From the Department of Genetics, General and
Molecular Biology, University of Naples "Federico II," via
Mezzocannone 8, Napoli 80134 and the
Department of Clinical and
Biological Sciences, S. Luigi Hospital, Orbassano, Torino 10043, Italy
Received for publication, July 31, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The INK4a gene, one of the
most often disrupted loci in human cancer, encodes two unrelated
proteins, p16INK4a and p14ARF (ARF) both
capable of inducing cell cycle arrest. Although it has been clearly
demonstrated that ARF inhibits cell cycle via p53 stabilization, very
little is known about the involvement of ARF in other cell cycle
regulatory pathways, as well as on the mechanisms responsible for
activating ARF following oncoproliferative stimuli. In search of
factors that might associate with ARF to control its activity or its
specificity, we performed a yeast two-hybrid screen. We report here
that the human homologue of spinophilin/neurabin II, a regulatory
subunit of protein phosphatase 1 catalytic subunit specifically
interacts with ARF, both in yeast and in mammalian cells. We also show
that ectopic expression of spinophilin/neurabin II inhibits the
formation of G418-resistant colonies when transfected into human and
mouse cell lines, regardless of p53 and ARF status. Moreover,
spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic
expression is ineffective, somehow results in a synergic effect. These
data demonstrate a role for spinophilin in cell growth and suggest that
ARF and spinophilin could act in partially overlapping pathways.
The INK4a gene, one of the most frequently disrupted loci in
human cancer (1-3) gives rise to two distinct transcripts from different promoters (4). Each transcript has a specific 5'-exon, E1 The induction of ARF by oncoproteins such as Myc, E1A, Ras, and
v-Abl (22-25) highlights its role in sensing hyperproliferative signals in incipient cancer cells, and because ARF is also induced by
E2F (26) it biochemically connects the pRb and p53 pathways.
Furthermore, although it has been clearly demonstrated that ARF
inhibits cell cycle via p53 stabilization, very little is known about
the involvement of ARF in other cell cycle regulatory pathways (27,
28), as well as on the mechanisms responsible of ARF activation by
oncoproliferative stimuli.
To identify and isolate proteins important for conferring functional
specificity to ARF, we employed a yeast two-hybrid screen. In this
paper we report the isolation of the human homologue of the rat
spinophilin (also known as neurabin II), a regulatory subunit of
protein phosphatase 1 catalytic subunit (PP1c) (29-31), and
demonstrate the specific interaction between ARF and spinophilin. PP1
is one of the major serine/threonine-specific protein phosphatases in
eukaryotic cells (32) involved in controlling diverse cellular functions, including the exit from mitosis and splicing of mRNAs (33, 34). PP1 has been implicated in the mitotic dephosphorylation of
pRb (35), as well as in the dephosphorylation of specific residues of
p53 (35).
We also show that spinophilin is able to inhibit the formation of
G418-resistant colonies when transfected into human and mouse cell
lines regardless of p53, pRb, and ARF status. These data suggest a role
for spinophilin in cell growth.
Plasmids
Yeast Two-hybrid Screening--
The
EcoRI/SalI fragment encoding the entire ARF (132 amino acids) was excised by p19 plasmid (3) and cloned into the yeast two-hybrid bait vector pBTM116 (34) to generate pBTM-ARF.
Exon 1 ARF Deletion Mutants--
pBTM-ARF-(1-38) was obtained by
cutting with EcoRI/NarI pBTM-ARF and cloning the
114-bp fragment in pUC19 cut by EcoRI/AccI (pUC19-ARF-(1-38)). The insert EcoRI/PstI was
cloned in pBTM digested with the same enzymes. pBTM-ARF-(39-132) was
obtained by cutting with NarI/HindIII pBTM-ARF
and cloning the 436-bp fragment in pUC19 cut with
AccI/HindIII (pUC19-ARF-(39-132)). The
EcoRI/HindIII (filled-in) fragment, excised from
pUC19-ARF-(39-132) was inserted in pBTM cut by
EcoRI/SalI (filled-in).
Full-length Spinophilin Constructs--
The
BamHI/EcoRI fragment (1100 nucleotides), encoding
the C-terminal portion of spinophilin, was excised by clone B2 and
cloned in pGEM (pGEM-B2). The amplification from a human
cDNA brain library (CLONTECH, Palo Alto, CA) of
the N-terminal region was done using two oligonucleotides ATGSP
(5'-GCTCCAAGCTTCATGATGAAGACGGAG-3') and RSPBAM
(5'-ACCAGGAGATCGTTCACTTGGATCCT-3'), designed on the published sequence
of the rat spinophilin (GenBankTM accession number AF016252). The
1650-bp-amplified fragment was digested with
HindIII/BamHI, releasing a
HindIII/BamHI fragment (1450 bp containing the
spinophilin ATG) and a BamHI/BamHI fragment (200 bp, located downstream the 1450-bp fragment). The 1450-bp fragment was
cloned in pGEM-B2 cut by HindIII/BamHI
(pGEM-N-spino-BamHI-C-spino). The 200-bp fragment was cloned
in the plasmid PGEM-N-spino-BamHI-C-spino cut by
BamHI (pGEM-Spinophilin, 2500 bp). The plasmid
pGEM-Spinophilin was used for coupled transcription/translation
in vitro using the TnT T7 Quick-Coupled
transcription/translation system (Promega). pGEM-Spinophilin was
sequenced, and the sequence was submitted to the Nucleotide Sequence
Data base at EMBL (EMBL accession numbers AJ401189 and HSA401189).
Spinophilin Deletion Mutants--
All the C-terminal
mutants were obtained by inverse PCR using as template pGAD10-SpinoC
(encoding aa 605-813) and the common GAD10c primer
(5'-CGTCTAGATATGAATCGTAGATACTGAAAAACCCCGCAAGTTC-3'). The GAD10c primer
was used together with the NH-AB primer
(5'-CGTCTAGATTACTCCGACTCCTCCAGAACCCGACGCTG-3') to obtain pGAD-nH-AB
(encoding aa 605-787); with the NH-A primer (5'-CGTCTAGATTAACCCCAGTAGCCTTCCAGTTTCTCCATGCG-3') to obtain pGAD-nH-A (encoding aa 605-728); with the NH primer
(5'-CGTCTAGATTACAGCTTCTCGGGCTCCATGTCCACAGG-3') to obtain pGAD-nH
(encoding aa 605-668). Inverse PCR was performed using the Long Range
PCR kit (Roche Molecular Biochemicals) following the condition
suggested by the manufacturer. Amplified mutants cut by
XbaI were purified, ligated, and electroporated as described previously (35).
CFE Experiments and Coimmunoprecipitation--
The
HindIII/EcoRI (filled-in) fragment, encoding the
full spinophilin, derived by pGEM-Spinophilin was cloned in pRcCMV (3) cut by HindIII/NotI (filled-in)
(pCMV-Spinophilin). The fragment HindIII
(filled-in)/XbaI was excised from pCMV-Spinophilin and cloned in pcDNA3-HisA (CLONTECH, Palo Alto, CA)
cut by EcoRI (filled-in)/XbaI (pcDNA-Spinophilin). The EcoRI/SalI fragment
derived from pBTM-ARF was also cloned in pcDNA3-HisA
(pcDNA-ARF). To obtain the N-terminal region of spinophilin
(SpinoN) encoding aa 1-473 the pCMV-Spinophilin was cut by
BamHI and HindIII (filled-in). The SpinoN
fragment was cloned in pcDNA3-HisA cut by EcoRI
(filled-in)/BamHI (pcDNA-SpinoN). The C-terminal region
of spinophilin (SpinoC) encoding aa 605-813 was obtained by
EcoRI digestion of pGAD10-SpinoC and cloned in pcDNA3-HisA cut by EcoRI (pcDNA-SpinoC). PGAD-nH-AB,
pGAD-nH-A, and pGAD-nH were cut by EcoRI/XbaI,
and the DNAs encoding, respectively, aa 605-787, aa 605-728, and aa
605-668 were cloned in pcDNA3-HisA cut by
EcoRI/XbaI to generate pcDNA-nH-AB,
pcDNA-nH-A, and pcDNA-nH. The human MDM2 (HDM2) in
pcDNA3-His was a kind gift of B. Vogelstein.
Cellular Localization Experiments--
The
HindIII/EcoRI fragment excised from
pGEM-Spinophilin was cloned in pEGFP-C2 cut with the same restriction
enzymes (pEGFP-Spinophilin). The EcoRI/SalI
fragment, excised from pBTM-ARF, was cloned in pEGFP-C2 (pEGFP-ARF).
pEGFP-SpinoN was obtained cloning the
HindIII/BamHI fragment, encoding amino acids
1-473 of spinophilin, in pEGFP-C2 cut with the same restriction
enzymes. To obtain pEGFP-SpinoC encoding the amino acids 552-813 of
spinophilin, the HindIII/BglII fragment (amino
acids1-813), excised from pCMV-Spinophilin was cloned in pEGFP-C3 cut
with HindIII/BamHI. Subsequently
pEGFP-Spinophilin was cut with BglII/BamHI,
purified, and religated.
In Vitro Protein-Protein Interaction--
The 636-bp fragment
encoding the C-terminal region of spinophilin (SpinoC) was cloned in
pGEX-4T1 (Amersham Pharmacia Biotech) to obtain pGEX-SpinoC
plasmid. EcoRI/SalI fragment excised from p19
plasmid was cloned in pMAL-c2 (New England Biolabs, Hitchin, Hertfordshire, Great Britain) to obtain the plasmid pMAL-ARF. The
EcoRI/HindIII fragment encoding the full PP1c
coding sequence was excised from pYES-PP1c plasmid (36) and cloned in
pBAD-HisA (Invitrogen) plasmid (pBAD-PP1c).
Yeast Two-hybrid Screen
pBTM-ARF construct was used to screen a human brain cDNA
library cloned into the pGAD10 vector (CLONTECH,
Palo Alto, CA). The yeast strain L40 (34) was sequentially transformed
with the pBTM-ARF vector and the library. An estimated 107
transformants were screened. Yeasts containing interacting proteins were identified by growth on selective media lacking leucine, tryptophan, and histidine and confirmed by Anti-ARF Antibody Preparation and Purification
The MBP::ARF fusion protein was obtained by expression
in TG1 Escherichia coli strain transformed with pMAL-ARF
plasmid and purified on amylose-agarose (Amersham Pharmacia Biotech)
following the procedure suggested by the manufacturer. The protein was
further purified by gel-filtration on S-300 (Amersham Pharmacia
Biotech). Anti-ARF polyclonal antibodies were raised in rabbit using
purified MBP::ARF fusion protein. Antibodies were purified by
caprilic acid precipitation (38). To remove anti-MBP antibodies, the antibodies were subsequently loaded on MBP coupled to Sepharose 4B
(Amersham Pharmacia Biotech). The anti-ARF antibodies were finally
purified and concentrated on protein A-Sepharose (Amersham Pharmacia
Biotech) following the procedure suggested by the manufacturer. Anti-ARF antibodies show a detection limit of 5 and 50 ng,
respectively, for ARF and MBP (data not shown).
ARF, Spinophilin, and PP1c in Vitro Interaction
The GST::SpinoC fusion protein was obtained by
expression in TG1 E. coli strain transformed with
pGEX-SpinoC plasmid and purified by affinity chromatography on
glutathione Sepharose 4B (Amersham Pharmacia Biotech) followed by gel
filtration, in PBS buffer, on Superdex 200 HR (Amersham Pharmacia
Biotech). MBP and MBP::ARF were expressed in E. coli and purified by affinity chromatography on amylose-agarose
(New England Biolabs, Hitchin, Hertfordshire, Great Britain) followed
by gel filtration, in PBS buffer, on S-300 (Amersham Pharmacia
Biotech). Approximately 3 µg of purified MBP or MBP::ARF
were mixed with the same amount of GST or GST::SpinoC in TENN
buffer (50 µl, 50 mM Tris-HCl, pH 7.4, 1 mM
EDTA, pH 8, 10% NaCl, 0.1% Nonidet P-40, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1%
bovine serum albumin) and (25 µl of gel volume) amylose-agarose (New
England Biolabs, Hitchin, Hertfordshire, Great Britain) was added (20 min at 4 °C). The beads were collected by centrifugation and washed three times with TENN buffer containing 1 M urea
and twice with TENN buffer. Samples were loaded onto 10% SDS-PAGE, blotted on nitrocellulose paper (Schleicher & Schuell), and probed with
anti-GST antibody (1:2000; Sigma Chemical Co., St. Louis, MO) followed
by horseradish peroxidase (HRP) anti-goat immunoglobulin G (1:1000;
Sigma). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, UK).
His6::Xpress::PP1c was obtained by expression in
TOP10 E. coli strain (Invitrogen) transformed with pBAD-PP1c
plasmid. Induction and purification on nickel chelating resin
(Invitrogen) was performed as suggested by the manufacturer. PP1c and
MBP::ARF coprecipitation experiment was performed essentially
as described above.
Far Western
Two aliquots of purified MBP::ARF were separated on
10% SDS-PAGE, transferred to nitrocellulose, and blocked in PBS
containing 3% dried skimmed milk (blocking buffer) for 1 h at
37 °C. Filters were separately incubated with GST::SpinoC
and GST (50 µg/ml, 18 h, 4 °C), followed by anti-GST
polyclonal antibody (1:2000, 2 h, 37 °C) and by HRP-anti-goat
immunoglobulin G (1:1000, 2 h, 37 °C). Immunoreactive bands
were visualized by enhanced chemiluminescence.
Cell Culture, Transfection, Coimmunoprecipitation, and Cell
Imaging
All cells were cultured in a 37 °C incubator with 5%
CO2 in Dulbecco's modified Eagle's medium supplemented
with 2 mM glutamine, penicillin/streptomycin 100 units/ml
each and 10% fetal bovine serum except for NIH3T3 cultured in the same
medium supplemented with 10% calf serum.
For coimmunoprecipitation assay, COS-7 cells were seeded into a 6-well
multiplate (2.5 × 105 cells per well) and transfected
using the Superfect reagent according to the manufacturer's
instruction (Qiagen). Cells were washed twice with ice-cold PBS, lysed
into Nonidet P-40 lysis buffer (50 mM NaCl, 150 mM Tris-HCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and
protease inhibitor). The lysate was passed through a 21-gauge needle,
debris were removed by centrifugation (13,000 × g, 10 min, 4 °C), and the total amount of proteins was quantified (Bio-Rad
protein assay). Equal amounts of lysates were precleared with protein
A-Sepharose beads (Repligen, UK; 8 h at 4 °C) and subsequently
incubated with anti-ARF antibody (dilution 1:100, 2 h at 4 °C)
followed by addition of protein A-Sepharose beads (50 µl). Beads were
collected by centrifugation, and the immunoprecipitates were washed
three times with lysis buffer (4 °C), solubilized in SDS-PAGE sample
buffer, loaded on 8% SDS-PAGE and analyzed by immunoblotting with
anti-Xpress antibody (1:2000, 3 h, at room temperature;
Invitrogen) followed by incubation with the HRP-anti mouse antibody
(1:1000, 1 h, at room temperature; Amersham Pharmacia Biotech,
UK). Immunoreactive bands were visualized by enhanced chemiluminescence.
For imaging analysis NIH3T3 and COS-7 cells were grown on a sterilized
glass coverslip (5 × 104 cells per slip) into 6-well
multiplates and transfected using the Superfect reagent (Qiagen)
according to the manufacturer's instructions. Twenty-four hours after
transfection, cells were washed twice with PBS and fixed with 4%
para-formaldehyde in PBS (30 min). Glass coverslips were subsequently
washed twice with PBS and mounted on microscope slides with 50%
glycerol in PBS. Images were acquired using a confocal microscope
(Axiovert 100M Zeiss). A krypton-argon gas laser provided excitation at
488 nm with a 522/32 emission filter for green fluorescence.
For the CFE assay (colony formation efficiency assay) U2OS, Saos-2
(1 × 105 per well), and NIH3T3 (2 × 104 per well) cells were seeded into 6-well multiplates and
transfected with 2 µg of total plasmid DNA using the standard calcium
phosphate method (39). Forty-eight hours after transfection the cells were replated in a 100-mm dish and selected with the appropriate concentration of Geneticin (G418, Calbiochem) for 2 weeks. Saos-2 and
NIH3T3 cells were selected with 1 mg/ml G418 and U2OS cells with 600 µg/ml G418. Medium was replenished every 3 days. Cells were fixed
with 10% methanol/10% acetic acid and stained with crystal violet 15 days post-transfection. The colonies were counted. Each experiment was
repeated at least four times.
Identification of Spinophilin as an ARF-binding Protein--
In an
attempt to identify human proteins that interact with ARF, we have used
the yeast two-hybrid system (36). The entire ARF cDNA was fused to
the GAL4 DNA-binding domain (BD). As detailed under "Experimental
Procedures" this construct was used as bait to screen a human brain
cDNA library. From a screen of ~107 yeast
transformants, 30 colonies were scored as positive for reporter gene
activity (His+LacZ+). Among 19 clones scored
positive in secondary screening assays, 18 encoded a 212-amino acid
polypeptide (pGAD-SpinoC), which was identified in a data base search
as the human homologue of the C-terminal portion of rat
spinophilin/neurabin II (31, 37). The last clone (pGAD-B2) differed in
length only by 177 bp at the 5'-end (Fig.
1A). The full-length cDNA
(EMBL accession numbers AJ401189 and HSA401189), reconstructed as
detailed under "Experimental Procedures," encodes a 813-amino acid
long polypeptide showing 95% amino acids identity (Fig. 1B)
with the rat spinophilin/neurabin II (GenBankTM accession number
AAB72005). Performing a BLAST search on the GenBankTM data base we
have also identified a cosmid derived from the human chromosome 17 (GenBankTM accession number AC002401) containing the gene sequence of
human spinophilin, which consists of 12 exons spanning over a region of
15 kb. The so-called spinophilin was first identified in rat as a
cellular partner of type 1 protein phosphatase (PP1), which is one of
the main eukaryotic serine/threonine protein phosphatases involved in
the control of cell cycle progression. Rat spinophilin is characterized by an N-terminal domain (aa 1-295), in which are present various putative Src homology 3 binding motifs, and by a C-terminal domain being a protein phosphatase 1 (PP1c)-negative regulator (295). Spinophilin sequence contains various protein-protein interaction signals spread throughout the whole sequence (Fig. 1A): an
F-actin binding domain (ABD), a PP1c binding site
(R/K)(V/I)XF (gray triangle), a PDZ domain, and a
myosin-like left handed In Vitro Binding Assays and Coimmunoprecipitation--
To
confirm the ARF/spinophilin interaction we performed coprecipitation
and coimmunoprecipitation experiments. We expressed ARF as MBP fusion
(MBP::ARF) using the pMal-c2 system and the 212-amino acid
C-terminal region of spinophilin (SpinoC) as glutathion S-transferase fusion (GST::SpinoC) using the pGEX
system. Purified MBP::ARF or MBP were incubated with either
purified GST::SpinoC or GST, and amylose-agarose beads were
added. After extensive washing the bound proteins were separated on
SDS-PAGE, blotted, and analyzed using anti-GST polyclonal antibodies.
As shown in Fig. 2A,
GST::SpinoC coprecipitated with MBP::ARF
(lane 1), but not with GST (lane 2), and MBP did
not bind to GST::SpinoC (lane 3). The
GST::SpinoC interaction with MBP::ARF was further
confirmed by far-Western blotting. Two aliquots of purified
MBP::ARF were analyzed on SDS-PAGE and blotted onto
nitrocellulose. Ponceau S staining (data not shown) confirmed the
presence of an equal amount of proteins on the blots. The blots were
probed, respectively, with an equal amount of GST::SpinoC and
GST followed by incubation with anti-GST antibodies. As shown in Fig.
2B the anti-GST antibody only binds to the filter probed
with GST::SpinoC (lane 1).
Although the in vitro coprecipitation experiments clearly
indicate that the C-terminal region of spinophilin is able to bind ARF
we also confirmed the interaction in intact cells. COS-7 cells, a
mammalian cell line known for its robust expression of recombinant proteins, were transfected with mammalian expression plasmids encoding
Xpress-tagged spinophilin, or Xpress-tagged human MDM2, and/or ARF. The
cellular lysates were immunoprecipitated with anti-ARF antibodies. The
immunoprecipitates were blotted and probed with anti-Xpress antibodies.
Coimmunoprecipitation of spinophilin (Fig. 2C, lane
3) as well as human MDM2 (Fig. 2C, lane 4),
which has been reported to interact with ARF (7), occurred only when each of these proteins was coexpressed with ARF, because the complexes were not found when either protein alone was introduced into the cells
(Fig. 2C, compare, lanes 3 and 4 to
lanes 5 and 6). Furthermore, in a similar
experiment in which plasmids encoding ARF and/or, respectively, the
N-terminal region (SpinoN) or the C-terminal region of spinophilin
(SpinoC) (Fig. 2D) were transiently transfected in COS-7
cells, only the C-terminal region of spinophilin coimmunoprecipitated with ARF (Fig. 2D, lane 6) confirming that the
ARF-binding domain is localized in the C-terminal region of spinophilin.
ARF-Spinophilin Minimal Interaction Requirements--
To identify
the minimum region of ARF essential for the interaction with
spinophilin, two deletion mutants of ARF were assayed for the
interaction with spinophilin in yeast (Fig.
3). Our yeast two-hybrid analysis showed
that region 1-65 of ARF, corresponding to the region encoded by exon
1
To identify the minimum region of the C-terminal part of spinophilin
required for interaction with ARF we have created various deletion
mutants. The mutants design was based on the prediction, using the
COILS program (38), of the coiled-coil region (673) already
observed in the rat spinophilin (31). The C-terminal region of
spinophilin (605) encoded by the pGAD10-SpinoC is divided in four
regions: non-helix (605-673, black rods), helix A
(674-726, white rectangles), helix B (727-788, black
rectangles), and helix C (789-813, gray rectangles).
We have sequentially deleted the helices from C to A and analyzed the
ability of the mutants to bind to ARF. Our yeast two-hybrid data showed
that helices C (Fig. 3A, lane e) and B (Fig.
3A, lane f) are not required for the interaction with ARF whereas the C-terminal mutant, which also lacks the helix A
(Fig. 3A, lane g), failed to interact with ARF.
To obtain an independent evaluation of the protein-protein interaction
suggested by the two hybrid results, we examined the ability of the
described deleted peptides to coimmunoprecipitate with ARF in cell
extracts. COS-7 cells were transfected with mammalian expression
plasmids pcDNA-nH-AB (aa 605-787), pcDNA-nH-A (aa 605-728),
and pcDNA-nH (aa 605-668) and/or pcDNA-ARF. As shown in Fig.
3B (lanes 5-7) only the shorter mutant failed to
interact with ARF, suggesting again a key role for helix A in the
contact between spinophilin and ARF.
Biological Activity of Spinophilin--
The interaction between
ARF and spinophilin, a regulatory subunit of protein phosphatase 1, raises the question of the biological significance of this interaction.
PP1 is one of the major serine/threonine-specific protein phosphatases
in eukaryotic cells (32), which has a crucial role in the cell cycle
progression (33-35). In principle, a direct interaction could be
possible between PP1 and ARF. Careful examination of the ARF sequence
revealed the lack of the PP1c pentapeptide motif
((R/K)(R/K)(V/I)XF) conserved in all the PP1 regulatory subunits (29). Moreover, coprecipitation experiments were performed using purified MBP::ARF and purified His6::PP1c,
and we did not observe any specific interaction between these two
proteins (data not shown), although we cannot exclude that a trimeric
complex could exist.
Because ectopic expression of ARF results in growth suppression, to
assess a possible role of the ARF/spinophilin interaction, we analyzed
the effect of spinophilin in growth suppression by colony formation
efficiency assay (CFE) using different human and mouse cell lines.
Saos-2 cells express ARF and are p53 and pRb null (9), whereas U2OS
cells express wild type p53 and pRb and show a near undetectable level
of ARF expression, and the NIH3T3 cell line bears a deletion of the
INK4a locus. We transfected expression vectors encoding ARF or the
entire spinophilin, into all three cell lines. After completion of drug
selection, growth suppression was quantified by comparing the relative
number of drug-resistant colonies obtained with each construct to that
obtained with the empty vector. As already reported (3) ARF strongly inhibits the G-418R colony formation only in the U2OS and
NIH3T3 cell lines, which express both p53 and pRb (Fig.
4). Interestingly, spinophilin appears to
reduce the number of resistant colonies with an efficiency similar, if
not higher, to that exhibited by ARF, in both cell types. A more
complex picture derives from the experiments in Saos-2 cells, where ARF
ectopic expression is ineffective, with spinophilin reducing the number
of G-418R colony up to 50% with respect to the control
only when the higher amount of expression vector was used (Fig. 4).
These data indicate a role for spinophilin in cell growth that is
independent from the status of p53, pRb, and ARF.
We have also analyzed the effect of cotransfection of the two plasmids
and the results show that simultaneous expression of both proteins in
human cells resulted in a stronger effect on inhibition of colony
formation. The data in U2OS cells can be explained as the result of an
additive effect of the two proteins. In Saos-2 cells, however,
transfection of 1 µg each of spinophilin and ARF, which alone have
little or no effect on colony formation, reduces the number of
colonies significantly, suggesting a synergy between the two proteins
in this cell line (Fig. 4).
Transfection of expression vectors encoding, respectively, the
C-terminal region or the N-terminal region of spinophilin resulted in a
number of colonies almost similar to that of the control vector,
suggesting that the integrity of the protein in all cell lines is
required for the biological activity (data not shown). On the other
hand, the effect of the transfection of 1 µg of ARF did not vary when
either the C-terminal region or the N-terminal region of spinophilin
where coexpressed (data not shown).
Cellular Localization--
Spinophilin/neurabin II is ubiquitously
expressed and has been shown to localize in rat at the level of the
dendritic spines (31). It has been shown that in neuronal tissues rat
spinophilin/neurabin II is involved in the binding of various proteins
usually found in the cytoplasm, F-actin (39), TGN38 (40), D2 dopamine
receptor (41), and Lin-10 (42). ARF has a preferential localization in
the nucleolus, but, when overexpressed with MDM2 and p53, it has also
been found in "nuclear bodies" within the nucleoplasm (16). Because
our results clearly show an interaction in vitro and in
intact cells between ARF and spinophilin, we decided to assess the
intracellular localization of spinophilin in mammalian cell lines.
Consequently, we transfected COS-7 and NIH3T3 cell lines with GFP
fusion constructs of either ARF or spinophilin. To verify that addition
of the GFP did not alter the biological activity of these proteins, we
performed CFE experiments using GFP fusion constructs of either ARF or
spinophilin. The results were similar to that obtained with the
expression vector lacking the GFP, indicating that addition of the
reporter protein did not alter the biological activity of these
proteins (data not shown).
Our results show that spinophilin, at least in COS-7 and NIH3T3 cells,
is expressed both in the nucleus and in the cytoplasm (Fig.
5C). To define the minimum
region of spinophilin needed for the nuclear localization, two deletion
mutants of GFP::Spinophilin were constructed and assayed in
COS-7 and NIH3T3 cells. Our localization data suggest that the
spinophilin region necessary for nuclear localization is the region
encompassing amino acids 552-813, because this region is deleted in
the GFP::SpinoN mutant (1), which is clearly excluded
from the nucleoplasm (Fig. 5E). On the other hand, the
GFP::SpinoC mutant bearing the region encompassing amino acids 552-813 is localized both in the nucleus and in the cytoplasm (Fig. 5D).
Biochemical evidence supporting the genetic interaction between
ARF and p53 comes from the finding that ARF physically interacts with
MDM2 and consequently stabilizes p53, but the molecular pathway by
which ARF stabilizes p53 is not clear at present (15-17). Moreover, very little is known about the involvement of ARF in other cell cycle
regulatory pathways, as well as on the mechanisms responsible for the
activation of ARF by oncoproliferative stimuli. However, the existence
of additional ARF-interacting proteins was clear from our preliminary
data,3 and in this paper we
have described the identification of a new partner of human ARF,
spinophilin. Our studies have shown that an intact ARF N-terminal
region (aa 1-65) is necessary for this interaction, because deletion
of either the last 27 or the first 1-38 amino acids of this domain
impairs the interaction. Although further experiments are necessary for
a more refined definition, these results suggest that the ARF portions
interacting with spinophilin and MDM2 are partially overlapping (43).
We have also shown that the PDZ and the other protein-protein
interaction domains present in the N-terminal region of spinophilin are
not involved in the contact with ARF as, instead, only part of the
coiled-coil region present in the C-terminal region of spinophilin (aa
605-726) is required in yeast and in mammalian cells for an efficient interaction.
It has been shown that in neuronal tissues rat spinophilin/neurabin II
is involved in the binding of various proteins usually found in the
cytoplasm (39-42). However, in our localization experiments, we were
able to demonstrate that, at least in the cell types we used (COS-7 and
NIH3T3), spinophilin localizes both in the nucleoplasm and in the
cytoplasm, and that deletion of the C-terminal portion of the protein
resulted in the exclusion of the protein from the nucleus. On the other
hand, the analysis of spinophilin with the psort server on the
Web, using Reinhardt's method for cytoplasmic/nuclear discrimination (44) suggested an 89% probability of nuclear localization, although the program failed to identify canonical nuclear
localization signals. It is possible that subcellular compartmentalization of spinophilin could in some way be
tissue-specific, and/or could depend, at least in part, on interaction
with different partners. Colocalization experiments between ARF and
spinophilin will help to clarify this point.
Protein phosphorylation and dephosphorylation regulates many cellular
functions. Protein kinases and phosphatases have multiple substrates
in vivo, which enables several responses to physiological stimuli. However, their broad substrate specificity suggests the need
for mechanisms to restrict the action of these enzymes in vivo. Recent evidence reported that some protein phosphatases and
kinases are regulated by targeting subunits (45). This class of protein
not only acts to restrict the location of kinases and phosphatases but
also modifies their catalytic and regulatory properties and allows
their activity to be regulated by extracellular signals. Spinophilin
belongs to this class of regulators, because it negatively regulates
PP1 activity. PP1 is one of the main eukaryotic serine/threonine
protein phosphatases involved in the control of cell cycle progression
(reviewed in Ref. 35), and it has been implicated in the mitotic
dephosphorylation of pRb (35), as well as in the dephosphorylation of
specific residues of p53 (35, 46). PP1 is found associated with pRb in
the G1 phase and during mitotic exit (47). This temporal
association between PP1 and pRb appears to have a functional
significance in that it coincides with the reactivation of pRb-mediated
growth suppression (48). The observation that PP1d isoform containing
the greatest pRb-directed activity is found associated with a 110-kDa
interacting protein (49) again underlines the importance of the
interacting subunits of phosphatases.
Before PP1 activity was ever implicated in the regulation of pRb, it
was known to have a role in the regulation of mitosis and cell
division. PP1 mutations in Drosophila (50), yeast (51), and
fungi (52) displayed varied mitotic defects and different degrees of
lethality. Mitotic blocks were observed upon microinjection of
PP1-neutralizing antibodies (53) and PP1 inhibitors such as okadaic
acid (54, 55). In addition to this, the distribution of PP1 changes
with progression of the cell cycle, accumulating at the nucleus to
associate with chromatin during G2 and M phase (53).
Potential targets for PP1 in the nucleus are the mitotic cyclin B/cdk1
substrates that include histone H1, lamins, microtubule-associated proteins, and perhaps other proteins that have yet to be identified.
Strikingly, spinophilin appeared to be a strong growth suppressor in
CFE assays in both human U2OS and mouse NIH3T3 cells that are wild type
for both p53 and pRb but do not express ARF proteins. In U2OS cells
coexpression of ARF and spinophilin resulted in a stronger effect on
inhibition of colony formation, suggesting an additive effect between
the two proteins. Surprisingly, in Saos-2 cells, where ARF is unable to
suppress colony formation, as already reported (3), spinophilin
inhibited growth of G418-resistant colonies up to 50% respect to the
control, suggesting that it could act in a pathway that is at least in
part, p53-independent. Moreover, as coexpression resulted in a
remarkable level of inhibition of colony formation, it might be
speculated that ARF enforced the activity of spinophilin or, vice
versa, expression of spinophilin activated a p53-independent ARF
activity. Interestingly, it has been very recently reported (28) that
triple-knockout mice nullizygous for ARF, p53, and MDM2 (TK0) develop
multiple tumors at a frequency greater than those observed in animals
lacking both p53 and MDM2 or p53 alone, demonstrating that ARF can act
independently of the MDM2-p53 axis in tumor surveillance. Moreover,
reintroduction of ARF into TK0 mouse embryo fibroblasts arrested the
cell division in the G1 phase. We conclude that, in the
absence of MDM2 and p53, ARF interacts with other unknown proteins to
inhibit cell proliferation. Thus, it might be possible that only in a
p53 null context could overlapping pathways become unmasked.
To clarify these points, further experiments are necessary, in
particular to elucidate whether a cell cycle arrest or the activation
of apoptotic or senescence pathways is responsible for inhibition of
proliferation induced by spinophilin.
Our data also indicate that the ARF-binding domain of the spinophilin
does not have any effect on cell growth, regardless of the status of
endogenous ARF. Moreover cotransfection of the ARF-binding domain of
the spinophilin and ARF again does not result in any effect on the
biological activity of ARF.
In conclusion our experiments suggest that the physical interaction
between human oncosuppressor ARF and a PP1-binding protein might result
in a functional interaction, depending on the genetic context. In
particular, the results in Saos-2 cells allowed us to suppose that ARF
and spinophilin could act in partly overlapping pathways. One possible
scenario is that this interaction could function to target PP1 on ARF
and/or on other molecules involved in the same pathway. We are now
investigating the possibility that phosphorylation and
dephosphorylation could play a role in the regulation of ARF biological activity.
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or E1
, and two common exons E2 and E3. The E1
-containing transcript encodes p16INK4a, which acts as an inhibitor of
cyclin-dependent kinases 4 or 6 and prevents the
phosphorylation of pRb (5), thereby maintaining an active pRb and
blocking the exit from the G1 phase. The
E1
-containing transcript encodes ARF (a 14-kDa polypeptide in
humans, 19 kDa in mouse). Mouse and human
ARF1 proteins are 45%
identical through their exon 1
segments and 50% identical overall
(6). ARF inhibits cell growth by interacting with MDM2 (7-10), which
is a multifunctional protein that negatively regulates p53 in several
ways. First, its binding interferes with p53's ability to
transactivate target genes (11). Second, MDM2 has an intrinsic
ubiquitin ligase activity that most likely contributes to p53
degradation (12). At least in vitro, ARF can interfere with
this reaction (13), but whether this is central to ARF actions in
vivo is unknown. Third, MDM2 relocalizes p53 from the cell nucleus
to the cytoplasm where it undergoes proteosomal degradation (14). Both
mouse and human ARF are nucleolar proteins. When coexpressed with MDM2
or induced by conditional Myc expression, ARF relocalizes MDM2 to the
nucleolus, preventing MDM2 shuttling and stabilizing p53 in the
nucleoplasm, thereby prompting cell cycle arrest (15-17). In
principle, ARF may antagonize any or all of the MDM2 functions. On the
other hand, the antagonism of MDM2 by ARF could potentially affect
functions of proteins other than p53, such as E2F-1 (18), pRb (19),
p300/CBP (20), and other p53 family members
(21).2
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Construct--
The exon 1
was excised by
EcoRI digestion from pN-p19 plasmid (3) and cloned in
pBTM116 cut by EcoRI (pBTM-ARF-(1-65)).
-galactosidase activity. Isolated plasmids were retransformed into L40 with the negative control
plasmid pBTM-galactin (a gift of L. Chiariotti) and with pBTM-ARF and tested again for growing on the selection media. Those
that were negative for interaction with galactin were sequenced and DNA
sequences were used to search the non-redundant GenBankTM data base
using the BLAST search algorithm (37) available from the National
Institutes of Health on the Web.
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-helix. Both pGAD-SpinoC and pGAD-B2 encode
the predicted coiled-coil region (Fig. 1A) observed in rat
spinophilin (31) and pGAD-B2 encodes also a portion of the PDZ domain
(Fig. 1A). Both clones showed interaction with ARF in our
yeast two-hybrid system, leading us to draw the conclusion that the
coiled-coil region of spinophilin is the binding domain for ARF.
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Fig. 1.
A, schematic illustration of full-length
human spinophilin/neurabin II (817 aa) and of the isolated clones
(B2 and SpinoC). Indicated are the position of
the F-actin-binding domain (ABD), the PP1c binding site
(gray triangle), the PSD-95/discs large/ZO-1
(PDZ) domain, and the predicted coiled-coil motif at the
C-terminal region. B, comparison between the human and the
rat sequence (amino acids substitutions only are indicated). The ABD
domain is almost identical between the human and rat
(underlined sequence) showing 96% amino acids identity. The
PP1c binding site, (boldface sequence) as the PDZ domains,
(boldface italics sequence) are 100% identical to each
other. The coiled-coil motif at the C-terminal region shows an amino
acids identity of 98% between human and rat. The human spinophilin
sequence was deposited in EMBL with the accession numbers AJ401189 and
HSA401189.
View larger version (43K):
[in a new window]
Fig. 2.
In vitro/in vivo
interaction between ARF and spinophilin. A,
coprecipitation experiments were performed using purified proteins.
Approximately 3 µg of MBP::ARF or MBP were mixed with the
same amount of GST::SpinoC or GST, and amylose-agarose was
added. The beads were washed, and the samples were analyzed by
immunoblotting with anti-GST antibody. MBP::ARF
co-precipitates efficiently GST::SpinoC (lane 1)
but not GST (lane 2). Furthermore, MBP does not
coprecipitate GST::SpinoC (lane 3). The correct
size of GST::SpinoC fusion protein is defined by an aliquot
of GST::SpinoC-purified protein (lane 4).
B, far-Western blotting: Two aliquots of MBP::ARF
were loaded on 10% SDS-PAGE, blotted on nitrocellulose paper, and
separately probed with either GST::SpinoC or GST protein.
Filters were subsequently incubated with anti-GST antibody.
MBP::ARF binds only to GST::SpinoC (lane
1), whereas no interaction was observed using as tracer GST
(lane 2). C, co-immunoprecipitation of
full-length spinophilin with ARF: COS-7 cells were transfected with
mammalian expression plasmids encoding Xpress-tagged spinophilin or
Xpress-tagged human MDM2, and/or ARF. Cellular extracts were incubated
with anti-ARF antibody and precipitated with protein A-Sepharose beads.
Samples were analyzed by immunoblotting with anti-Xpress antibody. The
full-length spinophilin and MDM2 were detected after
co-immunoprecipitation only when ARF was coexpressed (compare
lanes 3 and 4 to lanes 5 and
6). D, co-immunoprecipitation of spinophilin
C-terminal domain with ARF: COS-7 cells were transfected with mammalian
expression plasmids encoding Xpress-tagged spinophilin or Xpress-tagged
SpinoN (1) or Xpress-tagged SpinoC (aa 605-813), and/or ARF.
Cellular extracts were incubated with anti-ARF antibody and
precipitated with protein A-Sepharose beads. Samples were analyzed by
immunoblotting with anti-Xpress antibody. Anti-ARF antibodies were
unable to immunoprecipitate spinophilin and the N-terminal and
C-terminal deletion mutants without the co-expression of ARF
(lanes 2-4). Only the C-terminal domain of spinophilin and
the full-length protein could be detected after co-immunoprecipitation
when ARF was coexpressed (lanes 6 and 7).
(1-65 aa, Fig. 3A, lane b), is required for
the interaction with the C-terminal region of spinophilin. The deletion
of either the last 27 amino acids (Fig. 3A, lane c) or of the amino acids 1-38 of exon 1
encoded region (Fig. 3A, lane d) impairs the interaction with
spinophilin, suggesting that the entire region encompassing amino acids
1-65 is essential for the binding to spinophilin.
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Fig. 3.
Mapping the interaction domains between ARF
and spinophilin. Expression vectors encoding ARF, the deletion
mutants ARF-(1-65), ARF-(1-27), and ARF-(38-132) were probed in the
yeast two hybrid system for interaction with expression vector encoding
the C-terminal part of spinophilin (aa 605-813, lanes
a-d). Expression vectors encoding, respectively, amino acids
605-787 (nH-AB), 605-728 (nH-A), and 605-668 (nH) of spinophilin
were probed in yeast two hybrid system for interaction with the
ARF-(1-65) (lanes e-g). Interaction between ARF and
spinophilin deletion mutants was qualitatively identified by histidine
auxotrophy (HIS-growth panel) and quantitatively by
-galactosidase activity measurement (
-gal
Activity panel). Values represent the means of three independent
experiments. The standard deviation for each value is also given.
B, co-immunoprecipitation of C-terminal deletion mutants of
spinophilin with ARF: COS-7 cells were transfected with mammalian
expression plasmids pcDNA-nH-AB (aa 605-787), pcDNA-nH-A (aa
605-728), pcDNA-nH (aa 605-668), and/or pcDNA-ARF. Cellular
extracts were incubated with anti-ARF antibody and precipitated with
protein A-Sepharose beads. Samples were analyzed by immunoblotting with
anti-Xpress antibody. Anti-ARF antibodies were unable to
immunoprecipitate deletion mutants without the co-expression of ARF
(lanes 2-4). Only the mutants pcDNA-nH-AB (aa 605-787)
(lane 7) and pcDNA-nH-A (aa 605-728) (lane
6) could be detected after coimmunoprecipitation in presence of
ARF.
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Fig. 4.
Growth suppression by ARF and spinophilin
expression. Saos-2, U2OS, and NIH3T3 cell lines were seeded into
6-well multiplates and transfected with the indicated amounts of ARF
and spinophilin expression vectors. Forty-eight hours after
transfection the cells were replated in a 100-mm dish and selected with
the appropriate concentration of G418 for 2 weeks. The cells were fixed
and stained with crystal violet, and the colonies were counted. The
graphic represents the percentage of colonies obtained with the
indicated plasmids relative to that detected on the pcDNA3
transfected plates. Values represent the mean of four independent
experiments. Standard deviation for each value is also given.
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Fig. 5.
Subcellular localizations. COS-7 and
NIH3T3 cells were seeded on glass coverslips and transfected
with expression vectors encoding, respectively, the fusion proteins
GFP::ARF (A), GFP (B),
GFP::spinophilin (C), GFP::spinoC
(D), and GFP::spinoN (E). Cells were
fixed 24 h after transfection, and images were acquired using a
confocal microscope (Axiovert 100M Zeiss).
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ACKNOWLEDGEMENTS |
---|
We thank R. Terracciano for skillful technical help and Dr. B. Peraccino for help in confocal images acquisition. We are grateful to Drs. B. Vogelstein, S.-L. Tan, and L. Chiarotti for generously providing some of the plasmids used in this study, Dr. P. Vito for the human brain cDNA library and Prof. P. Delli Bovi for helpful comments and suggestions.
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FOOTNOTES |
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* This work was supported in part by grants from the Italian Association for Cancer Research and Ministero della Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche (to G. L. M.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ401189 and HSA401189.
§ Both authors contributed equally to this work.
¶ Recipients of a predoctoral fellowship cofinanced by the Fondo Sociale Europeo (European Economic Community), and this work is in partial fulfillment of the requirements for the doctoral degree in Genetics at the University of Naples.
** Present address: DNAX Research Institute, 901 California Ave., Palo Alto, CA 94304.
To whom correspondence should be addressed: Tel.:
39-081-7903432; Fax: 39-081-5527950; E-mail: lamantia@unina.it.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M006845200
2 V. Calabrò et al., submitted.
3 V. Calabrò, G. Mdnsueto, T. Parisi, M. Vivo, R. Calogero, and G. La Mantia, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: ARF, protein p14ARF; PP1, protein phosphatase 1, PP1c, protein phosphatase 1 catalytic subunit; bp, base pair(s); PCR, polymerase chain reaction; aa, amino acid(s); PBS, phosphate-buffered saline; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; CFE, colony formation efficiency; kb, kilobase(s); ABD, F-actin-binding domain; GFP, green fluorescence protein; MBP, maltose-binding protein; PDZ, PSD-95/discs large/ZO-1 domain.
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