Download Profilin regulates the activity of p42 , a novel Myb

Research Article
331
Profilin regulates the activity of p42POP, a novel Mybrelated transcription factor
Marcell Lederer, Brigitte M. Jockusch and Martin Rothkegel*
Cell Biology, Zoological Institute, Technical University of Braunschweig, 38092 Braunschweig, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 18 October 2004
Journal of Cell Science 118, 331-341 Published by The Company of Biologists 2005
doi:10.1242/jcs.01618
Journal of Cell Science
Summary
Profilins, regulators of cytoplasmic actin dynamics, also
bind to several nuclear proteins but the significance of these
interactions is mostly unclear. Here, we describe a novel
Myb-related transcription factor, p42POP, as a new ligand
for profilin and show that profilin regulates its activity.
p42POP comprises a unique combination of domains and is
widely expressed in mouse tissues. In contrast to many
other Myb proteins, it contains only one functional
tryptophan-cluster motif. This is followed by an acidic
domain, a leucine zipper that mediates dimerization and
functional nuclear import and export signals that can
direct p42POP to either the nuclear or the cytoplasmic
compartment. Binding to profilins is mediated by a proline-
rich cluster. p42POP-profilin complexes can be precipitated
from cell lysates. In transfected cells displaying p42POP in
the nucleus, nuclear profilin is markedly increased. When
p42POP is anchored at mitochondrial membranes, profilin
is targeted to this location. Hence, in a cellular
environment, p42POP and profilin are found in the same
protein complex. In luciferase assays, p42POP acts as
repressor and this activity is substantially reduced by
profilins, indicating that profilin can regulate p42POP
activity and is therefore involved in gene regulation.
Introduction
Profilins (Carlsson et al., 1976) are small (14-17 kDa)
ubiquitous proteins with multiple functions. Originally, they
were described as G-actin-binding proteins, and it was
therefore postulated that they are mainly responsible for Gactin sequestration in cells with a high content of
nonfilamentous actin, but a wealth of in vitro and in vivo data
collected more recently suggest that profilins instead promote
cellular actin-filament assembly (Kang et al., 1999; Pantaloni
and Carlier, 1993; Wolven et al., 2000). This is particularly
important for actin-filament dynamics at the plasma
membrane, where profilins might form complexes with G-actin
and the actin-related protein Arp2, a component of the Arp
nucleator complex (Blanchoin et al., 2001; Machesky, 1997).
At this location, profilins might also bind to phospholipids
such
as
phosphatidylinositol-4,5-bisphosphate
and
phosphatidylinositol-3,4,5-trisphosphate (Haarer et al., 1993;
Lambrechts et al., 1997; Lassing and Lindberg, 1985;
Machesky et al., 1990). Because the binding of profilin to Gactin and phospholipids is mutually exclusive (Lassing and
Lindberg, 1985), the interaction with phosphatidylinositol-4,5bisphosphate might disrupt the profilin-actin complex and link
profilin-dependent actin dynamics to signal transduction (Sohn
and Goldschmidt-Clermont, 1994) (for review, see Schluter et
al., 1997). Consistent with its suggested role in membraneassociated actin dynamics, fluorescence microscopy revealed
profilin at the periphery of lamellipodia (Buss et al., 1992;
Geese et al., 2000), focal adhesions (Geese et al., 2000;
Mayboroda et al., 1997) and surface ruffles (Wittenmayer et
al., 2000). However, a contribution to actin dynamics at the
plasma membrane is clearly only part of profilin’s multifaceted
activities. Recently, it was shown that binding of profilin to
nuclear actin is a prerequisite for the exportin-dependent
transport of actin to the cytoplasm (Stuven et al., 2003).
Furthermore, the actin-binding site of profilins is not
exclusively confined to actin binding: gephyrin, a scaffolding
protein linking receptors to the submembrane cytoskeleton of
neuronal cells and a profilin ligand (Mammoto et al., 1998),
occupies the same binding motif (Giesemann et al., 2003).
Hence, profilins make use of their actin-binding site in diverse
cellular processes, cytoplasmic as well as nuclear.
Similarly, cytoplasmic and nuclear activities have been
described for the polyproline-binding motif of profilins
(Mahoney et al., 1999). Among the many polyproline-clustercontaining proteins that have been identified as ligands for this
binding site are proteins also involved in plasma-membraneassociated actin dynamics and actin organization, such as the
Ena/VASP proteins (Gertler et al., 1996; Reinhard et al., 1995),
members of the diaphanous subclass of the formin homology
proteins (Chang et al., 1997; Evangelista et al., 1997; Krebs et
al., 2001; Watanabe et al., 1997) and the Wiskott-Aldrich
syndrome protein (WASP) (Suetsugu et al., 1998). However,
profilin also interacts with the proline-cluster protein survival
motoneuron protein (SMN) (Giesemann et al., 1999), which is
involved in RNA processing (Fischer et al., 1997). Consistent
with the view that profilin is engaged in RNA processing, it
was localized to intranuclear bodies such as speckles, Cajal
bodies and gems (Giesemann et al., 1999; Skare et al., 2003).
Here, we report a novel interaction partner of profilin in the
nucleus. We show that profilin directly binds to a hitherto
Key words: Cytoskeleton, Gene regulation, Leucine zipper, Myb
protein, Nuclear profilin
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Journal of Cell Science 118 (2)
unknown transcription factor, termed p42POP, which is widely
expressed in mouse tissues. It comprises a unique combination
of a single Myb-like domain, an acidic domain, nuclear import
and export signals, a leucine zipper and a proline cluster
mediating profilin binding. Profilin-p42POP complex formation
is seen in vitro and in transfected cells. In luciferase assays,
p42POP represses transcription, and this activity is substantially
altered by the interaction with profilin. Hence, this study
expands our knowledge of nuclear ligands and nuclear
activities of profilins and suggests a functional engagement of
profilins in gene regulation.
Journal of Cell Science
Materials and Methods
Yeast two-hybrid system and cDNA screening
Using the Matchmaker Yeast Two-Hybrid System (Clontech,
Heidelberg, Germany), we screened a mouse 17-day embryo cDNA
library (Clontech) with mouse profilin 1 cDNA (Widada et al., 1989)
as a bait. Positive colonies were selected and analysed according to
the manufacturer’s protocols (Clontech). Quantitation of interactions
was performed with liquid assays measuring β-galactosidase activity,
using O-nitrophenyl-β-D-galactoside as a substrate, according to the
manufacturer’s advice (Clontech).
A mouse 17-day embryo cDNA library (ML5000b in λgt-11,
Clontech) was screened with the ∆POP1 cDNA fragment using
standard protocols (Sambrook et al., 1989). Two positive clones were
obtained and the DNA sequences of the respective inserts were
analysed in a model 310 DNA sequencer (Applied Biosystems,
Weiterstadt, Germany).
Tissue-specific expression
Tissue-specific expression of POP was analysed by quantitative PCR
using normalized mouse multiple tissue cDNA (MTC™) panel I
(Clontech), following the manufacturer’s protocol, and was
subsequently verified by Southern hybridization.
Plasmid constructs
cDNA fragments encoding p42POP (Fig. 1c) were subcloned into the
vectors pGBKT7, pGADT7 and pEGFP-C2 (Clontech), pcFLAG
(Sigma-Aldrich, Deisenhofen, Germany), pcDNA3-BiPro (Rudiger et
al., 1997), pGEX-4T1 (Amersham Biosciences, Freiburg, Germany),
pQE-30 (Qiagen, Hilden, Germany) and pcDNA3-MOM-BiPro∆POP6 (Kaufmann et al., 2000). Mouse profilin 1 and profilin 2a
cDNAs were subcloned into the vectors pGBTB7, pGADT7
(Clontech), pET21c (Novagen, Schwalbach, Germany) and pcDNA3
(Invitrogen). To construct the reporter plasmid pGL3-mre-luc, the
DNA sequence 5′-CGCTCGAGATCGACACATTATAACGGTTTTTTAGCG-3′, comprising the Myb recognition element within the mim
promoter (mreA) (Miglarese et al., 1996) was synthesized and inserted
into the pGL3-promoter vector (Promega, Mannheim, Germany). The
c-Myb expression vector pcDNA-cMyb (Oelgeschlager et al., 1995)
served as a control in luciferase assays. The plasmid pCMV-nls-lacZ,
encoding β-galactosidase, was used to monitor transfection efficiency
(Winter et al., 1993). p42POP mutants were generated using the
QuikChange™ site-directed mutagenesis kit (Stratagene, Amsterdam,
The Netherlands) with appropriate oligodeoxynucleotides and verified
by DNA sequencing.
Protein expression, purification, crosslinking, pull-down assays
and immunoblotting
Recombinant profilin 1 and profilin 2a were expressed in Escherichia
coli and purified by poly-L-proline affinity chromatography as
described (Giehl et al., 1994; Wittenmayer et al., 2000). In vitro
translation of p42POP fragments was performed according to the
manufacturer’s instructions (TNT®T7 coupled transcription/
translation system, Promega). p42POP and its deletion and point
mutants were expressed in E. coli. Proteins were equipped with a His
tag or fused to glutathione-S-transferase (GST) for purification on NiNTA/Sepharose or on glutathione-Sepharose, respectively. For
monitoring these p42POP variants in pull downs and immunoblots,
they were equipped with GST, green fluorescent protein (GFP), the
FLAG tag or the BiPro tag (Rudiger et al., 1997). To study interactions
between different p42POP fragments in pull-down assays, E. coli
extracts containing GST-tagged fragments were mixed with extracts
containing GFP-tagged p42POP fragments. The mixtures were
incubated for 16 hours at 4°C in buffer A (50 mM NaH2PO4, 300 mM
NaCl, 10 mM imidazole, 1 µM pepstatin A, 50 µM pefabloc SC, 0.46
µM aprotinin, pH 8.0) and subsequently incubated with glutathioneSepharose for 1 hour at 4°C. Sepharose beads were sedimented by
centrifugation for 5 minutes at 500 g. After five washes with buffer B
(50 mM NaH2PO4, 150 mM NaCl, 0.5% Triton X-100, 50 µM
pefabloc SC, 0.46 µM aprotinin, pH 8.0), the Sepharose pellet was
resuspended in sample buffer and the precipitated proteins were
subjected to SDS-PAGE and subsequent immunoblotting with
antibodies against GFP (Clontech) or GST (Amersham Biosciences).
For profilin pull-down assays, profilin 1 coupled to Nhydroxysuccinimide (NHS)-activated Sepharose was incubated for 4
hours at 4°C with in-vitro-translated [35S]-labelled p42POP mutants in
PBS supplemented with 0.1% Triton X-100, 0.5% bovine serum
albumin (BSA) and 20 mM β-mercaptoethanol. Sepharose beads were
sedimented and washed with PBS, PBS + 150 mM NaCl and PBS +
0.2% Triton X-100. The precipitated proteins were resuspended
in sample buffer, separated by SDS-PAGE and analysed by
autoradiography. To analyse p42POP dimerization and the binding of
profilin to monomeric or dimerized p42POP, purified FLAG-tagged
p42POP fragments were subjected to chemical cross-linking with 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide/NHS (EDC/NHS) as
described (Mullins et al., 1997), in the absence or presence of profilin
1, subjected to SDS-PAGE and to immunoblotting with monoclonal
antibodies against the FLAG tag (Sigma) and profilin (Mayboroda et
al., 1997).
Electrophoretic-mobility-shift assays
Electrophoretic-mobility-shift assays (EMSAs) were performed
essentially as described previously (Miglarese et al., 1996). Briefly,
either in-vitro-translated p42POP or purified deletion mutants were
incubated with [32P]-end-labelled double stranded (ds) synthetic
oligodeoxynucleotides (5′-GGTCGATCGACACATTATAACGGTTTTTTAGC-3′) containing the mreA sequence. For competition assays,
the in-vitro-translated protein was preincubated with different
amounts of unlabeled competitor oligodeoxynucleotides at 30°C for
30 minutes and subsequently with the labelled oligodeoxynucleotide
for 20 minutes. A random ds oligodeoxynucleotide sequence served
as control. The DNA-protein complexes were separated on native 5%
polyacrylamide gels and analysed by autoradiography.
Cell culture, transfection and fluorescence microscopy
PtK2 and HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% foetal bovine serum. For transient
expression of recombinant proteins, cells were transfected with
vectors coding for either BiPro- or GFP-tagged p42POP proteins, using
calcium-phosphate precipitation. For fluorescence microscopy, cells
were grown on coverslips, fixed 24-48 hours after seeding by
treatment with 4% formaldehyde, and permeabilized with 0.2% Triton
X-100. For indirect immunofluorescence, the cells were rinsed three
times in PBS, incubated with antibodies against the BiPro tag
(Wiedemann et al., 1996) or a monoclonal (Mayboroda et al., 1997)
or a polyclonal (Buss and Jockusch, 1989) against profilin for 1 hour,
Profilin regulates p42POP
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Journal of Cell Science
Fig. 1. Deduced amino acid sequence and molecular anatomy of
p42POP. The various motifs identified by the program PSORTII are
indicated. The sequence of ∆POP1, identified in the yeast two-hybrid
analysis as a profilin ligand, is underlined.
washed three times in PBS, and further incubated with the appropriate
secondary antibodies conjugated with fluorescein, rhodamine (Sigma)
or Alexa-488 (Molecular Probes). Mitochondria were stained by
MitoTracker™ (Molecular Probes, Göttingen, Germany) and cell
nuclei by 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma). After three
washes, the cells were mounted in Mowiol (Hoechst, Frankfurt,
Germany). Fluorescence images were taken with either a conventional
fluorescence microscope (Axiophot, Zeiss, Oberkochen, Germany)
equipped with a digital CCD camera (MicroMax, Princeton
Instruments, Trenton, NJ) and processed by MetaMorph Imaging
software (Visitron Systems, Puchheim, Germany) or a confocal laserscanning microscope (LSM510, Zeiss). Quantitation of nuclear
profilin was performed with PtK2 cells expressing p42POP, ∆POP4 or
∆POP6 fused to GFP and stained with the monoclonal anti-profilin
antibody. Images were taken by confocal laser-scanning microscopy,
nuclei were identified by DAPI stain and the nuclear profilin content
was determined by quantitation of the fluorescence signals by
LSM510 Meta version 3.0 (Zeiss). The ratios of the nuclear profilin
contents of transfected cells to those of untransfected cells were
compared by variance analysis (Student-Newman-Keuls; P<0.05)
using Statview 5.0 (SAS Institute, Cary, NC).
Luciferase assay
16-20 hours after seeding, HeLa cells were co-transfected with the
reporter plasmid pGL3-mreA-luc and different p42POP constructs and
pcDNA-cMYB; also, to monitor transfection efficiency, the cells were
transformed with pCMV-nls-lacZ, which encodes β-galactosidase.
Luciferase activity was monitored with the Steady Glo Luciferase
Assay (Promega), according to the manufacturer’s instructions. The
values obtained were normalized to β-galactosidase activity,
according to standard protocols.
Results
p42POP, a multidomain protein, is a novel ligand for
profilins
In a yeast two-hybrid assay, we screened a mouse embryo
cDNA library for novel profilin ligands, using mouse profilin
1 as a bait. This search yielded an unknown polypeptide of 309
amino acids (∆POP1; Fig. 1, underlined). To identify the fulllength coding sequence, a mouse-embryo cDNA λ phage
library was screened with the ∆POP1 cDNA. One of the
Fig. 2. p42POP is a widely produced nuclear protein with functional
NLS and NES. (A) Expression pattern of p42POP obtained by
quantitative PCR and subsequent Southern-blot analysis with
standardized samples. p42POP is produced in all adult mouse tissues
tested, with high expression levels in brain and testis, and is already
present at embryonic stage E7. (B,C) Subcellular localization of
BiPro-tagged p42POP (B) and ∆POP6 (C) in PtK2 cells transfected
with the appropriate constructs, as revealed by immunofluorescence
with anti-BiPro antibody. (D) Compiled data for all constructs tested
in analogous assays, using either BiPro- or GFP-tagged probes. Both
sets of experiments gave identical results. Whereas p42POP was seen
exclusively in the nucleus, deletion of the C-terminal NLS resulted in
an exclusively cytoplasmic location of the appropriate fragments
∆POP6 and ∆POP9. Bar, 10 µm.
isolated clones contained a candidate DNA of 1913 bp. The
longest open reading frame within this sequence starts with an
ATG codon in position +174 and encodes a hitherto-unknown
protein of 393 amino acids with a calculated molecular mass
of 41,890 kDa, which was termed p42POP (partner of profilin;
GenBank AF364868; Fig. 1).
Amino acid sequence comparisons in databases revealed
p42POP as a novel protein. The N-terminus contains a
tryptophan cluster motif, similar to the DNA-binding domain
(DBD) of Myb-related transcription factors, but only a single
copy (Fig. 1). This is followed by an acidic region (Fig. 1)
reminiscent of the transcriptional activation domains present in
several Myb proteins. Furthermore, p42POP displays a leucinezipper motif in its C-terminus (Fig. 1) and two nuclear location
signals (NLSs) in its N- and C-termini (Goldfarb et al., 1986;
Kalderon et al., 1984). The N-terminal NLS overlaps with the
DBD (Fig. 1).
Additional motifs were identified in the C-terminal half of
p42POP. A consensus nuclear export sequence (NES) (Wen et
al., 1995) was recognized (Fig. 1), followed by three discrete
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Journal of Cell Science 118 (2)
Journal of Cell Science
proline-rich regions (Fig. 1). Two of these contain consecutive
proline sequences (P5 in the first, P12 in the second of these
regions, Fig. 1), the third harbours a putative SH3-binding
module (KPLPPAP; Fig. 1) with homology to the class-I SH3binding motif R/KPLPPψP (where ψ is a hydrophobic residue)
(Mayer and Gupta, 1998; Rickles et al., 1995).
covalently linked to Sepharose beads and in-vitro-translated
p42POP fragments (Fig. 3B). Only the complete molecule and
the fragment C-POP (containing the polyproline stretches P5
and P12) precipitated with profilin, whereas the N-terminal
fragment N-POP did not. Addition of actin to profilin-p42POP
mixtures did not significantly alter these results, which is
consistent with the assumption that the actin-binding site does
not interfere with the polyproline-binding site of profilin
(Schluter et al., 1997).
p42POP is a widely expressed nuclear protein
p42POP expression was analysed by using quantitative PCR and
subsequent Southern blot on standardized samples of various
embryonic stages and adult mouse tissues. POP transcripts
were identified in all adult tissues tested and were already
present in mouse embryos at day 7 (E7; Fig. 2A). Localization
of p42POP was monitored by fluorescence microscopy. PtK2
cells were transfected with a gene encoding p42POP fused to
either GFP or the BiPro tag, for detection with an antibody to
BiPro. As expected for a putative transcription factor, p42POP
was localized in the nucleus (Fig. 2B). To evaluate the
significance of the NLS and NES motifs identified in p42POP
for cellular localization, several GFP-fused truncated mutants
were expressed in PtK2 cells. As shown in Fig. 2D, GFPp42POP and the truncations ∆POP4, ∆POP7 and ∆POP8 were
localized to the nucleus, indicating that the N-terminal as well
as the C-terminal NLS can direct the polypeptides to the
nucleus, in the absence of the putative NES. By contrast,
∆POP6 and ∆POP9, which include the NES, were found
exclusively in the cytoplasm (Fig. 2C,D), whereas ∆POP5,
which contains both the C-terminal NLS and the NES,
partitioned equally between the cytoplasm and the nucleus
(Fig. 2D).
Dimerization of p42POP interferes with profilin binding
Calculation of the secondary structure elements of p42POP by
the program Lasergene (DNAStar, Madison, WI) reveals an
amphipathic helix between amino acids 250-290, including
the putative leucine-zipper motif (Fig. 1). Initial dot-overlay
experiments indicated self-association of p42POP (data not
shown). To confirm these results and to determine the role of
the putative leucine zipper in such a process, we again used
a yeast two-hybrid analysis. Using different p42POP deletion
constructs, we could localize the self-association region to
amino acids 203-329, which contain the leucine zipper (Fig.
1, Fig. 4A). Furthermore, glutathione-Sepharose pull downs
with recombinant GST-∆POP14 (which also includes the
entire leucine zipper) and GFP-tagged p42POP fragments
revealed that only polypeptides containing this motif could
be precipitated from bacterial extracts (Fig. 4B). The
contribution of individual leucines to this homodimer
formation was quantified with point mutations in a liquid
yeast two-hybrid assay. Mutants of the deletion fragment CPOP with either a single mutation of leucine residue L281 or
Profilin-p42POP interaction is mediated by proline
clusters
Because eight consecutive proline residues are sufficient for
effective profilin binding (Domke et al., 1997; Machesky and
Pollard, 1993), we speculated that the proline-rich regions
[especially the stretch of 12 consecutive prolines identified in
the p42POP sequence (Fig. 1)] might be responsible for the
interaction with profilin seen in the initial yeast two-hybrid
analysis. Using the yeast two-hybrid system again, we tested
the interaction between various truncated p42POP variants
(Fig. 3) with mouse profilin 1 and also with profilin 2a,
because this neuronal isoform has also been shown to bind to
proline-cluster proteins (Di Nardo et al., 2000; Lambrechts et
al., 2000; Witke et al., 1998). As shown in Fig. 3A, all
constructs containing the consecutive proline motifs gave a
positive reaction, whereas all constructs lacking the
polyproline stretches did not. A fragment comprising only the
most C-terminal proline-rich region (∆POP8) but devoid of the
consecutive proline stretches also failed to interact with
profilins 1 and 2a in this assay. The positive result obtained
with ∆POP9, which is a short fragment mainly reduced to the
proline clusters within amino acids 203-329, indicates that
these clusters are necessary and sufficient for interaction with
the polyproline-binding motif of profilins. These results were
supported by dot-overlay assays with in-vitro-translated
p42POP fragments (data not shown). The importance of the
proline clusters for complex formation could also be
demonstrated in pull-down assays with recombinant profilin
Fig. 3. The proline-rich domain of p42POP mediates profilin binding.
(A) Yeast two-hybrid analysis with profilins 1 and 2a (PFN1/2a) as a
bait and different p42POP fragments as prey. Only fragments
containing the proline clusters gave a positive reaction (+).
(B) Autoradiograms obtained after SDS-PAGE of pellets (P) and
supernatants (S) derived from pull-down assays with profilin-1coupled Sepharose (PFN1) or BSA-coupled Sepharose (control).
Both types of Sepharose bead were incubated with in-vitrotranslated, radioactively labelled full-length p42POP or the N-terminal
(N-POP) or C-terminal (C-POP) fragment. Only C-POP was
precipitated by PFN1 beads.
Journal of Cell Science
Profilin regulates p42POP
335
Fig. 4. p42POP can dimerize via the
leucine zipper but profilin binding is
restricted to the monomer. (A) Yeast
two-hybrid analysis with full-length
p42POP as a bait and various p42POP
fragments as prey. Only fragments
containing the leucine-zipper motif
give a positive reaction (+), indicating
dimerization. (B) Immunoblots
derived from p42POP pull-down
assays. Crude extracts of E. coli,
transformed with plasmids coding for
GFP-tagged ∆POP10, ∆POP12 or
∆POP13, respectively, displayed the
respective fragments in immunoblots
with a GFP antibody (e). They were
then incubated with glutathioneSepharose, in the presence (+) or
absence (–) of extracts expressing the
GST-tagged ∆POP14 or GST only (c,
control). After centrifugation, pellets
were subjected to SDS-PAGE and
immunoblotting with the anti-GFP
antibody. Only ∆POP10 and ∆POP12,
both harbouring the leucine zipper, were precipitated. (C) Liquid yeast two-hybrid assays to analyse the importance of an intact leucine zipper
for p42POP dimerization. Full-length p42POP was incubated with samples containing C-POP and C-POP variants containing single or double
point mutations within the leucine zipper, as indicated. β-Galactosidase activity was monitored for quantitation of the interaction. Bars indicate
the standard deviations of four independent experiments. Replacement of two leucines within the leucine zipper with either alanine or proline
abolishes dimerization. (D) Dimerization of FLAG/C-POP and failure of the mutant FLAG/C-POP-LLPP to dimerize as demonstrated by
chemical cross-linking. The recombinant fragments were exposed to the cross-linker EDC/NHS followed by SDS-PAGE and immunoblotting
with an anti-FLAG antibody. Whereas C-POP is revealed as a monomer (~25 kDa) and a dimer (50 kDa), POP-LLPP cannot form dimers.
(E) Reactivity of profilin 1 with monomeric or dimeric POP variants. Profilin 1 was incubated with the cross-linker EDC/NHS in the absence
(lane a) or the presence of either FLAG/C-POP (lanes b,b′) or FLAG/C-POP-LLPP (lanes c,c′). Samples were subjected to SDS-PAGE and
immunoblotting with monoclonal antibodies against profilin 1 (a-PFN1) or FLAG (a-FLAG), respectively. Free profilin is seen at a position
corresponding to ~14 kDa. In the samples containing C-POP (b) or C-POP-LLPP (c), it is also present in the band at 39 kDa, which
corresponds to the heterodimer of profilin with either C-POP or C-POP-LLPP (arrow). The immunoblot with the FLAG antibody reveals that
only C-POP, but not C-POP-LLPP, has formed dimers (at ~50 kDa, b′,c′, arrowhead) but profilin is not found complexed to C-POP dimers
(b,b′). Hence, profilin binds only to the monomeric p42POP.
double mutations of leucine residues L274 and L281 to
alanine or proline were tested and compared with the
unmodified C-terminal fragment (C-POP). A substantial
decrease in complex formation was obtained with the single
substitutions C-POP-LA and C-POP-LP, respectively, and the
double mutations C-POP-LLAA and C-POP-LLPP abolished
the binding to p42POP almost completely (Fig. 4C). These
results demonstrate that p42POP can dimerize, probably via
the leucine zipper.
The relation between profilin binding and dimerization was
analysed in cross-linking experiments with p42POP fragments
that were able or unable to dimerize. As expected from the
yeast two-hybrid data (Fig. 4A,C), full-length p42POP was
easily cross-linked into dimers, as revealed by SDS-PAGE and
immunoblots with the FLAG antibody, whereas the mutant CPOP-LLPP (with a nonfunctional leucine zipper) did not
dimerize (Fig. 4D). When recombinant profilin 1 was added to
the p42POP proteins before incubation with the cross-linker,
subsequent SDS-PAGE and immunoblotting with anti-profilin
and anti-FLAG antibodies revealed heterodimer formation
between profilin and the mutant C-POP-LLPP, whereas the
dimers formed by C-POP did not contain profilin (Fig. 4E).
These results demonstrate that profilin exclusively interacts
with the p42POP monomer.
p42POP binds to the Myb DNA consensus sequence
To confirm that the single tryptophan cluster identified in the
N-terminal region of p42POP is indeed a Myb-related DBD, we
first performed computer-assisted analysis of a range of Myb
proteins from different organisms. Fig. 5A shows the relevant
consensus sequence in comparison with tryptophan-cluster
modules from Myb proteins identified in mammals, insects and
plants. This analysis revealed the high degree of conservation
shared between the tryptophan cluster domains of p42POP and
respective Myb proteins throughout the eukaryotic kingdom.
Remarkably, p42POP contains three insertions within this
sequence, which comprise 5-11 amino acid residues and are
not found in other analysed Myb examples with the exception
of the Drosophila protein Zeste (Mohrmann et al., 2002).
Because of these differences and because the Myb-related
motif is only present as a single copy in p42POP, we analysed
the DNA-binding ability of p42POP directly in an EMSA with
double-stranded oligodeoxynucleotides containing the Myb
recognition core motif PyAACG/TG in the context of the mim1A Myb response element (mreA) (Miglarese et al., 1996).
In-vitro-translated p42POP proteins were incubated with
double stranded [32P]-labelled mreA oligodeoxynucleotides
and complex formation was monitored by EMSA. As seen
in Fig. 5B, p42POP forms a complex with the mreA
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Journal of Cell Science 118 (2)
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Fig. 5. p42POP is a Myb-like transcription factor.
(A) Comparison of the p42POP N-terminus with
DBDs of Myb-related proteins. Bold characters
indicate conserved residues indicative of the Myb
repeat consensus. Sequences are from mouse (MM cMyb: AAB59713), human (HS A-Myb: CAA57552),
potato (MybSt1: AAB32591), Arabidopsis (AT
MybL2: S71283), maize (ZM MYB1: S04898; ZM
MYB38: S04899), barley (HV MYB1: P20026) and
Drosophila (Zeste: P09956). (B) DNA-binding
ability of p42POP as revealed by EMSA. In-vitrotranslated p42POP was seen to bind to doublestranded [32P]-labelled mreA oligodeoxynucleotides
[left, without (–) and with (+) p42POP]. (centre)
Competition of complex formation by an identical
unlabelled probe. (right) Control with a random
DNA sequence. (C) The N-terminal fragment of
p42POP (N-POP) is sufficient for complex formation
with mreA. Purified recombinant N-POP and the
mutants N-POP-S25D and N-POP-R83E/W84P were
incubated with the mreA sequence as described in
(B). Whereas N-POP displays the electrophoretic
shift indicating binding, single and double point
mutations within the Myb-like repeat (asterisks in A)
abolish this interaction. Arrows indicate the positions
of the complexes.
deoxynucleotide sequence. The specificity of this interaction
was analysed in competition assays. Preincubation of in-vitrotranslated p42POP with unlabelled mreA (Fig. 5B) decreased the
binding of the [32P]-labelled oligodeoxynucleotides. At a 100fold molar excess of unlabelled competitors, binding was
completely abolished, whereas random oligodeoxynucleotides
had no effect. Band-shift assays showed that the N-terminal
p42POP fragment (N-POP, comprising amino acids 1-170),
which lacks the proline clusters and the leucine-zipper motif
and thus cannot form dimers or interact with profilin, binds to
mreA (Fig. 5C). Hence, neither dimerization nor the profilinbinding motif are required for this complex formation. In
accordance with this result, the addition of profilin did not
influence p42POP-mreA complex formation (data not shown).
However, substitution of even a single conserved amino acid
within the DBD (a serine replaced by aspartic acid) interfered
severely with p42POP-mreA complex formation, supporting
the conclusion that DNA binding depends critically and
specifically on an intact Myb-related DBD (Fig. 5C). These
findings confirm the conclusions drawn from the computerbased sequence analysis (Fig. 1) that p42POP is indeed a Mybrelated protein with a functional DBD.
p42POP interacts with endogenous profilin in cells
The interaction of endogenous profilin with p42POP was studied
in PtK2 cells. These were transfected with GFP-fused p42POP,
∆POP4 and ∆POP6. Expression and localization of the
recombinant proteins and endogenous profilin were analysed in
optical sections through the nucleus, using a confocal laserscanning microscope. As already seen in Fig. 2, p42POP and
∆POP4 (which lacks the polyproline cluster) localized to the
nucleus, whereas ∆POP6 was found in the cytoplasm. We then
determined the amount of endogenous profilin in the nuclei in
these cell populations. In untransfected cells, profilin was clearly
seen in the nuclear compartment after immunostaining (Fig. 6D),
confirming previous results (Giesemann et al., 1999; Mayboroda
et al., 1997; Skare et al., 2003). After overexpression of p42POP,
the profilin signal was markedly increased and doublefluorescence images revealed a fine granular distribution of both
proteins. Some, but not all, of these granules stained for both
profilin and p42POP (Fig. 7A). Quantitation of the nuclear
profilin level in untransfected and transfected cells revealed that
cells expressing full-length p42POP contained significantly more
(17%) profilin in their nuclei than the untransfected controls,
whereas the expression of the mutant lacking the proline-rich
region (∆POP4) or of the fragment residing in the cytoplasm
(∆POP6) did not alter the amount of the endogenous profilin
(Fig. 7B). These results indicated that nuclear p42POP, via its
proline cluster motif, can recruit profilin and form complexes
with it in the nuclear compartment.
We could also confirm the cellular interaction between
profilin and p42POP in another, unrelated, analysis using a
mitochondrial recruitment assay. PtK2 cells were again either
transfected with ∆POP6 (which includes the proline clusters)
or a control protein (E-cadherin). Both proteins were presented
as constructs coding for fusion proteins, equipped with the
BiPro tag for anti-BiPro staining and a short motif derived from
an outer-mitochondrial-membrane protein. We had already
shown that transfection with such constructs results in
anchoring of the proteins at mitochondrial membranes
(Kaufmann et al., 2000). Immunofluorescence with the antiBiPro antibody confirmed this result for the protein fragments
considered here: ∆POP6 and cadherin were both targeted
to and anchored at the mitochondrial membrane (Fig.
6A,A′,C,C′). When the distribution of endogenous profilin in
such cells was examined with an anti-profilin antibody,
mitochondrion-immobilized ∆POP6 had recruited the PtK2
profilin, and the proteins colocalized (Fig. 6B,B′). By contrast,
membrane-bound E-cadherin, which is not a ligand for profilin,
had no effect on the subcellular distribution of profilin (Fig.
Profilin regulates p42POP
6D,D′). Hence, these results, together with the results seen for
nuclear recruitment of profilin by p42POP, suggest that profilin
and p42POP can be present in the same protein complex in a
cellular environment.
transfected β-galactosidase, and equal protein expression of the
p42POP constructs was verified by corresponding immunoblots
(Fig. 8A). Fig. 8B shows that, in such assays, the control
Myb protein c-Myb caused almost a fourfold increase in
transcription, whereas p42POP acted as a transcriptional
repressor, decreasing to ~15% of the basic values obtained with
the reporter plasmid alone. When ∆POP6 (the fragment
residing in the cytoplasm) was used instead of p42POP, no
inhibition of luciferase activity was observed, indicating that
the modulating effect of p42POP depended on its presence in
the nucleus (data not shown). By contrast, ∆POP4, the nuclear
fragment lacking most of the C-terminal half, including the
leucine zipper, induced a threefold increase (Fig. 8B). These
Journal of Cell Science
Profilin modulates the transcriptional activity of p42POP
As a Myb-related protein, p42POP should possess a regulatory
activity on transcription, and this activity might be modulated
by ligand proteins or dimerization. We tested this hypothesis
in a luciferase assay (Fig. 8). HeLa cells were transfected
with the reporter plasmid pGL3-mreA-Luc and a cDNA
encoding c-Myb, full-length p42POP or the fragment ∆POP4.
Transfection efficiency was monitored by the activity of the co-
337
Fig. 6. ∆POP6 and endogenous profilin localize to cytoplasmic
membranes in PtK2 cells in a mitochondrial targeting assay. Cells
were transiently transfected with vectors coding for a fusion protein
containing a mitochondrial outer membrane fragment (MOM) and
either BiPro-∆POP6 or BiPro-E-cadherin (control). Staining was
performed with MitoTracker™, a monoclonal antibody against the
BiPro tag and a polyclonal anti-profilin antibody. Doublefluorescence images show that both MOM fusion proteins target to
mitochondrial membranes (A,A′,C,C′) but only ∆POP6 recruits
endogenous profilin to this position (B,B′), whereas E-cadherin fails
to do so (D,D′). Bar, 10 µm.
Fig. 7. p42POP localizes with profilin 1 and recruits profilin 1 into the
nucleus. Fluorescence images obtained as optical sections by
confocal laser-scanning microscopy revealed that GFP-p42POP (A)
and profilin 1, seen by immunostaining with a monoclonal antibody
(A′), are both localized to the nucleus, in a fine granular pattern with
some overlap (A′′). Bar, 10 µm. (B) Quantitation of nuclear profilin
(PFN1) in PtK2 cells transiently transfected with GFP-fused p42POP,
∆POP4 or ∆POP6. The bars display the relative fluorescences of the
profilin-specific signals obtained for cells transfected with p42POP,
∆POP4 or ∆POP6 compared with values obtained with untransfected
cells on the same coverslip (defined as 1.0). Values were obtained for
150 cells and are shown with standard errors compared by variance
analysis (Student-Newman-Keuls; P<0.05). Expression of p42POP
raised the nuclear profilin level by 17% in comparison with
untransfected cells, whereas neither ∆POP4 (which lacks the proline
clusters) nor ∆POP6 (which does not target to the nucleus) (cf. Fig.
2) showed this increase in nuclear profilin.
Journal of Cell Science
338
Journal of Cell Science 118 (2)
Fig. 8. p42POP modulates transcriptional activity. HeLa cells were
transfected with the reporter plasmid pGL3-mreA-Luc and a cDNA
encoding c-Myb, BiPro-p42POP or BiPro-∆POP4. (A) Immunoblot of
cell extracts with the anti-BiPro antibody after transfection reveals
comparable expression of p42POP and the ∆POP4 fragment.
(B) Luciferase activity in extracts of cells transfected with the
reporter plasmid only (control) or with the reporter plus c-Myb,
BiPro-p42POP or BiPro-∆POP4. Whereas c-Myb acts as a
transcriptional activator in this assay, p42POP causes a decrease in
transcription to less than 15% of the control value. By contrast,
∆POP4, lacking the C-terminal modules, induces a threefold increase
in transcription.
data show that p42POP, like other Myb proteins, can modulate
transcription in the nuclear compartment and, again like other
Myb proteins, is regulated in this activity by its C-terminal half.
The consequence of an interaction between p42POP and
profilin on transcription was analysed in a luciferase assay.
HeLa cells were co-transfected with the reporter plasmid pGL3mreA-Luc and p42POP, and with either wild-type profilin 1
(PFN1) or a profilin mutant defective in polyproline binding, as
demonstrated by pull-down experiments with polyprolineSepharose (PFN1-S138D; Fig. 9A). As seen before (Fig. 8),
luciferase activity (as a measure of transcriptional activity) was
severely inhibited by p42POP (15% of the control). However, in
the presence of profilin 1, this decrease was markedly reduced
(60% of the control; Fig. 9B). This regulation of p42POP
transcriptional activity by profilin was strongly dependent on an
intact polyproline-binding site on profilin: when wild-type
profilin was replaced with PFN1-S138D in this assay, no rescue
of transcriptional activity was observed (Fig. 9B). Because the
expression levels of all proteins were comparable (Fig. 9C),
these differences must reflect complex formation of both
profilin variants with the p42POP transcription factor These
results indicate that profilin, via its polyproline-binding motif,
is able to interact directly and specifically with p42POP in HeLa
cells, and to regulate transcriptional activity.
Discussion
Here, we report the identification of a novel ligand for profilins
Fig. 9. Profilin modulates p42POP transcriptional activity, mediated
by its polyproline binding site. (A) Extracts of E. coli expressing
wild-type profilin (PFN1) or a profilin variant with a single point
mutation in the polyproline-binding site (PFN1-S138D) were
incubated with polyproline-Sepharose. Sedimented material was
subjected to SDS-PAGE and immunoblotting with the monoclonal
anti-profilin antibody. Although both proteins are present in the
bacterial lysates (extract), only PFN1 is precipitated by polyprolineSepharose (PLP), demonstrating that PFN138D is defective in
polyproline binding. (B) Luciferase assays with HeLa cells
transfected with pGL3-mreA-Luc, BiPro-p42POP and either cDNAs
encoding FLAG-tagged profilin or the empty reporter plasmid
(control). Analogous to the results shown in Fig. 8B, the presence of
p42POP (+) consistently induced a strong repression of the basic
transcriptional activity (left, –). When profilin was expressed with
p42POP, this repression was markedly reduced (centre). However,
when the profilin mutant PFN1-S138D was present, no such rescue
effect was seen (right). (C) Immunoblots with antibodies against the
BiPro or FLAG tag obtained from HeLa-cell extracts used in B.
Comparable protein expression is seen for all three analyses. Hence,
profilin markedly alters the transcriptional activity of p42POP and this
effect depends strongly on an intact polyproline-binding site.
that was initially found in a yeast two-hybrid screen.
Subsequent characterization of this protein, termed p42POP,
revealed that it was hitherto unknown and is widely expressed
in mouse tissues. Detailed domain analyses and biochemical
and functional assays were combined to characterize p42POP.
In a luciferase assay, with the transcription factor c-Myb as a
positive control, we could demonstrate that p42POP also acts as
a transcription factor. Computer-assisted analysis of p42POP
suggested that p42POP was indeed related to transcription
factors of the Myb family, because it displayed a tryptophan
cluster at its N-terminus and, indeed, binds to the consensus
DNA recognition motif indicative of Myb proteins
(PyAACG/TG) (Biedenkapp et al., 1988). However, the DBD
of p42POP includes only a single tryptophan cluster motif,
similar to several Myb-related proteins identified in plants
(Baranowskij et al., 1994; Kirik and Baumlein, 1996) and in
the Zeste protein found in Drosophila (Mohrmann et al., 2002),
whereas all mammalian members of the Myb transcription
factor family known so far display three of these repeats.
Furthermore, within the tryptophan cluster, the first tryptophan
is replaced by a phenylalanine, a substitution described for
several other Myb proteins (Kirik and Baumlein, 1996;
Journal of Cell Science
Profilin regulates p42POP
Marocco et al., 1989) and the DBD contains three insertions,
again similar to that found in Zeste (Mohrmann et al., 2002).
Yet these modifications obviously do not interfere with the
activity of p42POP as a transcription factor. An acidic stretch
adjacent the tryptophan cluster, which modulates
transcriptional activity in other Myb-related proteins
(Kalkbrenner et al., 1990; Sakura et al., 1989), is also present
in p42POP, as is a leucine-zipper motif, which allows
dimerization, analogous to the situation in c-Myb (Nomura et
al., 1993). Owing to the presence of DNA-binding, activating
and regulatory elements, we define p42POP as a Myb-related
transcription factor, albeit with some unusual and unexpected
properties for mammalian Myb proteins. Its ubiquitous
expression in embryonic and adult tissues of the mouse
suggests that it performs important functions in gene
regulation. To unravel the nature of such a presumed biological
role, one would need to obtain information about a putative
target promoter and the ability of p42POP to form heterodimers
with other Myb-like transcription factors. As observed for
many transcription factors, p42POP is not an abundant protein.
Although we could identify the widely expressed POP mRNA
by RT-PCR, the levels of endogenous p42POP were too low for
immunofluorescence analysis with p42POP-specific antibodies.
The proline-rich C-terminal part of p42POP, unlike other
Myb-like proteins, contains two stretches of five and 12
consecutive prolines. These are responsible for profilin
binding, as we have seen in various independent assays.
Complexes of purified profilin and the proline cluster
containing fragments of p42POP could be precipitated but, in
addition and more significantly, we could show that these two
proteins interact in a cellular environment in yeast and
mammalian cells. A significant recruitment of profilin to the
nucleus was seen in p42POP-transfected HeLa cells, and a
membrane-bound p42POP fragment was able specifically to
attract and accumulate profilin in the cytoplasm of PtK2
epithelial cells. Hence, profilin moves towards and combines
with p42POP in both cytoplasmic and nuclear compartments.
The combined information obtained from the cross-linking
experiments, the DNA-binding studies, the luciferase assays
and the cellular recruitment data shows that p42POP can
dimerize and that dimerization reduces its DNA-binding
capacity and thus its ability to modulate transcription.
Furthermore, profilin can bind only to the monomer and is
recruited by p42POP to the nucleus. However, from these data,
one cannot deduce whether, in cells, profilin might modulate
p42POP activity by interfering with or even preventing
dimerization. The mode of interaction between profilin and
p42POP is probably subject to the local concentration and
availability of both partners, and depends on the presence of
additional proteins, like other transcription factors that might
form heterodimers with p42POP via a leucine zipper. Thus,
although the data presented here indicate that profilin is
engaged in the regulation of transcription, the precise
mechanism of such an activity remains elusive.
Previous studies of nuclear profilin have presented evidence
that profilins participate in ribonucleoparticle processing
(Giesemann et al., 1999), mRNA splicing (Skare et al., 2003)
and nuclear export (Stuven et al., 2003). A role for profilins in
transcription, as proposed here, is also supported by
observations of its involvement in viral transcription. In the
case of respiratory syncitial virus (RSV), a clinically relevant
339
pneumovirus, transcription depends on actin (Burke et al.,
1998) and profilin was found to act as a transcriptional
enhancer (Burke et al., 2000). Hence, it was suggested that the
interaction of profilin with actin might be important for
optimizing RSV transcriptase activity (Burke et al., 2000), but
alternative roles for profilin that do not require a simultaneous
interaction with actin were not excluded. Furthermore, recent
data show that profilin also interacts directly with the RSVencoded phosphoprotein P, which is a viral transcription factor
(Bitko et al., 2003). Because we find an interaction of profilin
with p42POP in mammalian cells in the absence of viral
infections, such interactions are not dependent on viral
components. It is, however, conceivable that actin and profilin
act in concert or even in a physical complex in the nucleus to
modulate transcriptional activity. In this context, it should be
interesting to learn whether profilin bound to actin can
influence p42POP activity.
Functional association of cytoskeletal proteins other than
profilins with transcription factors has also been reported.
Nuclear actin has been shown to play a role in transcription in
Xenopus oocytes (Scheer et al., 1984) and in insects (Percipalle
et al., 2001). The microfilament-associated protein β-catenin
can translocate to the nucleus and modulate gene expression
by interacting with the transcription factor LEF-1 (lymphoid
enhancer-binding factor 1) (Behrens et al., 1996; Fagotto et al.,
1998). The thyroid-hormone-receptor-interacting protein
Trip6, a LIM-domain-containing protein, localizes to adhesion
plaques and is thought to transport intracellular signals from
the cell surface to the nucleus and to bind the transcriptional
activator v-Rel (Zhao et al., 1999). Furthermore, the zyxinrelated actin-associated protein LPP is also a dualcompartment protein that can enhance transcriptional activity
(Petit et al., 2000). These observations probably represent
isolated pieces of a large puzzle that is not even well defined.
Unravelling the molecular mechanisms governing the interplay
of microfilament components with transcription factors in cells
and tissues will probably require a combination of
biochemical, cell biological and genetic analyses.
We thank S. Illenberger (TU Braunschweig) for constructive
suggestions on the manuscript and R. Hänsch for excellent help with
the confocal laser-scanning microscope. K. Marquardt and S.
Berensmeier contributed to the discovery and first characterization of
∆POP1, and to data shown in Fig. 3, respectively. C. Wucherpfennig,
K. Schloen and T. Geisler are acknowledged for expert technical
assistance. This work was supported by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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