A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that o
Trang 1Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm
Yoshiko Katoh1,*, Hiroshi Takemori1, Li Min1, Masaaki Muraoka1,3, Junko Doi4, Nanao Horike1
and Mitsuhiro Okamoto1,2
1
Department of Biochemistry and Molecular Biology, Graduate School of Medicine (H-1) and2Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, Japan;3ProteinExpress Co., Ltd, Choshi, Chiba, Japan;
4
Department of Food and Nutrition, Senri Kinran University, Osaka, Japan
Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577
by protein kinase A in adrenocorticotropic
hormone-sti-mulated Y1 cells, and the phospho-SIK1 translocates
from the nucleus to the cytoplasm The phospho-SIK1 is
dephosphorylated in the cytoplasm and re-enters the nucleus
several hours later By using green-fluorescent
protein-tag-ged SIK1 fragments, we found that a peptide region (586–
612) was responsible for the nuclear localization of SIK1
The region was named the RK-rich region because of its
Arg- and Lys-rich nature SIK1s mutated in the RK-rich
region were localized mainly in the cytoplasm Because SIK1
represses cAMP-response element (CRE)-mediated
tran-scription of steroidogenic genes, the mutants were examined
for their effect on transcription To our surprise, the
cyto-plasmic mutants strongly repressed the CRE-binding
pro-tein (CREB) activity, the extent of repression being similar to
that of SIK1(S577A), a mutant localized exclusively in the
nucleus Several chimeras were constructed from SIK1 and
from its isoform SIK2, which was localized mainly in the
cytoplasm, and they were examined for intracellular
local-ization as well as CREB-repression activity A SIK1-derived
chimera, where the RK-rich region had been replaced
with the corresponding region of SIK2, was found in the
cytoplasm, its CREB-modulating activity being similar to
that of wild-type SIK1 On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB-repressing activity similar to that of the wild-type SIK2 Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cyto-plasmic TORC2 entered the nucleus, activating CREB The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormone-induced nuclear entry of green fluorescent protein-fused TORC2 This suggests that SIK1 may regulate the intra-cellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression
Keywords: cAMP; CRE; nuclear localization signal; SIK; transcription repression
cAMP response element (CRE)-binding protein (CREB), a transcription factor involved in numerous physiological processes, regulates gene expression in a phosphorylation-dependent manner [1–3] Ser133, the major transcription activation site of CREB, is phosphorylated by protein kinase A (PKA) [4,5], p38 mitogen-activated protein kinase (MAPK) [6], mitogen- and stress-activated protein kinase 1 [7], pp90rsk[8], protein kinase B [9] and calcium-calmodulin-dependent kinase II/IV [10,11], whereas Ser142, the negative regulation site, is phosphorylated by calcium-calmodulin-dependent kinase II [12,13] The phosphorylation of Ser residues alters the affinity of CREB to CREB-binding protein and p300, and results in a change of the transcrip-tion efficiency [3,14–17] Several kinases, such as CPG16 [18] and leucine zipper protein kinase [19], even though they do not phosphorylate CREB directly, are known to modulate CRE/CREB activity
Salt-inducible kinase-1 (SIK1), a Ser/Thr protein kinase cloned from high-salt diet-fed rat adrenals [20], and also from PC12 pheochromocytoma cells induced by membrane
Correspondence to M Okamoto, Department of Biochemistry and
Molecular Biology, Graduate School of Medicine (H-1), Osaka
University 2-2 Yamadaoka, Suita, Osaka, 565-0871 Japan.
Fax: +81 6 6879 3289, Tel.: +81 6 6879 3280,
E-mail: mokamoto@mr-mbio.med.osaka-u.ac.jp
Abbreviations: ACTH, adrenocorticotropic hormone; b-ZIP, basic
leucine zipper domain; C, cytoplasm; CRE, cAMP-response element;
CREB, CRE-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole;
FITC, fluorescein-5-isothiocyanate; FRAP,
FKBP12-rapamycin-associated protein kinase; GFP, green fluorescent protein; GST,
glutathione S-transferase; HA, haemagglutinin; MAPK,
mitogen-activated protein kinase; MK5, MAPK-mitogen-activated protein kinase 5;
N, nucleus; NES, nuclear export signal; NLS, nuclear localization
signal; PKA, protein kinase A; SIK, salt-inducible kinase; TORC,
transducer of regulated CREB activity.
*Note: Yoshiko Katoh is a research fellow of the Japan Society for the
Promotion of Science.
(Received 9 June 2004, revised 26 August 2004,
accepted 21 September 2004)
Trang 2depolarization [21], is a member of the sucrose
nonfer-menting-1 protein kinase/AMP-activated protein kinase
family [22–26] SIK1 was found to be expressed in
adreno-cortical cells at an early phase of the adrenocorticotropic
hormone (ACTH) stimulation [20,27] When overexpressed
in Y1 cells, SIK1 repressed the expression of steroidogenic
genes, such as side-chain cleavage cytochrome P450 and
steroidogenic acute regulatory protein [27,28] Promoter
analyses of these genes indicated that the CREs in these
promoters were the sites for SIK1-mediated transcriptional
repression and that SIK1 might repress CREB activity
[28,29] Although SIK1 seemed not to phosphorylate CREB
directly, it repressed CREB in a kinase activity-dependent
manner The results of studies with several Gal4–fusion
CREBs suggested that SIK1 repressed CREB by acting on
the basic leucine zipper (b-ZIP) domain of CREB [29]
SIK1 is found mainly in the nucleus of resting Y1 cells In
ACTH-treated cells, SIK1 is phosphorylated at Ser577 by
PKA, and the phospho-SIK1 moves to the cytoplasm within
10 min The phospho-SIK1 is then dephosphorylated, and
SIK1 re-enters the nucleus several hours later A period when
SIK1 was not present in the nucleus appeared to coincide
with that when the SIK1-dependent CREB-repression was
not detected A mutant SIK1 (having Ala577) was
exclu-sively present in the nucleus, and its CREB-repression
activity was higher than that of the wild-type SIK1 [28,30]
Based on these results, we conclude that the nuclear presence
of SIK1 is important for the repression of CREB
The recent isolation and characterization of an
adipose-specific isoform, SIK2 [31], forced us to reconsider the
relationship between intracellular localization and
CREB-repression activity of SIKs SIK2 is localized mainly in the
cytoplasm of mouse 3T3-L1 preadipocytes but represses
CREB activity, although the degree of repression by SIK2
seems to be lower than that by SIK1 Therefore, we decided,
first, to identify a domain(s) that determined the
intracel-lular localization of SIK1 and, second, to examine its role in
the CREB repression Our results indicated that a short
peptide stretch (comprising residues 586–612), the RK-rich
region, was important for the nuclear localization of SIK1
Surprisingly, the RK-rich region-defective SIK1 mutants,
seen mainly in the cytoplasm, were able to repress the
CREB-mediated transcription strongly Moreover,
chime-ras constructed from SIK1 and SIK2, having their RK-rich
region and the corresponding region exchanged between the
two enzymes, showed no correlation between their nuclear
localization and CREB-repression activities On the other
hand, both the nuclear and cytoplasmic SIK1 mutants
inhibited the ACTH-induced nuclear entry of a
CREB-specific co-activator, TORC2 (transducer of regulated
CREB activity 2) We therefore concluded that SIKs, even
present in the cytoplasm, could repress CREB-mediated
gene expression, and the RK-rich region of SIK1 was
important for not only the nuclear localization, but also the
attenuation of CREB-repression activity
Materials and methods
Cell culture and reporter assay
Y1 cells were maintained in DMEM (Dulbecco’s modified
Eagle’s medium) (Sigma), containing 10% (v/v) fetal bovine
serum and antibiotics, at 37C under an atmosphere of 5%
CO2/95% air 3T3-L1 cells, obtained from Japan Health Sciences Foundation (Osaka, Japan), were maintained in DMEM, as described previously [31] 4¢,6-Diamidino-2-phenylindole (DAPI) dilactate was from Molecular Probes, and fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG was from Funakoshi (Tokyo, Japan)
The method of reporter assays was as described previ-ously [29] To introduce plasmids into cells, Lipofect-AMINE 2000 (Invitrogen Corp., Carlsbad, CA, USA) was used in this study Luciferase activities were measured by using the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA) For the CRE-reporter assay, Y1 cells (1· 105per well) were transfected with the SIK1 expression plasmid (pIRES-SIK1, pIRES-SIK1 mutants or pIRES empty vector: 0.2 lg), CRE-luciferase reporters [pTAL-CRE or pTAL (empty reporter) alone: 0.2 lg], the PKA expression plasmid (pIRES-PKA or pIRES: 0.1 lg) and pRL-SV40 (internal standard: 0.03 lg) For the CREB reporter assay, cells were transformed with Gal4 DNA-binding domain-linked CREB expression vectors [pM-CREB(F), pM-CREB(S) or pM, empty vector: 0.15 lg], expression vectors for SIK1 mutants or empty expression vectors (pIRES-SIK1 or pIRES: 0.2 lg), the PKA expres-sion vector or empty vector (pIRES-PKA or pIRES: 0.1 lg), and reporter vectors [GAL4-linked luciferase reporter (pTAL-5x GAL-4: 0.15 lg) and an internal control (pRL-SV40: 0.03 lg) Transformation efficiencies were corrected by Renilla luciferase activities The specific tran-scriptional activities derived from the CRE and CREB were expressed as fold-expression of the reporter activity of the empty vector, pTAL and pM, respectively
For fluorescence microscopy observations, cells were cultured on poly-L-lysine coated coverslips (18 mm; Matsunami Co Ltd, Tokyo, Japan) in a 12-well dish Cells (< 1· 104) were transformed with 0.5 lg of GFP-SIK1 expression vector, incubated for 16 h and stimulated with or without ACTH (10)6M) for 1 h, fixed with 1 mL of 4% paraformaldehyde dissolved in NaCl/Pifor 15 min, stained with DAPI [1 ngÆmL)1in NaCl/Picontaining 0.01% (v/v) Triton X-100] for 5 min, and then washed with NaCl/Pifour times Cells on the coverslip were embedded onto a glass slide using 50% (v/v) glycerol On average, 20–30% of the cells showed detectable GFP-SIK1 signals in independent triplicate experiments The majority of localization patterns
of GFP-SIK1 was classified according to whether it was found extensively in the nucleus (N), was present at a higher level in the nucleus (N > C), was evenly distributed between the nucleus and the cytoplasm (N¼ C), was present at a higher level in the cytoplasm (N < C) or was found extensively in the cytoplasm (C), for more than 200 cells
To visualize haemagglutinin (HA)-tagged SIK1, rat anti-HA tagged IgG (Boehringer Mannheim Biochemicals Inc., Mannheim, Germany) and FITC-conjugated anti-rat IgG were used The transformation efficiency of GFP-tagged, as well as HA-tagged, proteins was always constant ( 20%) Plasmids
cDNA fragments for rat SIK1 [27] and mouse SIK2 [31] were cloned into the green fluorescent protein (GFP) expression vector, pEGFP-C [28] For the reporter assay, the nontagged
Trang 3expression vector, pIRES (B-E), was used The original
pIRESneo1 vector (Clontech Laboratories Inc., Palo Alto,
CA, USA) contained, in its cloning region, EcoRI and
BamHI sites in a reverse order to that of the pEGFP-C
vector So, BamHI and EcoRI sites in this order (B-E) were
created using oligonucleotides at the NotI/BamHI site of
vector pIRESneo1 The oligonucleotides used were 5¢-GGC
CGGATCCGAATTC and 5¢-GATCGAATTCGGATCC
cDNA fragments of C-terminal deletion SIK1s (1–740,
1–708, 1–632, 1–572, 1–341) were amplified by PCR using a
common forward primer (5¢-AAAGGATCCATGGTGA
TCATGTCGGAGTTC: the BamHI site is indicated by
the underlined region) and each specific reverse primer
(740R: 5¢-AAAGAATTCCTGTGGCAGGGGACCAGT
GG; 708R: 5¢-AAAGAATTCGGGCTGGAGGAGGGG
CGTTG; 632R: 5¢-AAAGAATTCCGGGGTGTGGAAG
GTACTCA; 572R: 5¢-AAAGAATTCCTCCTGGAAGC
TGACAGG; 341R: 5¢-AAAGAATTCGAGCAGGAGG
TAGTAAAT; the EcoRI site is indicated by the underlined
region) Similarly, N-terminal-deleted cDNA fragments were
prepared by a common reverse primer (5¢-AAAGAATTC
TCACTGTACCAGGACGAACGTCC: the EcoRI site is
indicated by the underlined region) and specific forward
primers (24F: 5¢-AAAGGATCCGTGGGCTTTTACGAC
GTGGA; 163F: 5¢-AAAGGATCCATCAAGCTGGCAG
ATTTTGGA; 342F: 5¢-AAAGGATCCGAGCGCCTCA
GGGAGCATCGA; 571F: 5¢-AAAGGATCCCAGGA
GGGACGGAGAGCG; 632F: 5¢-AAAGGATCCCCGG
CCCCAAGCTCAGGTCTG; the BamHI site is indicated
by the underlined region) Amplified cDNA fragments were
digested by BamHI/EcoRI and ligated into the BamHI/
EcoRI site of vector pEGFP-C To prepare internal deletion
mutants, cDNA fragments of the N-terminal regions and of
the C-terminal regions were separately amplified by PCR
and then ligated to these fragments To amplify
frag-ments containing residues 1–572, 1–585 and 1–612, the
common forward primer and specific reverse primers
(572R: 5¢AAAGGGCCCCTCCTGGAAGCTGACAGG;
CG; 612R: 5¢AAAGGGCCCACGGGCCAATCCTTT
GATCTTGTTCAG; the Bsp120I site is indicated by the
underlined region) were used cDNA fragments containing
residues 586–776, 613–776 and 632–776 were amplified
by using the common reverse primer and specific primers
613F: AAAGGGCCCCAGGTGTGCCAGTCCTCCATC;
632F: AAAGGGCCCGCCCCAAGCTCAGGTCTG;
the Bsp120I site is indicated by the underlined region) The
cDNA fragments for the N-terminal regions and the
C-terminal regions were digested with BamHI/Bsp120I
and by EcoRI/Bsp120I, respectively, mixed at a ratio of
1 : 1, and ligated into the BamHI/EcoRI site of vector
pEGFP-C
cDNA fragments encoding the RK-rich region were
amplified by PCR by using the BamHI-linked 586F primer
and EcoRI-linked 612R primer, digested by BamHI/EcoRI
and ligated into the BamHI/EcoRI site of vector pEGFP-C
GFP expression vectors, having T-antigen nuclear
localiza-tion signal (NLS) and Rev-nuclear export signal (NES)
sequences, were prepared by direct ligation of
oligonucleo-tides at the NotI site of the pEGFP-C vector T-antigen
NLS oligonucleotides were 5¢-GGCCATTCAAAGTA
oligonucleotides were 5¢-GGCCTCTGCAGCTCCCGC CACTGGAACGTCTTACCCTCGACA and 5¢-GGCC TGTCGAGGGTAAGACGTTCCAGTGGCGGGAG CTGCAGA T-antigen NLS- and Rev-NES-inserted D586-612 SIK1 mutants were prepared by ligation of the above sets of oligonucleotides in the Bsp120I site of D586-612
Site-directed mutagenesis was carried out by using a kit, GeneEditor (Promega), according to the manufacturer’s protocol The following primers were used: SIK-1 (R593A/
CGAGGACCAAG; SIK-1 (R597A/K599A), 5¢-CTAA GGAAAAACGCGGCGACCGCGGGGTTCCTGGGA CTG); SIK-1 (L602A/L604A), 5¢-AGGACCAAGGGG
CGCCGGCTTGGCCCGTCAGGTG); SIK-1 (I607A/ L610A), 5¢-CTGGGACTGAACAAGGCCAAAGGCGC CGCCCGTCAGGTGTGC)
To prepare chimeric SIK1 and SIK2 mutants whose domain 3Bs were exchanged, the Quick change Site-directed Mutagenesis Kit (Stratagene) was used Because the cDNA fragment of the domain 3B was too long to perform site-directed mutagenesis, it was separated into 5¢ and 3¢ regions, and the mutagenesis was performed on the respective fragments The 5¢ fragments of the domain 3B from SIK1 and SIK2 were mutated with 5¢-GATACGTCTCTCACT CAAGGGATTGTAGCCTTCCGGCAGCATCTACAG AATCTCGCGAGGACCAAGGGGTTCCTG/5¢-CAG GAACCCCTTGGTCCTCGCGAGATTCTGTAGAT GCTGCCGGAAGGCTACAATCCCTTGAGTGAGA GACGTATC and 5¢-GATACGTCCCTTACACAAG GACTTAAGGCATTTAGACAACAGCTTCGGAAG AATGCTAGAACCAAAGGATTTCTG/5¢- CAGAAA TCCTTTGGTTCTAGCATTCTTCCGAAGCTGTTG TCTAAATGCCTTAAGTCCTTGTGTAAGGGACG TATC, respectively The 3¢ fragments of domain 3B from SIK1 and SIK2 were mutated with 5¢-GCGAGG ACCAAGGGGATTCTGGAGCTGAACAAGGTGC AATTGTTGTACGAACAGGTGTGCCAGTCCTCC/ 5¢-GGAGGACTGGCACACCTGTTCGTACAATAA TTGCACCTTGTTCAGCTCCAGAATCCCCTTTGGC CTCGC and 5¢-CTTGCTAGAACCAAAGGATTTCT GGGGTTGAACAAAATAAAAGGGCTGGCTCGGC AAATGGGATCAAACGCAGAC/5¢-GTCTGCGTTTG ATCCCATTTGTCGAGCCAGCCCTTTTATTTTGT TCAACCCCAGAAATCCTTTGGTTCTAGCAAG The mammalian expression vector, pCMVspor6, con-taining full-length mouse TORC2 (Clone ID: 5345301) cDNA, was purchased from Invitrogen Site-directed muta-genesis was used to introduce a BglII site at the 5¢ terminus
of the cDNA The following primers were used: mTORC2 Bgl II-F, 5-GGCGGGGACGGACGCGGGAGATCTA TGGCGACGTCAGGG; mTORC2 Bgl II-R, 5-CCC TGACGTCGCCATAGATCTCCCGCGTCCGTCCCC GCC The resultant full-length mouse TORC2 cDNA was digested with BglII and NotI, and ligated into the BamHI/ NotI site of vector pEGFP-C
Trang 4Immunoprecipitation was performed as described
previ-ously [27] Briefly, cells (5· 105) plated on a 10 cm dish were
transformed with 3 lg of expression plasmids (pSVL-HA)
for HA-tagged wild-type and mutant SIKs using 8 lL of
LipofectAMINE 2000 After 36 h of incubation, cells were
lysed in 0.7 mL of lysis buffer [50 mMTris/HCl (pH 8.0),
300 mMNaCl, 5 mMEDTA, 5 mMEGTA, 2 mM
dithio-threitol, 50 mM b-glycerol phosphate, 50 mMNaF, 1 mM
NaVO4, 0.5% Triton X-100, and protease inhibitor]
HA-tagged SIK protein was immunoprecipitated by using
anti-HA-tag IgG (2 lg) and protein G-Sepharose (30 lL) The
aliquots of immunopurified SIK1 were subjected to Western
blot analyses with anti-SIK1 IgG and in vitro kinase assays
Purified SIKs were incubated with glutathione S-transferase
(GST)-Syntide2 in the presence 0.5 lCi (18.5 kBq) of
[32P]dATP[cP] at 30C for 30 min The kinase reaction
was stopped by adding 3· SDS sample buffer [150 mMTris/
HCl (pH 6.8), 6% (v/v) SDS, 30% (v/v) glycerol, and 0.1%
(v/v) bromophenol blue] and heating at 100C for 5 min
The aliquots were subjected to 15% SDS/PAGE, and
phosphorylated peptides were visualized by
autoradio-graphy
Results
Subcellular distribution of truncated SIK1s
By using the GFP-fusion technique, we previously reported
the phosphorylation-dependent nuclear export of SIK1 in
ACTH-stimulated Y1 cells [28] When SIK1 was present in
the nucleus, it was able to repress CREB activity However,
a cytoplasmic isoform of SIK, SIK2 [31], forced us to
reconsider correlation between the loss of CREB-repressing
activity and the cytoplasmic localization of SIK1 To
address to this problem, we decided to isolate SIK1
mutants, localized essentially in the cytoplasm, by
modify-ing a region determinmodify-ing the nuclear localization of SIK1
When GFP-fused full-length SIK1 (i.e SIK1 with GFP at
its N terminus) was expressed in unstimulated Y1 cells, the
majority of GFP signals formed speckles in the nucleus, and
the minority diffused into the cytoplasm (the left column in
Fig 1A) The nuclear GFP–SIK1, however, was completely
translocated to the cytoplasm after treatment with ACTH
(the right column in Fig 1A) When GFP alone was
expressed, the green fluorescence was present more
exten-sively in the nucleus than the cytoplasm, with or without
ACTH Although the GFP fusion technique would yield
rapid results regarding the distribution of target molecules,
we must consider, as a caveat, that the GFP protein tends to
accumulate in the nucleus In any case, in the hope of
determining domain(s) important for the intracellular
localization, we first attempted to investigate the
intracel-lular distribution of a number of GFP–SIK1 mutants with
N- and C-terminal deletions Figure 1B illustrates areas in
the primary sequence of SIK1 that altered the intracellular
distribution of GFP–SIK1 fragments
The distribution of SIK1(1–740) was similar to that of the
full-length SIK1 When the region containing residues 709–
776 was deleted, the resultant SIK1(1–708) was localized
only in the nucleus of Y1 cells and was not translocated to
the cytoplasm, even when the cells were stimulated with ACTH Similar results were obtained with SIK1(1–632) From these results, we surmise that the region containing residues 708–740 was important for the nuclear export of SIK1 When the C-terminal deletion reached residue 573, part of SIK1(1–572) was again found in the cytoplasm Similar results were obtained for SIK1(1–341) These results suggest that the region containing residues 572–632 might
be important for the nuclear localization of SIK1 Consid-ering that proteins of < 40–60 kDa molecular mass would enter the nucleus passively [32,33], the nuclear presence of GFP-SIK1(1–572) and GFP-SIK1(1–341), having mole-cular masses of 90 kDa and 66 kDa, respectively, suggest the presence of minor nuclear localization activities in the N-terminal fragment (1–572)
Next, the subcellular distribution of N-terminus-deleted mutants was investigated The distribution of SIK1(24–776) was similar to that of full-length SIK1 On the other hand, SIK1(163–776) and SIK1(342–776) were localized only in the nucleus, and they failed to move to the cytoplasm in response to ACTH SIK1(571–776) was localized exclu-sively in the nucleus, suggesting the presence of an active nuclear localization signal in the region comprising residues 571–776 When the N-terminal deletion reached residue 631, the resultant SIK1(632–776) was diffusely distributed all over the cell Taken together, these results indicate that
a major nuclear localization signal might exist in region 573–631
The region 573–631 does not contain a cluster of more than three successive basic residues, a feature often seen in typical nuclear localization signals, such as the unipartite basic cluster KKKRK of SV40 (simian virus 40) T-antigen [34] and the bipartite cluster RKR-Xn-RKRKR of T-cell protein tyrosine phosphatase [35] However, close examina-tion revealed the presence in the region of a peptide stretch, Lys586-Ala-Phe-Arg-Gln-Gln-Leu-Arg-Lys-Asn-Ala-Arg- Thr-Lys-Gly-Phe-Leu-Gly-Leu-Asn-Lys-Ile-Lys-Gly-Leu-Ala-Arg612 (basic residues shown in bold), in which basic and hydrophobic residues were interspersed We named this stretch the RK-rich region (586–612) It should be noted that Ser577, an important residue for the phosphorylation-dependent nuclear export of SIK1, exists near the RK-rich region
Region 586–612, the RK-rich region, determines the intracellular localization of SIK1
To investigate further the role(s) played by the region 573–
631 for determining the intracellular distribution of SIK1,
we created several mutants with deletions in this region Noting that the RK-rich region (586–612) was positioned
in the centre of 573–631, we decided to split the region 573–631 into three parts – an N-terminal region (573–585), the RK-rich region (586–612), and a C-terminal region (613–631) – and produced four deletion mutants, shown in Fig 2A
SIK1(D573–631), a mutant lacking the entire region, was distributed diffusely all over the cell with no response to ACTH SIK1(D573–585) was localized specifically in the nucleus with a weak response to ACTH However, SIK1(D586–612) (i.e the mutant minus the RK-rich region) was present mainly in the cytoplasm of resting Y1 cells,
Trang 5although found to a minor extent in the nucleus, and was
translocated completely to the cytoplasm after stimulation
with ACTH SIK1(D613–631) behaved similarly to
wild-type SIK1 Whether the RK-rich peptide alone has nuclear
translocation activity was tested by using the GFP-fused
RK-rich peptide As shown in Fig 2B, GFP-SIK(586–612)
was localized only in the nucleus (Note that GFP alone was
distributed diffusely all over the cell, as shown in Fig 1A.)
In the control experiments, the GFP-linked SV40 T-antigen
nuclear localization signal (NLS), KKKRK (basic residues in
bold) [34] was found only in the nucleus, while the
GFP-linked HIV-1 Rev protein nuclear export signal (NES),
LGLPPLERLTLD (hydrophobic residues in bold) [36],
was found only in the cytoplasm These results indicated
that the RK-rich region might be important for the nuclear
localization of SIK1
Basic residues in the RK-rich region (586–612) were
replaced with Ala, and the intracellular distribution of
mutants was examined (Fig 3A) SIK1(R593A/K594A),
SIK1(R597A/K599A) and SIK1(K606A/K608A) were
found mainly in the cytoplasm of resting Y1 cells When
the cells were treated with ACTH, those mutants were translocated to the cytoplasm These results suggested that the basic residues in the RK-rich region might be important for the nuclear localization of SIK1 Next, bulky hydrophobic residues, such as Leu and Ile, were replaced with Ala, and the intracellular distribution of mutants was investigated (Fig 3A) SIK1(L602A/L604A) was distri-buted diffusely both to the nucleus and to the cytoplasm
of resting Y1 cells On the other hand, SIK1(I607A/L610A) was localized mainly in the cytoplasm These findings suggest that these hydrophobic residues, too, might have an important role for the nuclear localization
Based on the above findings, we attempted to prepare SIK1 mutants that would be predicted to localize exclusively either in the nucleus or in the cytoplasm Hence, either canonical SV40 T-antigen NLS or Rev NES was inserted into the deleted part of SIK1(D586–612) (Fig 3B) SIK1(D586–612 + NLS) was accumulated more in the nucleus than the parent SIK1(D586–612), and, when the cells were stimulated with ACTH, SIK1(D586–
612 + NLS) was translocated to the cytoplasm (Fig 3A)
A
B
Fig 1 Intracellular distribution of green
fluorescent protein (GFP)-fusion salt-inducible
kinase 1 (SIK1) mutants (A) Y1 cells, cultured
on coverslips and transformed with
over-expression vectors for GFP-tagged SIK1
protein, were treated with or without
adrenocorticotropic hormone (ACTH)
(10)6M ) for 1 h and fixed for
fluorocyto-chemical analyses Green fluorescent signals of
GFP-SIK1 (upper), and blue fluorescent
sig-nals representing nuclear staining with
4¢,6-diamidino-2-phenylindole (DAPI) (middle),
are shown The intracellular localization of
GFP alone is shown in the bottom panels The
patterns of the intracellular distribution of
green fluorescent signals were classified into
five groups (N, N > C, N ¼ C, N < C and
C), as described in the Materials and methods,
and representative pictures are shown.
(B) C-terminal- and N-terminal-deleted SIK1
mutants were expressed in Y1 cells The
wild-type SIK1 (Full) contains amino acids 1–776.
The kinase domain (27–278) is in the
N-terminal half, whereas the region essential
for nuclear localization (573–631) is in the
C-terminal half The Arg/Lys-rich region
(RK-rich region) is shown as a black box.
Trang 6By contrast, SIK1(D586–612 + NES) was found
exclu-sively in the cytoplasm of both resting and ACTH-treated
Y1 cells Thus, the T-antigen NLS could be replaced with
the RK-rich region as the nuclear localization signal, while
the Rev NES, if inserted into SIK1(D586–612), could
function as the nuclear export signal
We also produced an unphosphorylatable SIK1(D586–
612) mutant, in which Ser577 was replaced with Ala As
expected, the nuclear accumulation of SIK1(D586–
612 + S577A) was higher than that of parent mutant
SIK1(D586–612) and was influenced a little by ACTH
treatment On the other hand, SIK1(S577A), the nuclear
export-defective mutant, was localized only in the nucleus,
as reported previously [28] A comparison between the
intracellular distribution of SIK1(D586–612/S577A) and
that of SIK1(S577A) again highlights the importance of the
RK-rich region as the nuclear localization signal
The RK-rich region of SIK1 and the corresponding region
of SIK2 determine the intracellular localization
The above results suggest that the intact RK-rich region is
an important determinant for the nuclear localization of
SIK1 Before proceeding to investigate the
CREB-repress-ing activity of these cytoplasmic SIK mutants, we focused
our attention on SIK2, an adipose-specific isoform of
SIK1 SIK2, having a similar, but distinct, amino acid
sequence in a region corresponding to the RK-rich region
(Fig 4A,B), was localized mainly in the cytoplasm of
3T3-L1 mouse preadipocytes [31,37] Alignment of the two
isoforms indicates that they have three highly conserved
domains (Fig 4A) Protein kinase structures are present in
domain 1, and the function of domain 2 remains to be
explored Domain 3 shares 73% identical amino acid
residues between the two isoforms Domain 3A, the
N-terminal half, contains a PKA-dependent
phosphory-latable Ser (Ser577 in SIK1 and Ser587 in SIK2, respectively), whereas domain 3B, the C-terminal half, corresponds to the RK-rich region and its equivalent (Fig 4B) The similarity of domain 3B between the two isofoms is lower than that of domain 3A Notably, five of nine basic residues present in the RK-rich region are replaced with other residues in the corresponding region
of SIK2 To examine whether or not the lower similarity
in domain 3B between the two isoforms contributes to the difference in their intracellular distribution, two chimeras, each having its domain 3B replaced with that of the other isoform, were constructed and expressed in Y1 cells (Fig 4C) Chimera 1, SIK1, in which RK-rich region had been replaced with domain 3B of SIK2, was localized less
in the nucleus than the wild-type SIK1 (see the difference between the first and second panels in Fig 4C) On the other hand, Chimera 2, SIK2, in which domain 3B had been replaced with the RK-rich region, was localized more
in the nucleus than the wild-type SIK2 (see the difference between panels 4 and 5 in Fig 4C) The treatment of Y1 cells with ACTH seemed to induce the nuclear export of these chimeras When Ser577 of Chimera 1 was mutated
to Ala, a substantial amount of the mutant was retained
in the nucleus after treatment with ACTH (see the difference between panels 2 and 3 in Fig 4C) In contrast, the similar SerfiAla mutation in Chimera 2 seemed not to influence the intracellular distribution of Chimera 2 (see the difference between panels 5 and 6 in Fig 4C) These results indicate that the RK-rich region, domain 3B of SIK1, plays an important role in the nuclear localization
of SIK1, but not for domain 3B of SIK2
Although several mutants, such as D573–585 (Fig 2A), I607A/L610A (Fig 3A), D586–612 + S577A (Fig 3B), Chimera 1 + S577A (Fig 4C) and Chimera 2 + S587A (Fig 4C), did not give unequivocal results as to the intracellular localization as well as the ACTH-dependent
B
A
Fig 2 The RK-rich region is essential for the nuclear localization of salt-inducible kinase 1 (SIK1) (A) An overall structure of green fluorescent protein (GFP)-SIK1 is shown at the top Ser577 is the residue that is phos-phorylated by protein kinase A (PKA) when the cells are stimulated The intracellular distribution of SIK1 mutants containing the deletion in the RK-rich region were investi-gated as described in the legend to Fig 1 and
in the Materials and methods (B) The GFP-fused RK-rich region nuclear localization sig-nal (NLS) (KKKRK) of SV40 (Simian virus 40) T-antigen, and nuclear export signal (NES) (LGLPPLERLTLD) of the HIV-1 Rev protein were expressed, and their intracellular distribution were investigated.
Trang 7intracellular translocation, we decided to investigate the
relationship between the intracellular localization and
CREB repression activity of SIK1 mutants
Even SIK1 mutants present in the cytoplasm repress
PKA-induced CRE activity
As reported previously, SIK1(S577A) was retained in
the nucleus, even after the activation of PKA, and its
transcriptional repression activity was higher than wild-type
SIK1, indicating that SIK1 present in the nucleus could
repress CREB activity [28] We decided to test the
transcriptional repression activity of SIK1(1–708), another
mutant localizing exclusively in the nucleus (shown in
Fig 1B) The SIK1(1–708) expression plasmid,
pIRES-SIK1(1–708), was co-introduced with the CRE–reporter
construct into Y1 cells in the presence or absence of the
PKA expression vector, and the SIK1-dependent repression
of CRE activity was examined As expected, the extent of
CRE repression by SIK1(1–708) was greater than that
induced by wild-type SIK1 (Fig 5A) The protein kinase
activity was required for SIK1(1–708) to exert this
repres-sion, because the repression was not seen by SIK1(K56M/
1–708) We further tested the CRE-repression activities of a
variety of other mutants, whose intracellular localizations
were examined in Fig 3B To our surprise, all the mutants
seemed to inhibit CRE activity more strongly than wild-type
SIK1 (Fig 5B) SIK1(D586–612 + NES), which was localized exclusively in the cytoplasm (Fig 3B), could also repress CRE activity as strongly as the nuclear resident SIK1(S577A) These results suggest that the nuclear localization of SIK1 might not be a prerequisite for its CRE repression
By using Gal4–CREB reporter systems, we previously demonstrated that SIK1 repressed PKA-induced CREB activation by acting on the b-ZIP domain of CREB [29], and the nuclear resident SIK1(S577A) repressed CREB more strongly than the wild-type SIK1 [28] Similar experiments were designed to test whether the b-ZIP domain of CREB was the target site for the repressive action of the cytoplasmic SIK1 As shown in the left panel
of Fig 5C, the cytoplasmic SIK1(D586–612 + NES) and the nuclear resident SIK1(S577A) similarly repressed the full-length CREB-dependent reporter activity On the contrary, neither mutant repressed the reporter activities
of b-ZIP-less CREB (the right panel of Fig 5C) The other cytoplasmic SIK1 mutants, such as SIK1(D586–612) and SIK1(D573–631), were also able to repress the full-length CREB-dependent reporter activity (data not shown) To verify the above results, the expression levels of exogenous mutant SIK1 protein and their kinase activity were exam-ined (Fig 5D) The results showed no significant difference
in their expression levels These results suggest that SIK1, when present in the cytoplasm, is able to repress the
A
B
Fig 3 Effect of alteration of the RK-rich
region on the intracellular distribution of
salt-inducible kinase 1 (SIK1) (A) Effect of
site-directed mutagenesis in the RK-rich
re-gion on the intracellular distribution of SIK1.
Basic and hydrophobic residues, indicated by
underlines and asterisks, respectively, were
mutated to Ala The intracellular distribution
of mutant SIKs was investigated as detailed in
the legend to Fig 1 (B) The RK-rich region
(586–612) was replaced with a nuclear
local-ization signal (NLS) of T-antigen or with a
nuclear export signal (NES) of the Rev
pro-tein To disrupt the nuclear export, mutation
S577A was introduced into wild-type SIK1
and SIK1(D586–612) These mutants were
expressed as GFP-fusion proteins, and their
intracellular distribution was examined as
detailed in the legend of Fig 1.
Trang 8PKA-induced CREB-activation through the b-ZIP domain
of CREB We also confirmed that the size of the tag
(GFP or HA) did not influence the intracellular distribution
of SIK1 and its mutants The intracellular distribution
of HA-tagged SIK1 and its mutants, S577A (nucleus) or
D586–612 + NES (cytoplasm), showed similar features to
those of their GFP-tagged counterparts (Fig 3B)
The RK-rich region and the corresponding region
of SIK2 modulate the CRE-repression activity
The chimeras produced from the two SIK isoforms were
tested for their CRE repression activity in Y1 cells Chimera
1, localizing mainly in the cytoplasm (Fig 4C), repressed
CRE activity to an extent similar to that of wild-type SIK1
(Fig 6A) On the other hand, Chimera 1, having an S577A
mutation, was a strong repressor To our surprise, both
Chimera 2 and wild-type SIK2 elevated, not repressed, the
PKA-dependent enhanced CRE activity (Fig 6B), whereas
Chimera 2, having an S587A mutation, was a strong
repressor Because we previously reported that wild-type
SIK2 repressed, not elevated, CRE activity in 3T3-L1 cells
[31], the experiments with Chimera 2 and wild-type SIK 2
were repeated by using 3T3-L1 cells Both Chimera 2 and
wild-type SIK2 repressed the CRE activity to a moderate
extent in 3T3-L1 cells
The kinase activities of SIKs were essential for their
CRE-repression activities, and some kinase-defective mutant
SIKs [SIK1 (K56M) and SIK2 (K49M)] have been shown
to influence the CRE activity in a dominant-negative
manner [31] To exclude the possibility that the enhance-ment of CRE activity by SIK2 in Y1 cells was a result of lower kinase activity, GST-tagged SIK1 and SIK2 were expressed in Y1 cells, purified using glutathione columns and subjected to an in vitro kinase assay The specific kinase activity of SIK2 in Y1 cells was similar to that of SIK1 Although we cannot presently discuss the molecular mech-anism underlying the up-regulation of CRE activity by SIK2 in Y1 cells, the RK-rich region of SIK1, and the corresponding region of SIK2, may play a critical role in modulating the CREB-repression activity of SIKs
Both nuclear and cytoplasmic SIK1s inhibited the ACTH-induced nuclear entry of TORC2 TORCs, ubiquitously expressed in a variety of cells, are CREB-specific co-activators, essential for both basal and induced CREB activity [38,39] Recently, cAMP-induced nuclear import of TORC2 was found in insulinoma cells [40], suggesting that the nucleo-cytoplasmic shuttling
of TORC2 was important for the regulation of the co-activation function of TORC2 We also showed that SIK2 phosphorylated TORC2 at Ser171 and that the phospho-TORC2 could not enter the nuclei in forskolin-stimulated cells [40] These findings prompted us to examine whether or not the nuclear import of TORC2 could occur in the SIK1 mutant-expressing Y1 cells
As shown in Fig 7, GFP-fused TORC2 was localized both in the nucleus and in the cytoplasm of resting Y1 cells, ACTH(–) When the cells were treated with ACTH,
A
B
C
Fig 4 Difference between salt-inducible kinase
1 (SIK1) and SIK2 with special reference to domain 3 (A) Alignment of the primary sequence and the subcellular distribution of SIK1 (blue bar) and SIK2 (pink bar) Three highly conserved regions, domains 1–3, were depicted (B) Amino acid sequences of domain
3 are shown The protein kinase A (PKA)-dependent phosphorylation sites, Ser577 in SIK1 and Ser587 in SIK2, are indicated in bold The basic residues in domain 3B are indicated by underlines The similarity between SIK1 and SIK2 in domain 3A, the N-terminal half of domain 3, is higher than that in domain 3B, the C-terminal half (C) The intracellular distribution of chimeras
in Y1 cells.
Trang 9ACTH(+), GFP-TORC2 was found mostly in the nucleus,
indicating that the ACTH signalling induced the nuclear
import of TORC2, resulting in a TORC2-dependent CREB
activation When HA-tagged SIK1 was co-expressed with
GFP-TORC2 in these cells, most GFP-TORC2 signals were
localized in the cytoplasm In the ACTH-treated cells,
TORC2 seemed to move into the nucleus, although the
extent of the nuclear import seemed less than that in the
SIK1-nonexpressing control cells (–) When either the
nuc-lear resident mutant SIK1, S577A, or the cytoplasmic
mutant SIK1, D586–612 + NES, was co-expressed,
more TORC2 seemed to be retained in the cytoplasm, corroborating that these SIK1 mutants could constitutively repress CREB activity (Fig 5B)
Discussion
We reported previously that SIK1 could repress the CREB-mediated transcription activation in cultured cells, and the extent of CREB repression apparently correlated with the amount of SIK1 present in the nucleus [28] Based on the assumption that a certain region of the SIK1
A
C
D B
Fig 5 Both nuclear and cytoplasmic salt-inducible kinase 1 (SIK1) mutants repress cAMP-response element (CRE)/CRE-binding protein (CREB) activities (A) The protein kinase A (PKA)-induced CRE activity, and its repression by C-terminal-deleted nuclear SIK1, DC(708), and its kinase-defective mutant, K56M/DC(708), were investigated by using Y1 cells Y1 cells (1 · 10 5 /well) were transfected with SIK1 expression plasmid, CRE-luciferase reporter, PKA expression plasmid and pRL-SV40 (internal standard), as described in the Materials and methods After 15 h of incubation, cells were harvested for luciferase assay The specific transcriptional activities derived from the CRE were expressed as fold-expression
of the reporter activity of the empty vector, pTAL Mean values and SD are indicated (n ¼ 4) (B) The CRE activity of the RK-rich region mutants and S577A mutants (0.1 or 0.3 lg of SIK1-expression plasmid was used), as given in Fig 3B, were examined as described above Mean values and
SD are indicated (n ¼ 3) (C) The transactivation activities of full-length CREB [CREB (F): (left panel)] and basic leucine zipper domain (b-ZIP) minus CREB [CREB (S): (right panel)] were investigated by using the Gal4 DNA-binding domain-linked assay Cells were transformed with the Gal4 DNA-binding domain-linked CREB expression vectors, described above, the expression vectors for SIK1, and PKA and reporters (GAL4-linked luciferase reporter pTAL-5x GAL4 and internal standard pRL-SV40) The specific transcriptional activities of CREB were expressed as fold-expression of the empty Gal4 vector, pM Mean values and SD are indicated (n ¼ 3) Grey bars indicate CREB activities in Y1 cells overexpressing kinase-defective SIK1s Cells were transformed with expression plasmids for haemagglutinin (HA)-tagged wild-type and mutant SIKs After 36 h of incubation, cells were lysed, and HA-tagged SIK protein was immunoprecipitated by using anti-HA-tag IgG and protein G–Sepharose, as described in the Materials and methods The aliquots of immunopurified SIK1 were subjected to Western blot analyses with anti-SIK1 IgG (upper panel) and in vitro kinase assays (lower panel) GST-Syntide2 was used for a substrate The results were representative of experiments carried out in duplicate.
Trang 10peptide is responsible for the nuclear localization of SIK1
and that this region may play a crucial role in the
regulation of the SIK1-mediated transcription repression,
we first tried to identify a nuclear localization signal of
SIK1 Second, by utilizing SIK1 mutants with a defective
nuclear localization signal, we examined the relationship
between the nuclear localization and the transcription
repression activity of SIK1
The RK-rich region (586–612), containing nine basic
residues, was shown to act as a nuclear localization signal
(Figs 1–4) This region, however, was not typical of nuclear
localization signals [34,35,41–45] The importance of the
basic residues in the RK-rich region for the nuclear localization of SIK1 was also demonstrated by using chimeras of SIK1 and SIK2 (Fig 4) Hence, domain 3B
of SIK2, the region corresponding to the RK-rich region, has only four basic residues, and SIK2 was localized mainly
in the cytoplasm On the other hand, the SIK2-derived chimera, in which domain 3B was replaced with the RK-rich region, accumulated in the nucleus Conversely, the SIK1-derived chimera, containing domain 3B of SIK2, was localized to a significant extent in the cytoplasm The RK-rich region contains several hydrophobic resi-dues that intersperse in the peptide stretch, a feature typical
of the nuclear export signal (L-X2,3-L-X1,2-L-X-L) (hydro-phobic residues are shown in bold) [46–49] The experiments using SIK1 mutants with disrupted hydrophobic residues, especially those of SIK1(I607A/L610A), indicated the importance of these residues for the nuclear distribution
of SIK1
Using a variety of SIK1 mutants localized in the cytoplasm, we next examined CRE/CREB-repression activ-ity of these mutants The results indicated that the mutants, although localized mainly in the cytoplasm, could repress CRE-reporter activity more strongly than wild-type SIK1 (Fig 5) Moreover, SIK1(D586–612 + NES), SIK1 having its RK-rich region replaced with a strong nuclear export signal, was localized exclusively in the cytoplasm, and nonetheless it behaved as a strong CREB-repressor The extent of repression by SIK1(D586–612 + NES) was similar to that induced by the nuclear SIK1(S577A) The experiments using chimeras of SIK1 and SIK2 also supported no correlation between the intracellular distribu-tion and the CREB-repressing activity of SIKs (Fig 6)
Fig 7 Salt-inducible kinase 1 (SIK1) inhibits the nuclear translocation of TORC2 (transdu-cer of regulated CREB activity) The effects of SIK1 and its mutants on the subcellular localization of green fluorescent protein (GFP)-TORC2 were investigated as described
in the legend to Fig 1 and in the Materials and methods An expression plasmid for GFP-TORC2 was co-transfected without (–)
or with wild-type (WT) SIK1 or mutant SIKs (nuclear: S577A or cytoplasmic: D586-612 + NES) to Y1 cells, and cells were treated with (+) or without (–) adrenocorticotropic hormone (ACTH) for 30 min.
Fig 6 Effect of chimeric salt-inducible kinases (SIKs) on
cAMP-response element (CRE)/CRE-binding protein (CREB) activities The
CRE-repressing activities of SIK1 and chimera 1 (A) and of SIK2
and chimera 2 (B) were examined by using Y1 cells, as described in
the legend to Fig 5 To express SIK1 and SIK2, we used pIRES
(B-E) plasmid and pTarget plasmid, respectively The means and
SD values are indicated (n ¼ 3).