182 and kinase activities, depended on LKB1, but not on the forskolin treat-ment

When Ser577 is phosphorylated, the phospho-SIK1 moves to the cytoplasm Using this property, we exam-ined the LKB1-initiated autophosphorylation of SIK1

at Ser577 As shown in Fig 9B, in control HeLa cells (–), GFP–SIK1 was localized only in the nucleus When GFP–SIK1 was coexpressed with LKB1 or PKA, part of the SIK1 population moved to the cyto-plasm LKB1-induced nuclear export of SIK1 was abolished by the T182A substitution (Fig 9C) Substi-tution at Ser577 completely inhibited both LKB1- and PKA-induced nuclear export of SIK1

Finally, we tested the level of TORC phosphoryla-tion using wild-type and S577A-SIK1 (Fig 9D) In COS-7 cells, the Ser577 mutant SIK1 phosphorylated GST-fusion TORC2 more efficiently than the wild-type These observations suggested that the PKA-phos-phorylatable Ser577 also acted as the autophosphory-lation site, and that phospho-Ser577 might be a critical modulator of the TORC phosphorylation activity of SIK1 In this context, LKB1 might also play important roles in the attenuation step of the phosphorylation of TORC The SIK3 T163E-mutant having the additional mutation at Ser493, equivalent to S577A of SIK1, also suggested the importance of the Ser phosphorylation (Fig 8B)

Inhibition of kinase cascades activates CRE-dependent transcription constitutively The constitutive activation of CRE-dependent tran-scription in HeLa cells (Fig 6A,B) might be due to inactivation of the phosphorylation cascades from LKB1 to TORCs, suggesting, paradoxically, that inhi-bition of the kinase cascades could mimic impaired CREB regulation even in LKB1-positive cells To test this possibility, we performed CRE-reporter assays in HEK293 cells in the presence of various kinase inhibi-tors As shown in Fig 10A, no specific kinase inhibitor could activate the CRE However, staurosporine (STS;

10 nm), a nonspecific kinase inhibitor [23], induced CRE reporter activity to levels as high as forskolin Moreover, staurosporine upregulated transcription of the NR4A2 gene to a level higher than that elevated by forskolin treatment (Fig 10B)

A

B

C

Fig 8 The constitutive active SIK3 restores forskolin-induced CRE

activity without LKB1 in HeLa cells (A) HeLa cells were infected

with the SIK3 (full-length) adenoviruses After 72 h incubation,

TORC2 was analyzed by immunoprecipitation, and SIK3 and LKB1

were detected by western blotting using cell lysates The lower

band in the SIK3 panel might be degraded products (B) HeLa cells

were cotransformed with SIK3-adenoviruses and

reporter-adeno-viruses, and CRE activity was measured as described in Fig 1.

S493A of SIK3 is equivalent to S577A of SIK1 (C) HeLa cells were

infected with the SIK3 T163E adenovirus or its control virus, LacZ.

After 72 h incubation, cells were harvested to analyze the level of

phospho-CREB using western blotting These panels represent

experiments performed at least in duplicate.

Staurosporine has been classified as a PKC inhibitor

(Fig S1A) However, another PKC-specific inhibitor,

bisindolylmaleimide I (Bis) did not induce any CRE

reporter activity in our assay system (Fig 10A),

sug-gesting that PKC might not be the kinase responsible

for staurosporine-induced CRE activity

To investigate the phosphorylation status of TORC

and CREB in staurosporine-treated cells, GST-tagged

TORC2 and endogenous CREB were examined in

COS-7 cells (Fig 10C) Forskolin induced both the

dephosphorylation at Ser171 and the decrease in the

level of bound 14-3-3 It enhanced the

phosphoryla-tion of CREB at Ser133, of course As expected,

sta-urosporine completely inhibited the phosphorylation

of TORC2 and did not enhance the phosphorylation

of CREB

Because staurosporine significantly blocked

SIK1-mediated CRE repression (not shown), the efficacy of

staurosporine on SIK1 was estimated by measuring its

IC50 as regards the kinase activity The in vitro IC50

was
0.15 nm (Fig S1B) To evaluate SIK1 inhibition

in vivo, the difference between forskolin-induced

CRE-reporter activity and its activity in the presence of

SIK1 S577A was used An in vivo IC50 of

staurospo-rine against the exogenously expressed SIK1 S577A

mutant was
5.0 nm (Fig S1C) These results

sugges-ted that the kinase activity of endogenous SIK might

be inhibited by staurosporine at a dose as low as that against PKC

To examine whether staurosporine-induced dephos-phorylation of TORC2 was accompanied by its nuclear accumulation, GFP–TORC2 was expressed in HeLa cells in the presence or absence of the LKB1–SIK cas-cades, and the cells were treated with staurosporine (Fig 10D) Staurosporine inhibited the nuclear export

of GFP–TORC2 in all the cases tested These results, however, might indicate two possibilities, namely that staurosporine either inhibited SIKs directly or blocked the upstream cascades of SIKs, including LKB1

To clarify this point, COS-7 cells that had been expressing GST-tagged SIK1 were treated with sta-urosporine and GST–SIK1 protein was purified (Fig 10E) SIK1 enzyme purified from the staurospo-rine-treated cells was phosphorylated at Thr182 but did not show any kinase activities Because Thr172 of AMPKa1 (corresponding to Thr182 of SIK1) is also phosphorylated by LKB1, we performed the same experiment using GST–AMPKa1 (lower left) Neither the phosphorylation level at Thr172 nor the kinase activity of AMPKa1 was affected by staurosporine treatment Forskolin treatment did not alter the levels

of Thr phosphorylation or the kinase activity of SIK1

C

D

Fig 9 Ser577 is an autophosphorylation site of SIK1 (A) HeLa cells were transformed with the expression plasmid, pEBG-SIK1 for GST-fusion SIK1 (full-length), then transfected with the lacZ- or LKB1-adenovirus After 48 h incubation, the cells were treated with forskolin (20 l M ) for 30 min, and the GST–SIK1 protein was purified using a glutathione column (CP) The SIK1 protein was subjected to western blot analyses as well as kinase assays as described in Fig 7 (B) HeLa cells were transformed with the GFP–SIK1 expression plasmid with the LKB1- or the PKA-expression plasmid as described in Fig 2 (C) Mutant GFP–SIK1, T182A or S577A, was expressed in LKB1-expressing HeLa cells with or without PKA (D) COS-7 cells were transformed with the GST–TORC2 expression plasmid in the presence of the SIK1 expression plasmid, wild-type or S577A After 48 h incubation, the cells were treated with forskolin (20 l M ) for 1 h, and then the GST– TORC2 protein was purified.