Biochem. J. (2009) 418, 173–184 (Printed in Great Britain)
173
doi:10.1042/BJ20080170
Paxillin nuclear-cytoplasmic localization is regulated by phosphorylation
of the LD4 motif: evidence that nuclear paxillin promotes cell proliferation
Jing-Ming DONG*, Lei-Shong LAU*, Yuen-Wai NG*, Louis LIM*† and Ed MANSER*1
*GSK-IMCB Group, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore, and †Department of Molecular Neuroscience, Institute of Neurology,
University College London, 1 Wakefield Street, London WC1N 1PJ, U.K.
Paxillin, a major focal-adhesion complex component belongs to
the subfamily of LIM domain proteins and participates in cell
adhesion-mediated signal transduction. It is implicated in cellmotility responses upon activation of cell-surface receptors and
can recruit, among others, the GIT1 [GRK (G-protein-coupledreceptor kinase)-interacting ARF (ADP-ribosylation factor) GAP
(GTPase-activating protein)]–PIX [PAK (p21-activated kinase)interacting exchange factor]–PAK1 complex. Several adhesion
proteins including zyxin, Hic5 and Trip6 are also nuclear and can
exert transcriptional effects. In the present study we show that
endogenous paxillin shuttles between the cytoplasm and nucleus,
and we have used a variety of tagged paxillin constructs to map
the nuclear export signal. This region overlaps an important LD4
motif that binds GIT1 and FAK1 (focal-adhesion kinase 1). We
provide evidence that phosphorylation of Ser272 within LD4 blocks
nuclear export, and we show that this modification also reduces
GIT1, but not FAK1, binding; however, Ser272 phosphorylation
does not appear to be mediated by PAK1 as previously suggested.
Expression of nuclear-localized paxillin LIM domains stimulate
DNA synthesis and cell proliferation. By real-time PCR analysis
we have established that overexpression of either full-length
paxillin or a truncated nuclear form suppresses expression of
the parental imprinted gene H19, and modulation of this locus
probably affects the rate of NIH-3T3 cell proliferation.
INTRODUCTION
in NIH-3T3 cells; however, no PDLP was concentrated in cell
nuclei in Drosophila embryos at the stage tested. The expression
of Xenopus paxillin is cell-cycle-dependent, with increased levels
in S-phase. Xenopus paxillin has been shown to be translocated
to the nucleus of Xenopus cells grown on vitronectin, but not on
fibronectin [4].
Group 3 LIM-containing proteins such as Hic5 and zyxin
actively shuttle between the nucleus and cytoplasm where their
nuclear role has started to be elucidated [5–8]. Paxillin may
interact with PABP1 (polyA-binding protein 1) to facilitate
PABP1 export from the nucleus to the cytoplasm [9]. In this
way paxillin–PABP1 may serve as a chaperone that directs
the targeting of specific mRNAs to nascent focal adhesions,
where localized protein translation contributes to efficient cell
motility [9,10]. In prostate cancer cell lines, paxillin may
facilitate androgen receptor and glucocorticoid receptor import
to the nucleus thereby potentiating transactivation activity [11];
however, the mechanism underlying paxillin shuttling between
the nucleus and cytoplasm is still unclear.
In the present study we link paxillin nuclear activity to the
control of H19 transcription. H19 was the first identified imprinted
gene located within the H19-Igf2 (insulin-like growth factor 2)
locus that is at the distal region of mouse chromosome 7 and
human chromosome 11 [12]. The H19 gene encodes a 2.5 kb
fully capped, spliced and polyadenylated, but untranslated, RNA.
The H19 locus is paternally imprinted and only mono-allelically
expressed from the maternal chromosome, whereas its upstream
Igf2 gene is mono-allelically expressed from the paternal
Focal adhesions are multi-molecular structures formed at the sites
where integrins and the extracellular matrix interact. These are
not only sites where cytoskeleton proteins such as actin anchor
to maintain cell shape, but are also relay stations where outsidein and inside-out signal transduction takes place through the
dynamic recruitment of many structural and signalling proteins.
The adaptor protein paxillin is a major component of the
focal-adhesion complex. It plays a pivotal role in embryonic
development, cell attachment, spreading and motility [1]. Paxillin
belongs to a LIM-containing protein family that has been classified into three groups, based on sequence relationships among
LIM domains and on the overall structure of the proteins
[2]. It contains five leucine-rich LD (Leu-Asp) motifs in
its N-terminal region and four cystine-rich LIM domains in its
C-terminal region. While C-terminally located LIM domains
are required for paxillin to localize to focal adhesions, the
various LD motifs are responsible for recruiting proteins
such as vinculin, actopaxin, ILK (integrin-linked kinase), FAK
(focal-adhesion kinase) and GIT1 [GRK (G-protein-coupledreceptor kinase)-interacting ARF (ADP-ribosylation factor) GAP
(GTPase-activating protein)] [1].
In Drosophila a truncated paxillin transcript termed PDLP that
only contains three LIM domains is known [3]. PDLP expression
is concomitant with late events in specific tissues, including
myogenesis, cell migration and attachment. Interestingly a
fraction of PDLP was found in the cell nucleus upon transfection
Key words: cell proliferation, GRK-interacting ARF GAP (GIT1),
H19, nuclear–cytoplasm shuttle, paxillin.
Abbreviations used: BrdU, bromodeoxyuridine; CRM1, chromosome region maintenance 1; CRP, cysteine-rich protein; DMEM, Dulbecco’s modified
Eagle’s medium; FAK, focal-adhesion kinase; FCS, foetal calf serum; GFP, green fluorescent protein; GIT1, GRK (G-protein-coupled-receptor kinase)interacting ARF (ADP-ribosylation factor) GAP (GTPase-activating protein); GIT1-C, the C-terminal of GIT-1; GST, glutathione transferase; Igf2, insulin-like
growth factor 2; IMP1, Igf2 mRNA binding protein 1; LHX, LIM homeodomain protein; mGFP, monomeric GFP; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H -tetrazolium bromide; NES, nuclear export signal; NLS, nuclear localization signal(s); PABP1, polyA-binding protein 1; PAK1, p21-activated
kinase 1; PFA, paraformaldeyhde; β-PIX, β PAK-interacting exchange factor.
1
To whom correspondence should be addressed (email [email protected]).
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Figure 1
J.-M. Dong and others
The focal-adhesion complex protein paxillin can be nuclear
(A) N-terminal truncated paxillin translocates into the nucleus. HeLa cells were transiently transfected with a full-length paxillin (GFP–Pax1−577 ; a) or an N-terminal truncated paxillin (GFP–Pax320−557 ;
b). At 24 h after transfection, cells were fixed with 4 % (w/v) PFA and assessed for cytoplasmic, compared with nuclear, localization of GFP-fusion proteins under a fluorescent microscope.
(B) Blockage of nuclear export causes accumulation of paxillin into the nucleus. Control (a and c) or leptomycin B (10 ng/ml for 2 h)-treated (b and d) COS-7 cells (a and b) or HeLa cells (c and d)
were fixed and stained for endogenous paxillin with a rabbit anti-paxillin antibody.
chromosome. These two genes have very tightly co-ordinated
tissue and stage expression patterns during development. In spite
of significant interest, H19 gene function is not understood.
It has been suggested that H19 functions as a tumour suppressor in Wilms’ tumours, embryonic rhabdomyosarcoma and
the Beckwith–Wiedmann cancer predisposing syndrome [13–
15]; however, other studies suggest that H19 may promote
tumorigenesis [16,17].
c The Authors Journal compilation c 2009 Biochemical Society
In the present study, we demonstrate that paxillin shuttles between the nucleus and cytoplasm, and we uncover the core
NES (nuclear export signal) of paxillin within the LD4 motif.
We also provide evidence that phosphorylation of Ser272 within
LD4 blocks nuclear export, and show that this modification also
reduces GIT1, but not FAK1, binding. Ser272 phosphorylation
does not appear to be mediated by PAK1 (p21-activated kinase
1). Translocation of paxillin into the nucleus promotes DNA
Nuclear-localized paxillin promotes cell proliferation
Figure 2
175
Leptomycin B-induced paxillin nuclear translocation is reversible
The subcellular distribution of endogenous paxillin in HeLa cells was monitored as described in the legend for Figure 1(B) at different time points (as indicated) after the addition of leptomycin B
and after leptomycin B washout. Note that the optical focal plane is 5 μm above the slide surface for a better view of the nuclear and perinuclear area, thus adhesion complexes are not observed.
synthesis and cell proliferation, and its overexpression suppresses
a parental imprinting gene, H19.
EXPERIMENTAL
cDNAs, chemicals and antibodies
Full-length human paxillin cDNA was cloned into the mammalian
expression vector, pXJ40-GFP (where GFP is green fluorescent
protein) [18]. The various paxillin deletion constructs were
created by PCR and each construct was confirmed by sequence
analysis. Leptomycin B, rabbit IgG, BrdU (bromodeoxyuridine)
and a mouse anti-BrdU monoclonal antibody were purchased
from Sigma. A mouse anti-paxillin monoclonal antibody was from
Signal Transduction Laboratories. Alexa Fluor® 488- or
Alexa Fluor® 546-conjugated secondary antibodies were from
Invitrogen.
Cell culture, transfection and immunostaining
HeLa cells were maintained in MEM (minimal essential medium)
supplemented with non-essential amino acids, L-glutamine,
NaHCO3 and 10 % (v/v) FCS (foetal calf serum). COS-7
cells were cultured in DMEM (Dulbecco’s modified Eagle’s
medium) with high glucose supplemented with 10 % (v/v) FCS.
HeLa and COS-7 cells were subcultured on 18 mm × 18 mm
glass coverslips. Transfections were carried out overnight using
calcium phosphate precipitation. Precipitates were then washed
with PBS, and cells were cultured in fresh medium for 24 h.
Cells were fixed in 4 % (w/v) PFA (paraformaldeyhde)/PBS
at room temperature (25 ◦C) for 20 min, washed with PBS for
10 min and permeabilized in 0.2 % Triton X-100/PBS for 10 min.
GFP-expressing cells were either directly viewed after mounting
in Vectashield mounting solution (Vector Laboratories), or
processed with primary (at 37 ◦C for 1 h) and secondary (at
room temperature for 1 h) antibodies before mounting. Stable
cell lines were generated by co-transfection of respective GFPtagged constructs and a selection vector neomycin-resistant gene
at a 20:1 ratio followed by selection in G418-containing medium
(500 μg/ml).
Western blotting, immunoprecipitation and kinase assay
COS-7 cells in 60 mm NUNC culture dishes were transfected
using LipofectamineTM 2000 (Invitrogen) according to the
manufacturer’s protocol. After 8 h, fresh medium was added and
cells were cultured overnight. Total cell lysate was collected in icecold cell lysis buffer [500 μl of 50 mM Hepes (pH 7.3), 150 mM
NaCl, 1.5 mM MgCl2 , 1 mM EDTA, 20 mM β-glycerophosphate,
5 % glycerol, 1 % Triton X-100, 1 mM dithiothreitol and a
protease inhibitor cocktail (Roche)]. Cells were broken by
10 passages through a 29-gauge insulin syringe before clarification at 14 000 g for 10 min. Pellets were washed with lysis buffer
for 15 min on ice, repelleted, and suspended in an appropriate
volume of 1 × SDS sample loading buffer. For SDS/PAGE,
samples were heated (3 min at 95 ◦C), run immediately, and
transferred on to PVDF membranes for Western blot analysis
using a standard protocol. For immunoprecipitation, 200 μl of
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Figure 3
J.-M. Dong and others
Paxillin dynamically shuttles between the nucleus and the focal contacts
A mouse anti-paxillin monoclonal antibody and a rabbit non-specific IgG were co-injected into the nuclei of COS-7 cells. Cells were fixed 15 min, 2 h or 5 h after injection. The antibodies were
detected with Alexa Fluor® 546-conjugated goat anti-mouse (red) or Alexa Fluor® 488-conjugated goat anti-rabbit (green) secondary antibodies respectively.
lysate was passed through 25 μl of anti-FLAG M2–Sepharose
or reduced glutathione–Sepharose and the beads were washed
with 1 ml of lysis buffer before being subjected to SDS/PAGE.
GST (glutathione transferase)-fusion proteins encoding GIT1-C
(the C-terminal of GIT-1) or paxillin LD4 amino acids 250–
281 as indicated were expressed in COS-7 cells, isolated on
glutathione–Sepharose and approx. 0.5 μg of purified protein was
incubated with 0.1 μg of recombinant active PAK1 and 10 μCi
of [γ -33 P]ATP (approx. 6000 mCi/mmol), in kinase assay buffer
[50 mM Hepes (pH 7.3), 10 mM MgCl2 , 2 mM MnCl2 , 1 mM
dithiothreitol and 0.05 % Triton X-100] for 15 min at 30 ◦C. After
separation by SDS/PAGE, proteins were transferred on to PVDF
and subjected to autoradiography for 1 h at room temperature and
then the same filters were processed for Western blot analysis
using anti-GST antibodies.
c The Authors Journal compilation c 2009 Biochemical Society
Microinjection
Diluted mouse anti-paxillin Ig was first dialysed against injection
buffer [50 mM Tris/HCl (pH 7.0), 50 mM KCl and 5 mM MgCl2 ]
for 24 h at 4 ◦C and mixed with rabbit IgG (1 mg/ml). COS-7
cells were grown on glass coverslips to 70 % confluence.
Antibodies were injected into cell nuclei using an Eppendorf
micromanipulator/transjector apparatus. At 15 min, 2 h and 5 h
after injection, cells were fixed and immunostained as decribed
above.
Fluorescent microscopy
A Zeiss axioplan2 fluorescent microscope equipped with a
coolsnap HQ cooled CCD (charge-coupled-device) camera or
Bio-Rad radiance 2000 laser scanning confocal system that was
Nuclear-localized paxillin promotes cell proliferation
Figure 4
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Mapping the paxillin NES sequences
A series of C-terminal and N-terminal deletions of paxillin constructs (as illustrated) were created and fused to the C-terminal of GFP. These were expressed in HeLa cells and scored for nuclear
localization (when the nuclear signal was greater than cytoplasmic signal) of the various fusion proteins. The results shown are the means +
− S.D. from at least five independent experiments.
mounted on a Nikon Eclipse TE300 fluorescent microscope was
used to acquire all of the images. A 60 × oil NA1.4 lens was used.
For protein nuclear localization scoring, when the fluorescent
intensity of the nuclear area was higher than or equal to that of
the cytosol it was defined as nuclear localization.
BrdU-incorporation assay and MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2H -tetrazolium bromide] cell-proliferation assay
After cells were cultured on glass coverslips overnight, BrdU
was added to a final concentration of 32 μM and cultured
for 2 h before fixation, permeabilization and blocking. Cells
were then incubated with mouse anti-BrdU (1:200)/DNaseI
(1.5 units/μl)/BSA(1mg/ml) in PBS at 37 ◦C for 1 h, followed
by Alexa Fluor® 546-conjugated goat anti-mouse IgG antibody
(1:200) at room temperature for 1 h and counter-stained with
DAPI (4 ,6-diamidino-2-phenylindole) for nuclear staining.
Cells were seeded at 3 × 104 cells per 35 mm dish in DMEM
with 10 % (v/v) FCS. The MTT assay was performed 4 h and
30 h after plating in triplicate. MTT stock solution (5 mg/ml)
was from Promega. A 1:10 dilution of the stock was made as
a working solution in culture medium. Per 35 mm dish, 1 ml of
working solution was incubated with cells at 37 ◦C for 30 min and
then removed. The converted dye was solubilized with 1 ml of
acidic propan-2-ol (0.04 M HCl in absolute propan-2-ol). The
dye solution was centrifuged at 16 000 g for 2 min before
the absorbance of colour was measured at a wavelength of 570 nm
with background subtraction at 650 nm.
RNA microarray assay and real-time PCR analysis
Total RNA was isolated using TRIzol® solution (Invitrogen)
according to the manufacturer’s protocol. An RNA microarray
assay was performed on GeneChip Mouse Expression Array 430A
(Affimetrix). Real-time PCR was performed using SYBR Green
Supermix on a MX3005 QPCR system (Stratagene) with β-actin
as an internal control.
Luciferase assay
The promoter region of the mouse H19 gene (− 882 to + 13) was
PCR amplified from genomic DNA of NIH-3T3 cells (forward
primer, 5 -CTGAGTGGTCATGACTGG-3 ; reverse primer,
5 -TCCCACACCCGGTGCTTC-3 ) and cloned into the pGLbasic vector (Promega) in KpnI and HindIII sites to create
pGL-m-H19-P. NIH-3T3 cells were co-transfected with 0.4 μg
of pGL-m-H19-P, 0.3 μg of pCH-110 (Pharmacia) and 0.3 μg of
either pXJ–GFP, pXJ–GFP–Pax1−557 or pXJ–GFP–Pax320−557 using
3 μl of LipofectamineTM 2000 (Invitrogen) according to the
manufacturer’s protocol. Luciferase activities were measured
using a commercial kit (Promega) according to the manufacturer’s instructions. The activity was normalized with β-gal
activity.
RESULTS
In GFP–paxillin-transfected HeLa cells the tagged protein
localized at focal-adhesion sites and was largely excluded from
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Figure 5
J.-M. Dong and others
For legend see facing page
the nucleus, as for endogenous paxillin (Figure 1A); however, an
N-terminal truncated paxillin construct, GFP–Pax320−557 , which
contains the four LIM domains previously defined as the focaladhesion targeting motif [1] exhibited strong focal adhesion
and nuclear localization (Figure 1A). This suggested that the
N-terminal region of paxillin contains an NES. Indeed treatment
of cells with a nuclear export pathway blocker, leptomycin B,
allowed endogenous paxillin to accumulate in the nuclei. As
shown in Figure 1(B), both HeLa and COS-7 cells showed a
nuclear accumulation of endogenous paxillin after leptomycin
B treatment for 2 h. Paxillin started to accumulate in nuclei as
early as 15 min after leptomycin B treatment, and, following
leptomycin B wash-out, paxillin was redistributed back to the
cytosol (Figure 2).
To confirm that endogenous paxillin at steady-state can shuttle
between nuclear and cytoplasmic compartments, COS-7 nuclei
were co-injected with an anti-paxillin IgG compared with a
control IgG. Cells were fixed after microinjection at different
time points, and tested by anti-IgG staining. As shown in Figure 3,
15 min after nuclear microinjection, both anti-paxillin and control IgGs were confined to the nuclei, but after 2 h, the anti-paxillin
IgG, but not the control IgG, appeared in the cytoplasm. After 5 h,
the anti-paxillin IgG clearly decorated focal-adhesion sites, with
control IgG being retained inside nuclei throughout this time. The
appearance of the anti-paxillin IgG in the cytoplasm indicates that
paxillin shuttling facilitates export of its associated antibody from
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the nucleus. Endogenous paxillin in asynchronous growing cells
was largely excluded from the nucleus; we did not find an increase
in nuclear paxillin during S- or G2 -phases of the cell cycle (results
not shown). Similarly paxillin was not nuclear-accumulated when
cells were placed in suspension.
To identify the region responsible for paxillin nuclear export, a
series of C-terminal and N-terminal truncation mutants of paxillin
were fused to the GFP C-terminus. The constructs were expressed
in HeLa cells and assessed for nuclear localization of the GFPfusion protein. The quantified results are shown in Figure 4; results
were indistinguishable in COS-7 fibroblasts (results not shown).
Since GFP–Pax1−248 was nuclear-retained in all of the cells,
whereas GFP–Pax1−316 was essentially completely excluded from
the nucleus, we inferred that the NES was in or around the LD4
motif [19]. Conversely the N-terminal-deleted GFP–Pax281−557
showed 100 % nuclear retention, but GFP–Pax246−557 exhibited
only approx. 20 % nuclear retention; again the difference covers
the LD4 motif. The LD4 motif-deleted GFP–Pax238−293 exhibited a
predominantly nuclear signal (Figure 5A). Scoring of transfected
HeLa cells indicated >40 % of GFP–Pax238−293 -transfected cells
were scored as ‘nuclear GFP’ (i.e. with higher levels of nuclear
compared with cytosolic paxillin), whereas full-length GFP–
Pax1−557 was nuclear-enriched in only approx. 7 % of cells.
We noted that all cells expressing the paxillin lacking LD4
(GFP–Pax238−293 ) showed higher fluorescent intensity within the
nucleus, even if these were not scored as nuclear (Figure 5B);
Nuclear-localized paxillin promotes cell proliferation
Figure 5
179
The LD4 motif is essential for paxillin export from the nucleus
Full-length GFP–paxillin or GFP–paxillin lacking residues 238–293 were expressed in HeLa cells and scored for nuclear localization of respective proteins as described in Figure 4. (A) The results
shown are the means +
− S.D. from at least five experiments. The same constructs in COS-7 cells were assessed using a Bio-Rad Radiance2000 confocal system. Representative images for each
construct are shown (B) and the pixel intensity around nuclei was scanned as a line drawing for each image (C). (D) LD3 and LD4 motifs form a functional NES. Various LD motif deletion constructs
were created and expressed in HeLa cells and the nuclear localization of respective fusion proteins were scored. The results shown are the means +
− S.D. from at least five experiments. (E) The S272A
mutation potentiates NES activity of LD4 . LD4 –LD5 or LD4 constructs with a Ser272 point mutation were created and expressed in HeLa cells and the nuclear localization of respective fusion proteins
were scored. The results shown are the means +
− S.D. from at least five experiments.
we scanned the signal across the cytoplasm and nuclear region to
quantify this process. The scoring method used in this and other
Figures is outlined in the Experimental section.
As unfused mGFP (monomeric GFP) protein is nuclearenriched owing to intrinsic signals (Figure 5D), we conjugated
different combinations of N-terminal constructs of paxillin to
GFP (at its C-terminus) to assay the paxillin NES. As shown
in Figure 5(D), we found that curiously neither LD4 nor the
tandem LD4 and LD5 (Pax246−316 ) were able to drive the export
of nuclear GFP (approx. 80 % nuclear GFP–Pax246−283 ), but that
the tandem LD3 /LD4 (GFP–Pax213−283 ) could drive mGFP export,
as for GFP–Pax135−316 that contains LD2 , LD3 , LD4 and LD5 .
Since deletion of LD4 in the context of LD2 –LD5 (compare with
GFP–Pax135−316LD4 ) completely abolished this NES activity we
concluded that the LD4 motif was essential. The reason for the
differences described above is discussed below. Although the NLS
[nuclear localization signal(s)] could be ascribed to the LIMdomain-containing region (results not shown) we were not able to
further delineate the specific regions involved, probably because
NLS are often bipartite basic sequences that in this instance might
span more than one zinc finger.
Paxillin is a highly phosphorylated protein with several binding
partners [20]; previously a large number of sites have been
mapped by MS [21]. In the N-terminal half of paxillin, a
number of ‘LD motifs’ form docking platforms for partner
targets including actopaxin, vinculin and FAK [22], although
LD3 does not provide such a role [1]. Modification of LD4 (for
example by phosphorylation) might be a means of regulating
the nuclear import–export of paxillin. It has been suggested
that Ser272 in the LD4 motif is subject to phosphorylation
by PAK1, which is required for GIT1 binding [23]. In order
to test the effect of the phosphorylation of this site on the
nuclear retention of paxillin, the phospho-mimetic mutant of
S272D was introduced in GFP–Pax246−316S272D . As for the wildtype LD4/5 protein, this mutant could not drive efficient nuclear
export of GFP (Figure 5E); however, the non-phosphorylatable
S272A mutant (GFP–Pax246−316S272A ) exhibited good NES activity.
Furthermore, in the context of the LD4 alone, GFP–Pax246−283S272A
was also a functional NES (Figure 5E). We concluded that
phosphorylation of Ser272 in the LD4 motif has a major effect
on nuclear export. The most likely explanation for the restored
NES ‘activity’ of the LD3/4 construct might involve phosphatase
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Figure 6
J.-M. Dong and others
Phosphorylation of paxillin Ser272 is not required for GIT1 binding
(A) Paxillin LD4 is essential for GIT1 binding. Constructs encoding paxillin with various N-terminal deletions as indicated were co-transfected with GIT1-C and tested for their ability to co-precipitate.
Only the paxillin construct lacking LD4 (N281) failed to bind GIT1; the recovery of other paxillin constructs indicated that the LD1 and LD2 do not contribute positively to GIT1 binding. (B) Endogenous GIT and PIX levels associated with GST–paxillin LD4 (S272A) are slightly elevated relative to wild-type. COS-7 lysate expressing GST–LD4 was isolated and analysed for associated
endogenous β-PIX and GIT1. (C) Paxillin Ser272 phosphorylation is associated with loss of GIT1 binding. Full-length FLAG–FAK or FLAG–GIT-C was co-transfected with full-length GFP–paxillin
with amino acid substitutions as indicated. The input (25 μg of cell extract) and the immunoprecipitates (from 250 μg of cell extract) were analysed by Western blot using an anti-GFP antibody.
A representative result is shown; significantly less GFP–paxillin(S272D) was recovered with GIT-C compared with FAK (in two independent experiments). The molecular mass in kDa is indicated
on the left-hand side of the gel. (D) Paxillin Ser272 is not phosphorylated by PAK1. GST-fusion proteins encoding GIT1-C or Pax250−281 as indicated were expressed in COS-7 cells, isolated on
glutathione–Sepharose and incubated with recombinant active PAK1. Each lane contained approx. 0.5 μg of purified protein and 0.1 μg of recombinant GST–PAK1 and was incubated for 15 min at
30 ◦C. After separation by SDS/PAGE, proteins were transferred on to PVDF and subjected to autoradiography for 1 h at room temperature, and then the same filters were processed for Western blot
analysis using anti-GST antibodies. FL, full length; IP, immunoprecipitate; WB, Western blot; WT, wild-type.
recruitment by LD3 to dephosphorylate Ser272 . Probably paxillinS272A preferentially associates with the leptomycin-sensitive
export factor CRM1 (chromosome region maintenance 1). The
behaviour of full-length paxillin paralleled smaller constructs,
but much fewer cells exhibited higher nuclear paxillin compared
with cytoplasmic levels. Thus the percentage of cells showing
‘nuclear paxillin’ was 8.6 % for paxillin-S272D compared
with 1.8 % for paxillin-S272A (see Supplementary Figure S1
at http://www.BiochemJ.org/bj/418/bj4180173add.htm). These
numbers indicate that paxillin could be sequestered in the
c The Authors Journal compilation c 2009 Biochemical Society
cytoplasm (by abundant clathrin?), or the NLS could be affected
by modification and/or masking.
The role of Ser272 (equivalent to chicken Ser273 ) phosphorylation
is the subject of some controversy. It has been suggested that
PAK1-driven phosphorylation is required for GIT1 binding [23],
leading to cell spreading and Rac activation. More recently others
have reported that Ser272 modification is deleterious to GIT1
binding [24]. These results are complicated by the notion that LD2
might also contribute to GIT1 binding [25] which would mask any
effect of LD4 modification. We confirmed that LD1 and LD2 motifs
Nuclear-localized paxillin promotes cell proliferation
Figure 7 H19 gene expression is suppressed by the expression of a nuclearlocalized form of paxillin (PaxC)
Real-time PCR was performed using mouse H19 gene-specific primers on mRNA isolated from
stable NIH-GFP, NIH-PaxFL and NIH-PaxC cells. Expression was normalized to β-actin. The
representative real-time PCR traces using mRNA from the cell lines as indicated are shown
in Supplementary Figure S3 (at http://www.BiochemJ.org/bj/418/bj4180173add.htm). Relative
H19 gene-expression levels in different stable cell lines are shown as means +
− S.D. from at
least four experiments (A). The expression level of NIH-GFP was arbitrarily set as 1. Relative
luciferase activities from pGL-mH19-P in NIH-3T3 cells co-transfected with different constructs
as indicated are shown as means +
− S.D. from at least five experiments (B).
do not contribute towards GIT1 binding, using a series of paxillin
constructs lacking LD1 , LD1/2 or LD1/2/3/4 (Figure 6A). Both LD2
and LD4 , when complexed with the paxillin-binding domain of
FAK [which overlaps the FAT (focal-adhesion targeting) domain]
assume a helical structure [26]. First we tested the influence of the
serine/alanine substitution on the ability of the recombinant LD4
domain to recover endogenous GIT1–β-PIX (β PAK-interacting
exchange factor) complex (Figure 6B). The GST–LD4 or GST–
LD(S272A) associated equally well with endogenous GIT1–βPIX from COS-7 cell lysates, indicating that phosphorylation
is not required for GIT1 binding; indeed the recovery with the
LD4 (S272A) was consistently better. We then compared how these
interactions behaved with respect to full-length paxillin S272D
(i.e. the phospho-mimetic mutant) binding to either FAK or GIT1,
since these both compete for the paxillin LD4 motif [27,28]. Fulllength paxillin and FLAG–FAK compared with FLAG–GIT were
tested; the phospho-mimetic significantly weakened the paxillin–
GIT1, but not the paxillin–FAK, interaction (Figure 6C). As for the
kinase responsible for phosphorylation of paxillin Ser272 , although
GST–LD4 was weakly phosphorylated by recombinant PAK1, no
differences in 32 P-labelling were seen with wild-type compared
with S272A (Figure 6D). We conclude that PAK1 is not the kinase
that phosphorylates paxillin Ser272 , and within the LD4 construct
the most likely ‘PAK1 site’ could be Ser258 . Thus phosphorylation
of paxillin LD4 is not essential for GIT1 binding as previously
suggested [23], but rather negatively affects its binding. The
Ser272 -phosphorylated paxillin preferentially interacts with FAK
and perhaps clathrin [28], which could both serve to sequester the
protein in the cytosol via LD4 binding, as well as LD2 binding
181
[25]. This might explain why the LD4 -deleted construct is not
entirely nuclear localized. A model for this nuclear-cytoplasmic
shuttling is presented in Figure 8. Since FAK has been established
as a nuclear protein [29,30], we conclude that the two proteins
could form a complex at this site.
Cell adhesion is essential for cell-cycle progression of adherent
cells [31], and we were interested in whether nuclear-localized
paxillin might affect cell proliferation. This was examined
in two ways. First, NIH-3T3 cell lines expressing GFP–
Pax320−557 (NIH-PaxC) or GFP (NIH-GFP) were established. BrdU
incorporation in non-synchronized cells was assessed in these
NIH-PaxC and NIH-GFP cells. Under these conditions, BrdU
incorporation (over 2 h) was clearly higher in the NIH-PaxC
compared with the NIH-GFP cells (see Supplementary Figure
S2 at http://www.BiochemJ.org/bj/418/bj4180173add.htm). This
approx. 45 % increase in BrdU incorporation for NIH-PaxC cells,
compares with an NIH-3T3 line expressing the full-length paxillin
(NIH-PaxFL) which was essentially the same as for NIH-GFP
cells (Supplementary Figure S2). Secondly, cell proliferation, as
determined by the MTT assay, indicated that the cell-growth rate
of NIH-PaxC was faster than that of NIH-GFP and NIH-PaxFL
(Supplementary Figure S2).
We considered that these changes in proliferation rate probably
result from transcriptional changes brought about by elevated
levels of paxillin (C-terminal LIM domains) in the nucleus.
Gene expression was therefore assessed using mRNA microarray
analysis on Mouse Expression Array 430A (Affymatrix). Those
genes which scored for significant changes in expression are
shown in Supplementary Table S1 (at http://www.BiochemJ.org/
bj/418/bj4180173add.htm). Real-time PCR using individual
gene-specific primer pairs on mRNA isolated from the three
cell lines was then used to assess the reproducibility of the
microarray results (results not shown). The most consistent and
significant change involved a parental imprinting gene, H19
(GenBank® accession number: NM_023123.1), whose expression
was suppressed in the NIH-PaxFL or NIH-PaxC cells compared
with NIH-GFP cells (Figure 7A). Since this gene is wellestablished as being associated with cell proliferation, the mouse
H19 gene promoter region (− 882 to + 13) was amplified from
NIH-3T3 cell genomic DNA and cloned into a luciferase reporter
plasmid, pGL-basic, to give pGL-m-H19-P. The activity of this
reporter was then tested for the effects of various paxillin
constructs in transiently transfected NIH-3T3 cells using the
luciferase reporter. These experiments showed that elevated
paxillin levels can inhibit H19 gene expression (Figure 7B),
and point to changes in levels of H19 as being responsible for
promoting faster cell-cycle progression in the paxillin-expressing
NIH-3T3 cell line.
DISCUSSION
Paxillin is a member of LIM protein superfamily [2,32]. LIM
proteins have been classified into three groups [2]. Proteins
containing A and B class LIM domains in tandem, such as LHX
(LIM homeodomain proteins) belong to group 1. Proteins in
group 2 are largely composed of class C LIM domains, such
as CRIPs (cysteine-rich intestinal proteins) and CRPs (cysteinerich proteins). Group 3 includes proteins containing different
numbers of LIM domains located at the C-terminus, such as
paxillin, zyxin and PINCH. Some LIM proteins, such as the
LHX are exclusively nuclear and have clear transcriptional
roles during development [32]. Many LIM proteins are directly
or indirectly actin-associated, including paxillin, zyxin, FHL
(four-and half LIM) and CRP families [33]. It has become
c The Authors Journal compilation c 2009 Biochemical Society
182
Figure 8
J.-M. Dong and others
A model for paxillin nuclear–cytoplasmic shuttling
Both the phosphorylated paxillin (at Ser272 ) and non-phosphorylated version of the protein are present at focal adhesion; however, modification favours FAK association. Paxillin phosphorylation can
aid nuclear retention of paxillin, where it might also be associated with nuclear FAK.
apparent that many cytoskeleton-associated LIM proteins shuttle
between cytoplasmic and nuclear compartments. For example,
Hic5/ARA55, which is highly homologous with paxillin, shuttles
between focal adhesion and the nucleus through an oxidantregulated mechanism, and is reported to function as an adaptorlike nuclear receptor co-activator [5,6]. Paxillin is also suggested
to be able to shuttle between the cytoplasm and nucleus [9,10],
but the underlying mechanism is still unclear. Consistent with the
previous findings, we show that endogenous paxillin accumulates
in the nucleus upon the blockage of the CRM1/exportin 1-dependent nuclear export pathway with leptomycin B, indicating that
nuclear paxillin translocates to the cytoplasm through the exportin
pathway (Figures 1 and 2). An anti-paxillin antibody, when injected into the nucleus, allowed us to visualize the export of paxillin
in the absence of drugs. With this approach, we can detect
paxillin in the nucleus which, when complexed to the antibody,
undergoes CRM1/exportin 1-dependent export (Figure 3).
Zyxin is also reported to cycle between adhesion complexes and
the nucleus via an NES mapped to its N-terminal portion [7,8].
Although paxillin has no conventional export signal predicted
by NetNES [34], the present study revealed that the LD4 region
of paxillin indeed contains a functional NES (Figures 4 and
5). The LD4 region is essential for nuclear export of paxillin,
and its deletion leads to retention of paxillin inside the nucleus
(Figure 5). Curiously this region did not behave as a functional
c The Authors Journal compilation c 2009 Biochemical Society
NES for mGFP; however, the tandem LD3 and LD4 domains
have a potent NES activity (Figure 5). Since substitution of
Ser272 with alanine allows NES activity of LD4 (Figure 5E) we
infer that phosphorylation is the switch that blocks NES activity
and thus helps to retain nuclear paxillin, as indicated in the
model (Figure 8). The paxillin homologue Hic5 also has an
NES that overlaps the region of its LD4 motif [6]; this region
has similar protein partners including GIT1 [35]. We have not
detected the paxillin-associated endogenous GIT1 in the nucleus
(results not shown), suggesting that the bulk of nuclear paxillin is
phosphorylated. Modification of Ser273 (of chicken paxillin) can
increase cell migration, protrusion and adhesion turnover; the
suggestion that this occurs via enhancing paxillin–GIT1 binding
[23] is unlikely. Clearly modification of (human) Ser272 decreases
rather than increases the paxillin–GIT1 association (Figure 6), in
agreement with more recent reports [24]. Furthermore, PAK1 is
not the kinase that phosphorylates paxillin Ser272 (Figure 6D); the
primary sequence has no similarity with the profile of PAK1 target
sites [36], as PAK1 is a basic directed kinase. One of the important
roles of Ser272 phosphorylation is thus to allow nuclear retention
(Figure 5E). Interestingly, a phosphatase regulatory subunit, PP2A
B56, was reported to interact with paxillin and dephosphorylate
it at its serine site(s) [37]. Although this interaction with paxillin
has yet to be mapped, a working hypothesis is that LD3 might
provide a platform to promote Ser272 dephosphorylation. This
Nuclear-localized paxillin promotes cell proliferation
would explain why LD3 /LD4 has good NES activity, but the
LD4 does not. In the context of the full-length protein, other
compensatory phosphorylation events around the NLS in the LIM
domain may dampen this effect. Clearly the interaction with the
nuclear exporter CRM1 plays a critical role in regulating the level
of nuclear paxillin. Such modulation is reported for the highly
related Hic5, where nuclear export is regulated by the redox state
of the cells, and oxidants lead to Hic5 accumulation in the nucleus
[6]. Paxillin does not behave similarly (J.-M. Dong and E. Manser,
unpublished work and [6]). One proposal is that cysteine residues
near the LD2 region contribute to redox-sensitive NES [6], but are
missing in paxillin.
Cell adhesion via integrins strongly promotes cell division of
adherent cells [38,39]. Increased levels of paxillin (predominantly
cytoplasmic) are ineffective with respect to enhancing cell
proliferation (Supplementary Figure S2); however, the Nterminal-truncated paxillin, which is nuclear-enriched, both
enhances the number of cells entering the cell cycle (i.e.
undergoing DNA synthesis) and thus promotes cell proliferation
(Supplementary Figure S2). Paxillin is reported to be a carrier
for PABP1 that targets the mRNA-binding protein to the leading
edge of migrating cells [10]. Paxillin is also reported to function
as a co-activator for the androgen receptor and glucocorticoid
receptor in prostate cancer cell lines [11]. In the present study, we
uncover a role for paxillin in the suppression of the H19 gene, an
imprinted, maternally expressed gene [40]. In NIH-3T3-derived
cell lines the H19 gene expression was suppressed by full-length
paxillin and by a ‘nuclear paxillin’, Pax320−557 , lacking the Nterminal protein interaction domains (Figure 7). We confirmed
that paxillin modulates H19 expression at the transcriptional level
using a luciferase reporter containing an approx. 900 bp upstream
sequence from the H19 transcriptional start site (Figure 7B).
The negative effect of H19 on cell growth was revealed by
studies on H19-knockout mice lines [41,42]. Both H1913
and H193 mice showed 8–25 % overgrowth phenotype and
inappropriate reactivation of Igf2 gene expression. An effect of
H19 RNA on Igf2 expression is suggested by several previous
studies [43,44]. The H19 RNA can bind to the IMP1 (Igf2 mRNA
binding protein 1), so to sequester IMP1 in the cytoplasm. IMP1
binds to the 5 -UTR of the Igf2 mRNA and regulates its translation,
as shown by IMP1-deficient mice which exhibit Igf2 translational
down-regulation and dwarfism. In this context, it is possible
that suppression of H19 gene expression in ‘nuclear paxillin’expressing cells might also promote Igf2 expression at the
translational level and thus favour cell proliferation. Alternatively,
the enhanced cell proliferation we observed may be due to other
mechanisms, such as the ability of paxillin to potentiate steroid
hormone receptor activities as shown in prostate cancer cells [11].
These are worth further investigation in the future.
FUNDING
This work was supported by the GSK-Singapore Research Fund.
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doi:10.1042/BJ20080170
SUPPLEMENTARY ONLINE DATA
Paxillin nuclear-cytoplasmic localization is regulated by phosphorylation
of the LD4 motif: evidence that nuclear paxillin promotes cell proliferation
Jing-Ming DONG*, Lei-Shong LAU*, Yuen-Wai NG*, Louis LIM*† and Ed MANSER*1
*GSK-IMCB Group, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore, and †Department of Molecular Neuroscience, Institute of Neurology,
University College London, 1 Wakefield Street, London WC1N 1PJ, U.K.
Figure S1 Wild-type (w/t) or Ser272 mutants of full-length GFP–paxillin were
expressed in HeLa cells and scored for nuclear localization of respective
proteins
The results shown are the means +
− S.D. from at least four experiments.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
J.-M. Dong and others
Figure S2
Expression of nuclear-localized paxillin enhances BrdU incorporation and promotes cell proliferation
Stable NIH-3T3 cell lines designated as NIH-GFP and NIH-PaxC were plated on to coverslips overnight and then transiently labelled with 32 μM BrdU for 2 h. After fixation, cells were stained with
mouse anti-BrdU and counterstained with DAPI (4 ,6-diamidino-2-phenylindole). The images were acquired using a Coolsnap HQ cooled CCD (charge-coupled-device) camera and representative
4
images are shown (A). Quantified data are shown as means +
− S.D. from at least four experiments (B). Stable NIH-GFP, NIH-PaxFL and NIH-PaxC cells were plated at 3 × 10 cells per 35 mm dish.
An MTT cell-proliferation assay was performed 4 h (as a reference) and 30 h after cell plating in triplicate. The results shown are means +
− S.D. from at least five experiments (C). The absorbance
reading at the 4 h time point was arbitrarily set as 1. The results were generated from stable clonal cell lines; at least two different clones were tested for each experiment which yielded similar results.
c The Authors Journal compilation c 2009 Biochemical Society
Nuclear-localized paxillin promotes cell proliferation
Figure S3
H19 gene expression is suppressed by expression of a nuclear-localized form of paxillin (PaxC)
Real-time PCR was performed using mouse H19 gene-specific primers on mRNA isolated from stable NIH-GFP, NIH-PaxFL and NIH-PaxC cells. Expression was normalized to β-actin. The
representative real-time PCR traces using mRNA from the cell lines as indicated are shown.
Table 1
List of genes that show significant changes in expression by mRNA microarray analysis
Expression level
Microarray number
GenBank® accession number
Description
Decrease*
1448194_a_at
1419519_at
1433919_at
1437401_at
1424932_at
1423294_at
1450708_at
1452014_a_at
1427574_s_at
1420512_at
1426439_at
1427760_s_at
1438564_at
1422155_at
1418072_at
1452540_a_at
1425078_x_at
1418367_x_at
1436994_a_at
NM_023123.1
BC012409.1
AV302111
BG075165
AF275367.1
AW555393
NM_009129.1
AF440694.1
BF232848
NM_020265.1
AJ007376.1
X75557.1
BM507943
BC015270.1
NM_023422.1
M25487.1
BC007193.1
BC010564.1
BB533903
H19 fetal liver mRNA (H19), mRNA
Insulin-like growth factor 1, mRNA
Similar to ankyrin repeat and SOCS (suppressor of cytokine signalling) box-containing protein 4
Similar to nsulin-like growth factor 1 (exon 6)
Epidermal growth factor receptor (Egfr) mRNA
Similar to mesoderm-specific transcript
Secretogranin II (Scg2), mRNA
Insulin-like growth factor 1, mRNA
Similar to SH3 domain protein D19 mRNA
Dickkopf homologue 2 (Dkk2), mRNA
mRNA for DBY RNA helicase
mRNA for proliferin
Similar to Fanconi anaemia, complementation mRNA
Histone cluster 2, H3c2, mRNA
H2bc histone cluster1
H2B histone family, 3 end
Similar to intracellular pathogen resistance 1 (Ipr1) mRNA
Histone cluster 2, H2aa1, mRNA
Similar to histone cluster 1, h1c
Increase†
* Expression levels decreased more than 4-fold in either NIH-PaxFL or NIH-PaxC cells compared with NIH-GFP cells; the decrease was more in NIH-PaxC cells than in NIH-PaxFL cells.
† Expression levels increased more than 4-fold in either NIH-PaxFL or NIH-PaxC cells compared with NIH-GFP cells; the increase was more in NIH-PaxC cells than in NIH-PaxFL cells.
Received 22 January 2008/15 October 2008; accepted 6 November 2008
Published as BJ Immediate Publication 6 November 2008, doi:10.1042/BJ20080170
c The Authors Journal compilation c 2009 Biochemical Society