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The EMBO Journal Vol.16 No.3 pp.484–494, 1997
The mammalian profilin isoforms display
complementary affinities for PIP
2
and proline-rich
sequences
the tail of Listeria monocytogenes, a bacterial pathogen that
Anja Lambrechts, Jean-Luc Verschelde,
uses actin assembly to move forward (Theriot et al., 1994).
Veronique Jonckheere, Mark Goethals,
Profilin also binds phosphatidylinositol 4,5-bisphosph-
Joe
¨l Vandekerckhove and
ate (PIP
2
) (Lassing and Lindberg, 1985, 1988). Phospho-
Christophe Ampe
1
lipase C1 cannot hydrolyse profilin-bound PIP
2
in vitro,
Flanders Interuniversity Institute for Biotechnology, Department of
unless the phospholipase is phosphorylated (Goldschmidt-
Biochemistry, Faculty of Medicine, Universiteit Gent, 9000 Gent,
Clermont et al., 1990, 1991; Machesky et al., 1990).
Belgium
These experiments led to a model in which profilin plays
1
Corresponding author
an important role as mediator in cell signalling to the
cytoskeleton. PIP
2
-associated profilin is not able to bind
We present a study on the binding properties of the actin monomers but, after hydrolysis of PIP
2
, profilin is
bovine profilin isoforms to both phosphatidylinositol free and can diffuse into the cytoplasm to interact with
4,5-bisphosphate (PIP
2
) and proline-rich peptides actin (Lassing and Lindberg, 1985, 1988).
derived from vasodilator-stimulated phosphoprotein Profilins, with the exception of vaccinia virus profilin
(VASP) and cyclase-associated protein (CAP). Using (Machesky et al., 1994), bind poly-
L
-proline, but the
microfiltration, we show that compared with profilin significance of this interaction remained an enigma for a
II, profilin I has a higher affinity for PIP
2
.Onthe long time. However, several proteins associated with the
other hand, fluorescence spectroscopy reveals that pro- cytoskeleton or its dynamics contain such a sequence:
line-rich peptides bind better to profilin II. At micromo- cyclase-associated protein (CAP), a surface protein from
lar concentrations, profilin II dimerizes upon binding the cytotoxic pathogen L.monocytogenes (ActA) and the
to proline-rich peptides. Circular dichroism measure- vasodilator-stimulated phosphoprotein (VASP). Already in
ments of profilin II reveal a significant conformational 1991, Vojtek et al. proposed a functional link between
change in this protein upon binding of the peptide. We profilin and CAP, which is the 70 kDa subunit of the
show further that PIP
2
effectively competes for binding Saccharomyces cerevisiae adenylyl cyclase complex (Field
of profilin I to poly-
L
-proline, since this isoform, but et al., 1990). CAP is a bifunctional protein of which the
not profilin II, can be eluted from a poly-
L
-proline N-terminal domain is necessary and sufficient for a Ras-
column with PIP
2
. Using affinity chromatography on responsive adenylyl cyclase complex, and of which the
either profilin isoform, we identified profilin II as the C-terminal domain binds actin (Freeman et al., 1995) and
preferred ligand for VASP in bovine brain extracts. is important for normal cell morphology and responsive-
The complementary affinities of the profilin isoforms ness to nutrient extremes (Gerst et al., 1991). CAP
for PIP
2
and the proline-rich peptides offer the cell mutants that lack the C-terminal domain can be rescued
an opportunity to direct actin assembly at different by overexpression of profilin (Vojtek et al., 1991). Since
subcellular localizations through the same or different CAP has a proline-rich stretch in its middle domain, it
signal transduction pathways. may be a ligand for profilin (Goldschmidt-Clermont and
Keywords: actin cytoskeleton/phosphatidylinositol 4,5- Janmey, 1991).
bisphosphate/profilin isoforms/signal transduction/ Similarly, ActA contains three single stretches of four
vasodilator-stimulated phosphoprotein proline residues. A Listeria mutant which does not express
ActA on the surface is unable to form actin tails, to spread
intracellularly and to infect adjacent cells (Domann et al.,
1992), although host factors also seem to be required for
Introduction
actin tail formation (Theriot et al., 1994; Chakraborty
et al., 1995).
Profilin is a ubiquitous, small (12–15 kDa) actin-binding VASP was identified originally as a protein present in
protein that plays an important role in the regulation of human platelets which becomes phosphorylated by cAMP-
actin polymerization in a number of motility functions and cGMP-dependent protein kinases in response to both
(Carlsson et al., 1977; Stossel, 1993). In vitro experiments cAMP- and cGMP-elevating vasodilators and platelet
suggest that profilin can promote actin assembly from the inhibitors (Waldmann et al., 1986, 1987; Halbru
¨gge et al.,
G-actin–thymosin-4 pool when barbed filament ends are 1990; Nolte et al., 1991). The sequence reveals a central
free. In the presence of gelsolin, a barbed end capping domain with a proline-rich motif, with five consecutive
protein, profilin can act as an actin-sequestering protein proline residues, that occurs as a single copy and a 3-fold
resulting in actin depolymerization (Pantaloni and Carlier, repeat (Haffner et al., 1995). Only very recently, Reinhard
1993). This dual function of profilin is thought to be and co-workers (1995) showed that VASP interacts directly
essential for rapid filament turnover during cell motility with profilin.
processes, e.g. in the growing end of fast moving lamellipo-
dia of fibroblasts (Buss et al., 1992; Small, 1995) and at It was shown that human cells have two profilin isoforms
484
© Oxford University Press
Binding of profilins to PIP
2
and poly-
L
-proline
and, given the different expression levels of each isoform
in different tissues (Honore
´et al., 1993), researchers have
used, unknowingly, preparations containing different ratios
of profilin isoforms in the past, e.g. in microinjection
studies (Cao et al., 1992) and bio- and immunochemical
studies (Buss and Jockusch, 1989).
Human and bovine profilin I (Ampe et al., 1988;
Kwiatkowski and Bruns, 1988) and II (Honore
´et al.,
1993; Lambrechts et al., 1995) show only 65.5% identity.
Most of the mutated residues map at the exterior of the
known profilin I structure, suggesting that both isoforms
have a similar overall fold but display different biochem-
ical properties. Their affinity for actin is quite similar
(Lambrechts et al., 1995), consistent with the observation
that the residues of profilin, forming the interface with
actin, are conserved. However, one of our previous experi-
ments suggested that the isoforms display a different
affinity for poly-
L
-proline at acidic pH (Lambrechts et al.,
1995). To probe this difference further, we investigated
the interaction of each isoform with proline-rich model
peptides derived from VASP, CAP and ActA using fluor-
escence spectroscopy at more physiological pH. We also
searched for differences in interaction with the other
known profilin ligand, PIP
2
, using a microfiltration assay.
We show that profilin I has higher affinity for PIP
2
, while
profilin II has higher affinity for proline-rich sequences,
and can form dimers upon binding these sequences. In
addition we show that PIP
2
is an effective competitor for
Fig. 1. Microfiltration of profilin–PIP
2
complexes. (A) Samples
containing a constant amount of profilin (4 µM) and increasing
poly-
L
-proline binding of profilin I, but not of profilin II.
concentrations of PIP
2
were made. The flow-through was analysed by
Our data suggest that in cells profilin I may be preferen-
SDS–PAGE, of which only the 14–20 kDa region is shown. The molar
tially associated with PIP
2
, and profilin II with proteins
excess of PIP
2
is indicated. (B) Result of scanning the Coomassie-
such as VASP, which we show is recruited preferentially
stained spots shown in (A). Boxes represent the percentage of non-
bound profilin. White boxes are profilin I and black boxes profilin II.
from bovine brain extracts by profilin II.
The amount of profilin found in the flow-through when no PIP
2
is
present was set to 100%.
Results
Bovine profilin I has a higher affinity for PIP
2
than
(CD) spectra, as has been shown for profilin I
(Raghunathan et al., 1992) and the pleckstrin homology
the profilin II isoform
An important characteristic of profilins is their ability to domain of spectrin (Hyvo
¨nen et al., 1995). The CD
measurements were carried out for samples containingbind PIP
2
(Lassing and Lindberg, 1985, 1988). Our previ-
ous results showed that bovine profilin II binds PIP
2
, but 15 µM profilin with different concentrations of PIP
2
ranging from 0 to 300 µM (Figure 2). Profilin I and IInevertheless they hinted at a reduced affinity compared
with profilin I (Lambrechts et al., 1995). To investigate alone have different spectra. This is probably because of
the difference in their tyrosine, tryptophan and phenylalan-this potential difference between the profilin isoforms
further, we used an assay described by Haarer et al. ine content and the environment of these residues. The
ellipticity of profilin I in the presence of PIP
2
shows a(1993). In a microfiltration experiment, we incubated a
constant amount of profilin (I or II) with increasing large decrease with only a 10-fold molar excess of
PIP
2
and only a slight further decrease when the PIP
2
amounts of PIP
2
(0- to 100-fold molar excess over profilin),
and centrifuged them on a filter with a molecular weight concentration is doubled. In contrast, the decrease in
ellipticity of profilin II is smaller and nearly doubles whencut-off of 30 000. The flow-through contains profilin not
bound to PIP
2
and is shown in Figure 1A. The result of the PIP
2
concentration is doubled.
scanning the gels is given in Figure 1B. With a 25-fold
molar excess of PIP
2
over profilin, nearly all profilin I is
Profilin I and II display different affinities for
proline-rich peptides
bound to PIP
2
. On the other hand, even with a 100-fold
molar excess of PIP
2
, 60% of profilin II (relative to the Whereas profilin I has a higher affinity for PIP
2
, profilin
II binds more strongly to poly-
L
-proline (Lambrechts et al.,sample without PIP
2
) is still present in the flow-through.
This microfiltration assay clearly demonstrates the differ- 1995). Poly-
L
-proline as such is not present in living cells,
though several proteins have proline-rich sequences. Atences in affinity for PIP
2
between the profilin isoforms. the start of our investigations, no natural profilin ligands
with stretches of proline residues were identified. However,
PIP
2
binding induces a greater change in the
structure of profilin I than in profilin II
a number of proteins were likely candidates for a profilin
ligand. CAP contains six and five prolines in a rowLocal structure changes induced by phospholipids can be
observed in near UV (250–320 nm) circular dichroism separated by a single glycine residue (Matviw et al.,
485
A.Lambrechts
et al
.
in VASP (Haffner et al., 1995). A shorter sequence
containing only five proline residues is also found in
the VASP sequence. As these three proteins have been
implicated in actin dynamics or are associated with the
actin cytoskeleton, we chemically synthesized peptides
derived from human CAP and VASP and from Listeria
ActA (Table I) and studied their interaction with both
profilin isoforms. We incubated a constant amount of
profilin (1 µM) with an increasing amount of peptide (0–
55 µM) under physiological conditions and monitored
binding by spectrofluorimetry (Figure 3).
The ActA peptide, peptACTwt, which contains only
four proline residues, induces no significant increase in
fluorescence even at high peptide concentrations, indicat-
ing that it has a very low affinity for both of the profilin
isoforms (Figure 3A and B). This is consistent with the
finding of Zeile et al. (1996) that the ActA peptide does
not bind to profilin at a concentration of 100 µM. The
same is observed for peptVASPs, a short proline-rich
sequence derived from VASP. The other peptides studied,
peptCAPwt and peptVASPwt, show a change in the
fluorescence intensity which means they do bind to
profilin.
We tried to fit a hyperbolic curve to the measured
values but could not find a sufficiently good fit. Also,
attempts to linearize the data using the Surewicz and
Epand equation (1984) were unsuccessful. This suggests
that the binding is heterogenous, as will be demonstrated
further below. Nevertheless, the difference in the steepness
of the curves in the low concentration range in Figure 3A
and B suggests a difference in affinity of these peptides
for each of the profilin isoforms. They appear to bind
better to profilin II than to profilin I.
As peptCAPwt and peptVASPwt (with 11 and 15 proline
residues respectively) seem to have a comparable affinity
and peptACTwt (with only four prolines) showed no
binding, we synthesized increasingly shorter mutant CAP
peptides (see Table I) to assay the minimal length required
for binding (Figure 3C). PeptCAP∆1 lacking one proline
residue has almost the same affinity as peptCAPwt, while
peptCAP∆2 and peptCAP∆4, lacking two and four prolines
respectively, have a strongly reduced affinity compared
with wild-type CAP peptide.
Profilin II dimerizes upon binding of proline-rich
peptides
Modelling experiments suggest that one repeat of five
proline residues is sufficient to fill the hypothetical poly-
L
-
proline binding pocket (data not shown). We demonstrated
above that two repeats are necessary for profilin binding.
This prompted us to study whether dimers of profilin can
be formed on the proline-rich peptides. Since profilin II
appears to bind better to these peptides, we chose to study
this isoform. Profilin II elutes at 47 min on a Superdex
Fig. 2. Circular dichroism spectra in the near UV of 15 µM profilin
200 gel filtration column (Figure 4a). With a 1.2-fold
(thick line) I or II with 150 (....) and 300 µM(–––)PIP
2
. The molar
molar excess of peptVASPwt, profilin II shifts partly to
ellipticity per residue weight is shown as a function of the wavelength.
an earlier position (Figure 4b). When more peptide is
added in 12-fold molar excess over profilin II, a second
shift can be observed (Figure 4c) relative to the profilin1992). A repeat of four prolines preceded by a phenyl-
alanine occurs three times in ActA, each time separated II peak. In the simplest scenario, the first shift is a 1:1
complex of profilin and peptVASPwt and the second,by 30 amino acids (Domann et al., 1992), and, more
recently, a three times repeated sequence of five consecut- larger shift would then be a 2:1 complex consisting of a
dimer of profilin bound to one peptide. We observed theive proline residues separated by a glycine was identified
486
Binding of profilins to PIP
2
and poly-
L
-proline
Table I. Sequence of proline-rich peptides
Peptide Sequence Protein Reference
peptCAPwt Ac.SGPPPPPPGPPPPPVS.OH human CAP Matviw et al. (1992)
peptVASPwt Ac.GPPPPPGPPPPPGPPPPPGL.OH human VASP Haffner et al. (1995)
peptVASPs Ac.GGPPPPPGL.OH human VASP Haffner et al. (1995)
peptACTwt Ac.FPPPPTD.OH Listeria ActA Domann et al. (1992)
peptCAP∆1 Ac.SGPPPPPGPPPPPVS.OH CAP mutant
peptCAP∆2 Ac.SGPPPPGPPPPPVS.OH CAP mutant
peptCAP∆4 Ac.SGPPGPPPPPVS.OH CAP mutant
same shift with profilin II and peptCAPwt (data not amount of profilin is found in the flow-through (17%).
No profilin II is found in the wash, and the remainingshown), but observed no shift when an 18-fold molar
excess of the short peptVASPs is added to profilin II, 83% can only be eluted with 8 M urea. These results
show that profilin I has a higher affinity for PIP
2
than forindicating that no binding occurs (Figure 4d). poly-
L
-proline.
A proline-rich peptide induces a conformational
change in profilin II Identification of VASP as a ligand for profilin II but
not for profilin I
Local structural changes in proteins induced by peptide
binding can also be observed in CD spectra in the region Reinhard and colleagues (1995) identified VASP as a
ligand for profilins and, since our experiments with thefrom 250 to 290 nm. We measured spectra for profilin I
and II with three different concentrations of peptCAPwt VASP-derived peptide suggest that the profilin isoforms
have a different affinity for this ligand, we assayed the(Figure 5), and the results suggest that aromatic amino
acids are involved in peptide binding, consistent with binding of VASP to each isoform using profilin I or II
affinity columns. We first show that the monoclonalresults from mutational analysis of profilin I (Bjo
¨rkegren
et al., 1993). antibodies against human VASP also recognize bovine
VASP (Figure 7A).The first decrease in profilin II ellipticity is very large,
while subsequentdecreases at larger peptideconcentrations We loaded an equal amount of bovine brain extract on
the columns and, after washing, eluted the remainingare small. This suggests that a conformational change
accompanies the binding of profilin II to peptCAPwt. On proteins with a step gradient of the proline-rich peptide
derived from VASP (peptVASPwt) as indicated in Figurethe other hand, for profilin I, the ellipticity decreases more
gradually as the peptide concentration increases, consistent 7B. We tested the eluted fractions for the presence of
VASP on Western blot using a monoclonal antibodywith a lower affinity for the peptide. (Figure 7B). Interestingly the capacity of the profilin
isoforms to retain VASP appears different. While only a
The polyproline binding site of profilins is not
accessible when PIP
2
is present
barely noticeable amount of VASP is eluted from the
profilin I column, a much larger amount of VASP boundWe also investigated whether PIP
2
and poly-
L
-proline
compete for profilin binding. In the control experiment, to the profilin II column. The elution starts with 50 µM
peptVASPwt but the majority of the protein is eluted withprofilin I or II were bound to poly-
L
-proline and, after
washing, were eluted with 8 M urea. A small fraction of 500 µM peptVASPwt. This result confirms the other
in vitro experiments with purified proteins and peptidesprofilin I did not bind or bound very weakly to the poly-
L
-proline column. The majority of profilin I is in the early and indicates that profilin II rather than profilin I is a
possible ligand for VASP.eluting 8 M urea fractions (Figure 6A, lanes 5 and 6)
while the majority of profilin II elutes later (lanes 7–11).
This result is consistent with our previously published
Discussion
data where a similar experiment was carried out at a
different pH (Lambrechts et al., 1995) and again points In this study, we demonstrate that the profilin isoforms
have complementary affinities for two ligands. Profilin Ito a difference in affinity for poly-
L
-proline of these two
isoforms. interacts strongly with PIP
2
and more weakly with poly-
L
-proline sequences. On the other hand, profilin II has aWe next loaded the poly-
L
-proline affinity column with
each profilin isoform and, after washing, eluted them higher affinity for proline-containing peptides and binds
less tightly to PIP
2
.with increasing concentrations of PIP
2
(Figure 6B). We
observed that PIP
2
was capable of eluting profilin I from Our data on binding of profilin I to proline-rich peptides
are in agreement with previous results of Perelroizen andthe column although a significant amount still remained
bound to poly-
L
-proline. By contrast, PIP
2
was not able co-workers (1994). These authors postulated the optimal
length of the poly-
L
-proline sequence to be 15–20 residues.to elute profilin II.
In the reverse experiment, the profilin isoforms were However, we find that there is little difference in affinity
between peptCAPwt and peptVASPwt containing 11 andincubated with a 25-fold molar excess of PIP
2
micelles
prior to loading them onto the column. A large fraction 15 proline residues, and a slight decrease in affinity of a
mutant CAP peptide lacking one proline (peptCAP∆1).of profilin I is found in the flow-through and the wash
(combined, ~53%), though still ~47% is eluted with 8 M Shorter peptides, such as those that occur in the Listeria
surface protein ActA and the shorter repeats in VASP,urea (Figure 6C). In the profilin II sample, only a small
487
A.Lambrechts
et al
.
Fig. 4. Gel filtration of profilin peptide complexes. Only the relevant
part of the chromatograms between 40 and 50 min is shown.
(a) Profilin II alone at 33 µM, (b) profilin II (33 µM) and a 1.2-fold
molar excess of peptVASPwt, (c) profilin II (30 µM) and a 12-fold
molar excess of peptVASPwt, (d) profilin II (20 µM) and an 18-fold
molar excess of peptVASPs. Detection was at 280 nm, a wavelength
where the peptides do not absorb, but their presence was investigated
using reversed phase HPLC (data not shown). The original absorption
units to full scale are set at 0.1 for (a) and (b) and 0.2 for (c) and (d).
1995). The precise cellular function of VASP, a focal
adhesion protein, is not known but its interaction with
profilin, which is known from in vitro experiments, sug-
gests that it may be an anchor for profilin-mediated actin
polymerization (Pantaloni and Carlier, 1993; Reinhard
et al., 1995). This is exemplified by the fact that after
Listeria infection, VASP is recruited to the bacterial
surface and binds ActA, prior to actin polymerization
(Chakraborty et al., 1995), and that depletion of profilin
from extracts used for in vitro motility assays slows down
Listeria bacteria (Theriot et al., 1994).
So far, a direct binding of CAP to profilin has not been
demonstrated, although a functional link between the two
proteins exists in yeast (Vojtek et al., 1991). Over-
expression of profilin is able to rescue morphological
defects associated with deletion of the CAP C-terminal
domain. Interestingly the part of CAP still present in this
mutant contains the proline-rich sequence, used in the
present study. This suggests that the poly-
L
-proline site
Fig. 3. Binding of profilin I and II to different proline-rich peptides.
may be cryptic most of the time and only made accessible
The relative fluorescence change (rfc) is plotted against peptide
after an, as yet unknown, type of regulation. Cryptic sites
concentration. The best fitting logarithmic curves are drawn as full
have been identified in other cytoskeletal proteins (Menkel
lines. The profilin concentration is 1 µM in all samples, and the
peptide concentration ranges from 0 to 55 µM. (A) Profilin I and
et al., 1994; Turunen et al., 1994; Gilmore and
peptides peptCAPwt (r), peptVASPwt (u), peptVASPs (.) and
Burridge, 1995).
peptACTwt (n). (B) Profilin II and peptides peptCAPwt (r),
We also found that profilin II forms dimers upon binding
peptVASPwt (u), peptVASPs (.) and peptACTwt (n). (C) Profilin II
to proline-rich peptides. If this also occurs in vivo it gives
and CAP wild-type and mutant peptides peptCAPwt (r), peptCAP∆1
(e), peptCAP∆2(j), and peptCAP∆4(,).
the cell a more efficient machinery to direct localized
actin assembly. For each tetrameric VASP molecule
(Haffner et al., 1995) eight profilin molecules would behave very low affinity for both of the profilin isoforms.
This result suggests that the role of profilin in Listeria recruited at focal adhesions (or other sites) where VASP
is active. In view of the polymerization-promoting activitymovement is not by direct interaction with ActA, consistent
with recent results showing that VASP may be the host of profilin (Pantaloni and Carlier, 1993), this concentrating
effect could have a dramatic effect on the actin dynamicsfactor that links profilin to ActA (Chakraborty et al.,
488
Binding of profilins to PIP
2
and poly-
L
-proline
Fig. 6. Competitive interaction between PIP
2
and poly-
L
-proline for
the profilin isoforms. (A) Profilin I (white boxes) and II (black boxes)
are loaded onto a poly-
L
-proline column and eluted with 8 M urea.
Fractions 1 and 2, flow-through; 3 and 4, wash; 5–12, 8 M urea
elution. (B) Profilin I (white boxes) and II (black boxes) are bound to
a poly-
L
-proline column and eluted with PIP
2
. The PIP
2
gradient is
indicated by full lines. Fraction 1, flow-through; 2, wash; 3, 50 µM
Fig. 5. Circular dichroism spectra in the near UV. Profilin I or profilin PIP
2
;4,100µM PIP
2
;5,200µM PIP
2
;6,300µM PIP
2
;7,400µM
II at a concentration of 15 µM was measured in the absence (thick PIP
2
; 8, wash; 9–12, 8 M urea. (C) Samples containing profilin I
line) or presence of peptide peptCAPwt at a concentration of 20 (....), (white boxes) or II (black boxes), pre-incubated with a 25-fold molar
50 (– – –) or 100 µM (thin line). The molar ellipticity per residue excess of PIP
2
, are loaded on a poly-
L
-proline column and eluted with
weight is shown as a function of the wavelength. The profilin I and II 8 M urea. Fraction 1 and 2, flow-through; 3–10, buffer wash; 11–16,
spectra are somewhat different from those in Figure 2, due to a 8 M urea elution.
different buffer composition and dilution effects.
489
A.Lambrechts
et al
.
Fig. 7. Binding of VASP to profilin. (A) Immunoprecipitation of bovine VASP from brain extracts with monoclonal antibodies against human VASP.
(B) Profilin affinity chromatography and Western blotting using anti-VASP monoclonal antibody. Profilin I or II Sepharose columns are loaded with
bovine brain extracts and, after washing, eluted with increasing concentrations of peptVASPwt. The eluted fractions are precipitated in 10%
trichloroacetic acid and, after gel electrophoresis and Western blotting, tested for the presence of VASP using monoclonal antibodies.
at these subcellular localizations. This dimerization may calculation in vacuum), the N- and C-terminal α-helices
move apart, thus widening the hypothetical poly-
L
-prolinealso explain the slightly sigmoidal behaviour of the fluor-
escence data, from which it appears that profilin II has a binding groove formed by aromatic and hydrophobic
amino acids from these helices and from the underlyinghigher affinity for proline-rich peptides than has profilin
I. This is evidenced further by other experiments in the β-strands (Bjo
¨rkegren et al., 1993; Archer et al., 1994;
Metzler et al., 1994). Comparison of the crystal and NMRpresent study. An excess of peptCAPwt only induces a
small change in the near UV CD spectrum of profilin I, structure of human profilin I by Metzler and co-workers
(1995) shows that the N- and C-terminal helices are morewhile similar concentrations result in a large change in
the CD spectrum of profilin II. In addition, elution of closely packed in the NMR ensemble than in the crystal
structure, suggesting that this movement of the terminalprofilin I from poly-
L
-proline affinity columns requires
less urea than does profilin II (control experiment in α-helices may occur under certain conditions even in
profilin I. Moving these terminal helices would makeFigure 5 and Lambrechts et al., 1995). Finally, in brain
extracts, we could recover VASP only from profilin II– the hydrophobic patch more accessible, and this motion
appears energetically more favourable in profilin II.Sepharose columns (see alsobelow). Although the profilins
from eukaryotic organisms display this difference in affin- The location of the PIP
2
binding site is less clearly
defined. It is generally accepted that the polyanionicity, in Acanthamoeba both profilin isoforms appear to
have similar dissociation constants for poly-
L
-proline headgroups of this phospholipid contact basic amino acids.
An interesting observation is that bovine profilin I is more(Kaiser and Pollard, 1996).
We also show that, in contrast to poly-
L
-proline binding, basic than profilin II (Honore
´et al., 1993; Lambrechts
et al., 1995) and binds better to PIP
2
(this work). Aprofilin I has a higher affinity for PIP
2
than does profilin
II. Using a 50-fold molar excess of PIP
2
over profilin, all similar correlation is found for the Acanthamoeba profilin
isoforms where profilin II is more basic and binds moreof isoform I and only ~24% of isoform II was bound to
PIP
2
vesicles. The CD spectra after PIP
2
binding show a strongly to PIP
2
(Machesky et al., 1990). Some basic
amino acids implicated in PIP
2
binding are indicated inlarge decrease for profilin I and only a small decrease for
profilin II. This may indicate that profilin II binding to Figure 8. Mutagenesis of Arg72 to Glu in yeast profilin
decreased the affinity for PIP
2
(Haarer et al., 1993). ThisPIP
2
is not accompanied by structural changes or, more
probably, that only a small amount of profilin II is bound arginine corresponds to Arg74 in bovine profilin. Sohn
and co-workers (1995) recently showed that Arg88 into PIP
2
. The competition experiment shows that, under
the conditions used, PIP
2
is a competitor for poly-
L
-proline human profilin I also is important in PIP
2
binding, because
mutation to Leu resulted in decreased inhibition of phos-in the case of profilin I, but not of profilin II. In addition,
PIP
2
binding makes the poly-
L
-proline binding site inac- pholipase Cγ1 (PLCγ1) activity. However, these residues
are conserved between the profilin isoforms and, therefore,cessible on both profilin isoforms. Evidence from other
investigators (for references, see below) indicates that they cannot be responsible for the difference in affinity.
On the other hand, substitution of Ser56 in profilin I bypoly-
L
-proline and PIP
2
occupy different binding sites.
These are illustrated in Figure 8 where we show the Glu in profilin II may exert a negative effect on binding
of PIP
2
to profilin II, since it is only 11.06 and 16.36 Å awaycrystal structure of profilin I (Cedergren-Zeppezauer et al.,
1994) and a calculated structure of profilin II. Our CD from the basic residues Arg74 and Arg88 respectively.
Comparison of electrostatic potential calculations of pro-data and those from Raghunathan and co-workers (1992)
indicate that PIP
2
and proline-rich peptides alter the filin I and II show that residue 56 is located between two
ridges of positive potential (data not shown). An extraconformation of profilin. The competition between these
two ligands which we observed would, therefore, result negative charge in between these could interfere with
PIP
2
binding.rather from a conformational switch. Interestingly, in the
calculated profilin II structure (after molecular dynamics Our binding studies of the isolated isoforms indicate
490
Binding of profilins to PIP
2
and poly-
L
-proline
Fig. 8. Ribbon structure of bovine profilin I and II after molecular dynamic calculations in vacuum. The N- and C-terminal helices are shown in
white. The hydrophobic side chains of the residues in the postulated poly-
L
-proline binding pocket are shown in green (bottom of the figure). N and
C indicate the N- and C-terminal ends. The amino acids postulated to be in the PIP
2
binding site are also indicated. The basic residues Arg74 and
Arg88 are shown in blue and the acidic residue Glu56 in profilin II is shown in red, while the corresponding Ser56 in profilin I is shown in green on
top of the molecule.
that they both are capable of binding to proline-containing tion. It opens the way to differential regulation of these
peptides. One is also able to purify profilin I by poly-
L
-isoforms in vivo. This is especially relevant in view of
prolineaffinity chromatography (Tanakaand Shibata,1985; the fact that VASP seems to be downstream of a cAMP
Kaiser et al., 1989; Lambrechts et al., 1995). Nevertheless, or cGMP signal transduction pathway (Nolte et al., 1991)
VASP inbrainextracts doesnot bind toprofilin I–Sepharose, while PIP
2
is a substrate of PLCγ1 and downstream of
while it is retained by a profilin II column. This is not receptor tyrosine kinases (Rhee, 1991), and that in some
because the profilin I coupled to Sepharose is no longer able cells (e.g. platelets) these pathways work in an antagonistic
to interact with VASP; VASP from platelet extracts does manner (see Halbru
¨gge and Walter, 1993, and references
bind to these profilin I columns and can be eluted with therein). Alternatively, it may enable cells to direct actin
peptVASPwt (Reinhard et al., 1995, and data not shown). assembly at different subcellular localizations through the
Thefailure ofVASPinbrain extractsto interactwith profilin different profilin isoforms.
I columns may have several causes. VASP in brains may be
modified differentially (compared with VASP in platelets)
and this modification could interfere only with binding to
Materials and methods
profilin I. As pointed out by Reinhard et al. (1995), the
Protein preparation and peptide synthesis
phosphorylatedform ofVASPiscapable ofbinding profilin.
We purified profilin I and II from bovine spleen and brain respectively,
It is more probable that the binding reaction is regulated by
as described previously (Kaiser et al., 1989; Cao et al., 1992; Lambrechts
the balance of the concentrations of profilin isoforms
et al., 1995).
and their cellular ligands. Indeed, profilin II is the
Peptides were synthesized on a model 431A peptide synthesizer
(Applied Biosystems Inc., Foster City, CA) according to the manufac-
predominant isoform in brain (Honore
´et al., 1993;
turer’s instructions. Peptides were purified using reversed phase HPLC,
Lambrechtset al., 1995) while platelets are enriched for
and the mass and purity were assessed by electrospray mass spectrometry
profilin I (A.Lambrechts, J.-L.Verschelde, V.Jonckheere,
(VG-platform, Fisons, UK).
M.Goethals, J.Vandekerckhove and C.Ampe, unpublished
results). Thus, the fact that we do not observe profilin I
Microfiltration
binding to VASP in brain extracts may be due to the endo-
PIP
2
(Sigma) at a concentration of 1 mg/ml in H
2
O was sonicated for
genous profilin II which is abundant in this tissue and com-
5 min to obtain micelles. Increasing concentrations of these micelles
were incubated with either profilin I or II in 10 mM Tris–HCl, 75 mM
petes efficiently with the Sepharose-bound profilin I.
KCl, 0.5 mM dithiothreitol (DTT) pH 7.5 for 30 min on ice. Afterwards,
The fact that profilin I and II have complementary
the samples were loaded on a Millipore filter with a molecular weight
affinities for the phospholipid PIP
2
and for poly-
L
-proline-
cut-off of 30 000 and centrifuged for 1 min at 2000 g. The flow-through
containing sequences, such as they occur in VASP and
was analysed on a 20% SDS–polyacrylamide gel, stained with Coomassie
Brilliant Blue.
CAP, may have important implications for signal transduc-
491
A.Lambrechts
et al
.
Circular dichroism
branes applying 30 V for4hin50mMTris, 50 mM borate buffer pH 8.
The blots were blocked in phosphate-buffered saline (PBS; 0.14 MCD in the near UV region (250–320 nm) was done in a JASCO J-170
spectropolarimeter using a 1 cm pathway cell. The step resolution was NaCl, 2.6 mM KCl, 10 mM Na
2
HPO
4
, 1.7 mM KH
2
PO
4
, pH 7.4) 1
2% non-fat milk powder at room temperature for 1.5 h. The primary0.5 nm and the scan speed used was 20 nm/min. For each sample, the
average of nine scans was obtained. The sample composition for the and secondary antibodies were diluted in PBS 12% non-fat milk
powder to the appropriate concentration. In between each step, the blotspeptCAPwt binding assay is 15 µM profilin (I or II) in 20 mM Tris–
HCl, 0.7 mM EDTA pH 8.2 and 0, 20, 50 or 100 µM peptCAPwt. In were washed five times in PBS. The primary antibody used was IE310.
The secondary antibody was an alkaline phosphatase conjugate and thethe PIP
2
binding assay we used samples containing 15 µM profilin in
20 mM Tris–HCl pH 8.2, 0.7 mM EDTA with 150 or 300 µM PIP
2
. staining of the blots was done in 0.1 M NaHCO
3
, 0.1 M MgCl
2
pH 9.8,
nitroblue tetrazolium (Sigma) in 70% dimethylformamide and 5-bromo,4-PIP
2
was sonicated for 10 min prior to addition of profilin and
the mixture was incubated for 30 min at room temperature before chloro,3-indolylphosphate (Sigma) in dimethylformamide.
measurement.
Immunoprecipitation
Fluorescence spectroscopy
Bovine brain extract was incubated with an anti-VASP monoclonal
Perelroizen et al. (1994) established that binding of poly-
L
-proline to antibody (IE273) for 2 h at room temperature. The antibodies were
profilin results in a change in fluorescence. Because the increase in pelleted with protein G–Sepharose (Pharmacia). After three washes with
fluorescence is a saturation function of the peptide concentration, we buffer B, the Sepharose pellet was dissolved in sample buffer and loaded
made a series of samples with an increasing amount of peptide (0–60 on an SDS–polyacrylamide gel. Detection of VASP was done with a
µM) and a constant amount of profilin (1 µM) in a total sample volume second monoclonal antibody (IE310) after electroblotting and was
of 400 µl. The buffer used was 20 mM Tris–HCl pH 7.5, 150 mM NaCl, processed as described above.
5 mM DTT. The samples were incubated for2hatroom temperature.
Fluorescence was measured (excitation was at 275 nm and emission at
Model building of profilin II
320 nm) with a SFM 25 fluorescence spectrophotometer (Kontron We built a model of bovine profilin II starting from the known profilin
Instruments). Because the profilin isoforms have a different number of I three-dimensional structure (Schutt et al., 1993) using the software
tyrosines (profilin I has four and profilin II seven), they have a different Homology (BIOSYM, 1994). The sequence identity (65%) of the two
intrinsic fluorescence (F
0
), and therefore we calculated the relative bovine isoforms is sufficiently high to use this homology building
increase in fluorescence, defined as (F–F
0
)/F
0
.strategy. First, we aligned the primary sequence of profilin II with the
profilin I sequence; no gaps or insertions needed to be introduced. The
Gel filtration
coordinates of all backbone atoms and atoms of the conserved side chain
The Superdex 200 column (Pharmacia) was equilibrated with 20 mM atoms of profilin I were then assigned to profilin II. Non-conserved side
Tris–HCl, 150 mM NaCl, pH 7.5. Profilin II samples in the same buffer chains were positioned in their most probable conformation by using a
were reduced with 40 mM DTT prior to addition of peptVASPwt, rotamer library (Ponder and Richards, 1987). We successively minimized
peptCAPwt or peptVASPs. Profilin and peptide were mixed at the the energy of side chains in mutated loops, the mutated side chains in
concentrations and ratios indicated in the legend to Figure 4 and were structurally conserved regions and finally all side chains. We assigned
incubated for 30 min at room temperature and then loaded on the the AMBER force field to the profilin II model. Acidic and basic residues
column. The flow rate was 0.4 ml/min and detection was at 280 nm. were charged –1 and 11 respectively. A charge of 0.5 was assigned to
histidines. We used this model for molecular dynamics simulations
Poly-
L
-proline affinity chromatography
(‘DISCOVER’, Biosym 1994) in vacuum to investigate the possible
Samples (1 ml) containing 16 µM profilin were loaded on a poly-
L
-dynamic behaviour of the conformation of each isoform. We applied an
proline column (0.5 ml) equilibrated with buffer A (20 mM Tris–HCl, energy minimization using a conjugated gradient optimizer whereby a
1 mM DTT, 1 mM EDTA, pH 7.5). After washing the column with 5 ml non-bond cut-off algorithm smooths the interaction over a range from
of buffer A, the remaining profilins were eluted with 5 ml of 8 M urea 15 to 16 Å. Throughout, we used an internal distance-dependent dielectric
in buffer A (Figure 5A). In the parallel experiment, profilin was eluted constant of 2. The structures of both profilins were equilibrated by
with increasing concentrations of PIP
2
in buffer A, as indicated in running dynamics at 300 K during 10 ps. We explored the local
Figure 5B. conformation space during 300 ps at 300 K.
Samples (1 ml) containing 10 µM profilin and 250 µM PIP
2
micelles
in 10 mM Tris–HCl, 0.3 mM EDTA, 1 mM DTT, pH 7.5 were incubated
for 30 min on ice and loaded on the poly-
L
-proline column. The elution
Acknowledgements
protocol was the same as for the samples containing only profilin.
We thank Professor M.Rosseneu for use of the CD spectropolarimeter.
Profilin affinity chromatography
We acknowlegdge Professors Ju
¨rgen Wehland and Ulrich Walter for a
One milligram of bovine profilin I or II was coupled to 0.39 g of CNBr- generous gift of the monoclonal VASP antibodies. C.A. is a Research
activated Sepharose 4B (Pharmacia) according to the manufacturer’s Associate of the Belgian National Fund for Scientific Research (NFWO).
manual. In parallel, Sepharose columns were made without coupling This work was supported by EC grant CI1-CT93-0049 and grant
any protein to the resin. The column was equilibrated with buffer B 13.0008.94 of the F.G.W.O. to C.A. and grants GOA-91/96-3 and
50 mM NaCl (buffer B 520 mM NaH
2
PO
4
, 2 mM EDTA, 0.5 mM G.0060.96 of the N.F.W.O. to J.V.
DTT pH 6.8).
Bovine brain extracts (20 ml) in 20 mM Tris–HCl pH 8.1, 50 mM
NaCl, 5 mM DTT, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride
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Received on June 20, 1996; revised on October 10, 1996
494
