(Received for publication, May 23, 1995)
From the
Profilin is a small 12-15-kDa actin-binding protein, which
in eukaryotic organisms is ubiquitous and necessary for normal cell
growth and function. Although profilin's interactions with its
three known ligands (actin monomers, phosphatidylinositol
4,5-bisphosphate (PIP), and poly-L-proline (PLP))
have been well characterized in vitro, its precise role in
cells remains largely unknown. By binding to clusters of
PIP
, profilin is able to inhibit the hydrolysis of
PIP
by phospholipase C
1 (PLC
1). This ability is
the result of profilin's affinity for PIP
, but the
specific residues of profilin's amino acid sequence involved in
the binding of PIP
are not known. Using site-directed
mutagenesis, we sought to localize regions of profilin important for
this interaction by generating the following mutants of human profilin
(named according to the wild-type amino acid altered, its position, and
the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53I,
R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of
L10R, all of the mutants were successfully expressed in Escherichia
coli and purified by affinity chromatography on PLP-Sepharose.
Only Y6F and K25Q demonstrated moderately less stringent binding to
PLP, indicating that most of the mutations did not induce marked
alterations of profilin's structure. When tested for their
relative abilities to inhibit the hydrolysis of PIP
by
PLC
1, most of the mutants were indistinguishable from wild-type
profilin. Exceptions included D8A, which demonstrated increased
inhibition of PLC
1, and R88L, which demonstrated decreased
inhibition of PLC
1. To assess the importance of the region
surrounding residue 88 of human profilin, three synthetic decapeptides
selected to correspond to non-overlapping stretches of the human
profilin sequence were tested for their abilities to inhibit PLC
1.
We found that only the decapeptide that matched the peptide stretch
centered around residue 88 was able to inhibit PLC
1 activity
substantially and was able to do so at nearly wild-type profilin
levels. Taken together with the finding that mutating residue 88
resulted in decreased inhibition of PLC
1 activity, these data
provide strong evidence that this region of human profilin represents
an important binding site for PIP
.
Since the molecule's discovery 20 years ago(1) ,
profilin's interactions with its three ligands (actin monomers,
phosphatidylinositol 4,5-bisphosphate (PIP), (
)and PLP) have been well established through in vitro studies (for review, see (2) ). For example, by binding to
actin monomers in a 1:1 complex, profilin decreases the critical
concentration of monomeric actin in the presence of thymosin
4(3) , inhibits the spontaneous nucleation of actin
filaments(4) , and catalyzes the exchange of adenosine
nucleotides bound to actin monomers(5) . By binding to
PIP
and to a lesser degree its precursor PIP(6) ,
profilin prevents PLC
1 from hydrolyzing
PIP
(7) . However, when PLC
1 is phosphorylated
on specific tyrosine residues, such as it occurs when extracellular
growth factors bind to and activate receptor tyrosine kinases, it is
able to overcome the protective effects of profilin and hydrolyze
PIP
(8) . Since PIP
binding to profilin
precludes the formation of profilin-actin complexes, one can conjecture
that in resting cells, PIP
sequesters profilin from actin
and that upon growth factor-induced cell activation, profilin is
released from PIP
by the hydrolytic actions of
phosphorylated PLC
1 and diffuses freely to the actin cytoskeleton,
where it then exerts effects as a regulator of actin polymerization.
How PIP is able to displace actin so effectively remains
unclear, and indeed, efforts to localize a binding site for PIP
on profilin have been supplanted until only recently by the more
extensive efforts to identify the binding site for actin. The quest for
the latter began as early as 1982 with biochemical studies involving
peptidases applied to actin(9) , whereas the first mention of a
putative binding site for PIP
on profilin did not occur
until 1991 when Pollard and Rimm (10) noted that the charge
differences between Acanthamoeba profilin-I and -II occur
between residues 24 and 66 (corresponding to residues 25-69 of
human profilin) and that a polylysine region exists between residues 80
and 115 (corresponding to residues 88-126 of human profilin).
These regions were suspected to be involved in PIP
binding
because first, positively charged residues are assumed to be involved
in the binding of acidic head groups of PIP
and second, the
more positively charged isoform, profilin-II, has
100 times
greater affinity for PIP
(11) .
A year later, Yu et al.(12) implicated the region spanning residues
126-136 of human profilin as a binding site for PIP by proposing the sequence KXXXXXXHXRR to be a
modification of the KXXXKXKK and
KXXXXKXRR motifs of gelsolin, which by themselves
bind to PIP
(12) . These motifs are also found in
CapG, villin, cofilin, and the PLC family(12) , all of which
bind PIP
. Another region was implicated by Raghunathan et al.(13) , who used fluorescence spectroscopy to
show that binding of profilin to PIP
resulted in marked
fluorescence quenching of Tyr-3 and Tyr-31. However, they also showed
through circular dichroism spectroscopy that upon binding to
PIP
, profilin undergoes a significant conformational change
involving an increase in
-helical content from a base line of 5%
to one as high as 35%. This being true, it is difficult to know whether
the changes in the fluorescence of Tyr-3 and Tyr-31 are due simply to
the proximity of a binding site or due to local conformational changes
transmitted from a distant binding site.
Finally, Vinson et
al.(14) , in elucidating the three-dimensional structure
for Acanthamoeba profilin-I, proposed a
PIP-binding site consisting of the loop between
-strands 1 and 2, the loop between
-strands 6 and 7, and the
region immediately after
-helix 2 (see Fig. 1B).
However, Fedorov et al.(15) recently showed through
calculations of electrostatic surface potentials that a second distinct
region of positive potential present on Acanthamoeba profilin-II but
markedly less so on profilin-I is located on the opposite side of the
protein. The positively charged residues here include Arg-66, Arg-71,
Lys-80, Lys-81, and Lys-115, which are analogous to the residues
Lys-69, Arg-74, Arg-88, Lys-90, and Lys-125 of human
profilin(16) .
Figure 1:
Site-directed
mutagenesis of human profilin. A, the amino acid sequence for
human profilin. Substituted residues are listed below their
sites. Boldfacedresidues are strictly conserved
across Acanthamoeba, yeast, echinoderm, and vertebrate
profilins; asterisks indicate conserved positive charges. B, topology diagram for the three-dimensional structure of
human profilin. Mutated residues are matched with sequencing gels
verifying the corresponding single base pair changes. Note that both
terminal -helices are connected to their adjacent
-strands
via hairpin turns so that both the N- and C-terminal amino acids,
contrary to what is depicted for clarity, are in close proximity to the
central
-pleated sheet.
To further define the importance of the
residues proposed to be involved in PIP binding, we mutated
single base pairs distributed across the human profilin cDNA sequence,
corresponding to the substitution of several conserved residues on both
terminal
-helices (Tyr-6, Asp-8, Leu-10, His-119, Gly-121,
Lys-125), basic residues implicated by Vinson et al.(14) (Lys-25, Lys-53), and basic residues located on the
second region of positive potential identified by Fedorov et al.(15) (Arg-74, Arg-88, Lys-90). Here, we report the results
of our testing these mutations for the effects they have on
profilin's ability to inhibit the hydrolysis of PIP
by PLC
1, an effect that has been shown previously to
correlate precisely with profilin's affinity for PIP
(11) . We found that most of the mutations did not
significantly alter profilin's ability to inhibit PLC
1
activity. However, the mutation D8A caused a marked increase of
PLC
1 inhibition, and R88L resulted in a marked decrease of
PLC
1 inhibition. Based on our work, we propose that a crucial
binding site for PIP
on human profilin is contained within
five amino acids of residue 88 since this stretch, by itself, inhibits
PLC
1 activity as well as the entire molecule of profilin.
Actin was purified from
rabbit skeletal muscle(23) . G-actin was separated from
residual F-actin by size-exclusion chromatography on a Bio-Gel P60 gel
column after dialysis against G buffer (2 mM Tris, pH 7.5, 0.1
mM ATP, 0.5 mM DTT, 0.1 mM CaCl)
and used within 7 days. Some of the actin was labeled with
pyrenyliodoacetamide (24) and stored in G buffer.
Recombinant phosphoinositide-specific rat brain PLC1 was
purified from bacterial cell extracts using a three-amino acid
C-terminal tag (Glu-Glu-Phe) engineered into the PLC
1
cDNA(25) . The recombinant PLC
1 displayed calcium
dependence, pH sensitivity, and substrate specificity indistinguishable
from that of wild-type bovine PLC
1. Synthetic decapeptides
cross-linked to an octabranched matrix core were obtained from Research
Genetics(26) . The anti-human profilin antibody (JH44) was a
generous gift from Donald A. Kaiser and Thomas D. Pollard(26) .
Figure 2:
Purification of mutant profilins. A, elution of human profilin from PLP-Sepharose. Several
fractions were analyzed by SDS-PAGE. Lane1 contains
the fraction (0.5 µg of protein) corresponding to the small protein
peak seen at the beginning of the 3.5 M urea wash; lane2 contains a fraction (<0.1 µg of protein) at the
end of the 3.5 M urea wash; lanes3 and 4 contain the fraction (6.8 and 0.3 µg of protein,
respectively) corresponding to the large protein peak at the beginning
of the 7.5 M urea elution. Lanes1-3 were stained with Coomassie Blue, while lane4 was analyzed by Western blot analysis using an antibody specific
for human profilin (see ``Experimental Procedures''). All of
the mutant profilins purified by this method eluted in the same pattern
as wild-type profilin except for Y6F and K25Q, of which elution started
with the 3.5 M urea wash (data not shown). B,
wild-type profilin, D8A, K25Q, and R88L analyzed by SDS-PAGE (5
µg of protein per lane). As shown, K25Q migrated slightly
slower than the other profilins and was the only purified mutant to
migrate differently from wild-type profilin. Samples were loaded after
elution from PLP-Sepharose. The gels were stained with Coomassie
Blue.
Figure 3:
Inhibition of PLC1 activity. A, the abilities of wild-type profilin (opencircles), D8A (closedtriangles), and
R88L (closedcircles) to inhibit PIP
hydrolysis by PLC
1. PLC
1 activity is expressed as a
percentage of activity in the absence of profilins. Theoretical curves
based on the values for K
calculated in Table 1are superimposed on the data. B, inhibition of
PLC
1 activity by synthetic peptides. The amino acid stretches of
profilin on which the peptide sequences were based are as follows: P1 (closedcircles), residues 50-59; P2 (closedtriangles), residues 83-92; and P3 (closedsquares), residues 128-137. The datapoints and errorbars were
calculated from two separate experiments.
In
light of the work by Fedorov et al.(15) , we were
particularly interested in the reduction of profilin's affinity
for PIP caused by mutating residue 88 since this result
suggests that a region near the loop between
-strands 5 and 6 may
be involved in the binding of PIP
. To assess the importance
of this region, we tested three different synthetic decapeptides
containing sequences that matched that of three non-overlapping
10-amino acid stretches in the wild-type human profilin sequence for
their abilities to inhibit PLC
1 activity. Besides selecting the
peptide segment surrounding residue 88, we also selected segments
implicated by Vinson et al.(14) and Yu et al.(12) as being involved in PIP
binding. The
only decapeptide that had a significant effect on PLC
1 activity
was that which corresponded to the segment centered around residue 88,
spanning residues 83-92 (Fig. 3B). Furthermore,
the degree of inhibition observed was comparable to that of wild-type
profilin (K
0.63 ± 0.13
µM, calculated from peptide concentrations greater than 1
µM). Thus, it appears that this 10-amino acid segment, by
itself, could account for much of profilin's ability to inhibit
PLC
1 activity. These data implicate this region of human profilin
(residues 83-92) as a key binding site for PIP
.
Figure 4:
Effect of R88L on actin polymerization. A, critical concentration of actin. Steady-state F-actin
concentrations were measured in the absence and presence of 3.5
µM wild-type profilin or R88L. B, time course for
actin polymerization. Polymerization of 10 µM G-actin in
the absence and presence of 6.2 µM wild-type profilin or
R88L was initiated at time 0 by the addition of 2 µM
MgCl.
When we tested the effect of R88L on the rate of actin monomer adenosine nucleotide exchange, we found its effect to be greatly diminished compared to that of wild-type profilin, such that at least 25 times higher concentrations of R88L were required to achieve comparable levels of catalysis (Fig. 5).
Figure 5: Effect of R88L on actin monomer nucleotide exchange. Time courses for the exchange of eATP for ATP bound to 1.5 µM G-actin in the presence of varying concentrations (indicated on graphs) of wild-type profilin (A) and R88L (B).
Figure 6:
Confirmation that R88L is not globally
denatured. A, Scatchard plot analyses of wild-type profilin (closedcircles) and R88L (opencircles) binding to PLP in the absence (toprow) and presence (bottomrow) of 7.5 M urea. The values of K for
native wild-type profilin and R88L were calculated from the slopes of
the best-fit lines to be 4.6 and 2.7 µM, respectively. B, time courses for the digestion of wild-type profilin and
R88L by trypsin (see ``Experimental
Procedures'').
The many effects of profilin on its ligands have been well
worked out through numerous in vitro studies, but how these
effects are coordinated inside of cells to produce vital functions has
been more difficult to ascertain. For example, the precise manner in
which profilin's three effects on actin are balanced in vivo remains unclear. The relative contributions of these effects
likely depend on the ratio of profilin-to-actin concentrations, the
relative availabilities of ADP and ATP, and the concentration of
thymosin 4 and other sequestering proteins. Since these parameters
probably vary greatly between different subcompartments of the same
cell, profilin may actually inhibit actin polymerization in some
regions of a cell while promoting actin polymerization in
others(37) . Recent evidence suggests that even across species,
profilin's role may vary depending on its total intracellular
concentration and the relative availability of other actin monomer
sequestering proteins(38) . Indeed, a variety of elegant in
vivo studies including the microinjection, deletion, and
overexpression of profilin in
cells(26, 38, 39, 40, 41, 42, 43, 44) has
demonstrated the dramatic phenotypic changes caused by simply altering
the level of total profilin in cells. Unfortunately, the specific
mechanisms responsible for such changes fail to be revealed with any
certainty by these studies.
Mutagenesis offers an alternative and
more targeted approach for dissecting out profilin's functions
and functional domains(35) , made all the more feasible by the
recent elucidation of the three-dimensional structures for Acanthamoeba and bovine profilin(14, 16) .
Using this approach, we substituted a variety of residues in the
primary structure of human profilin to clarify further the importance
of various regions of the molecule in binding to PIP. In
particular, we tested the effects of point mutations on the ability of
human profilin to inhibit PLC
1 activity, a property of profilin
that is directly related to its ability to bind
PIP
(11) . We mutated residues in the proposed
binding sites for
PIP
(10, 12, 13, 14, 15) ,
as well as a number of other residues including several in both the N-
and C-terminal
-helices, which constitute the most highly
conserved regions of profilin.
If the binding site proposed by
Vinson et al.(14) were correct, we would expect the
mutations K25Q and K53I, if any, to decrease profilin's ability
to inhibit PLC1 activity since both of these are in the region
analogous to the proposed site, and they both involve substitution of a
positively charged residue analogous to one present in Acanthamoeba profilin-II but not in profilin-I. Contrary to this, neither
mutation had any observable effect on profilin's ability to
inhibit PLC
1, a result not entirely surprising since we now know
that the loop between
-strands 1 and 2 makes up part of
profilin's binding site for PLP (32, 33) and
that profilin can bind both PLP and PIP
simultaneously(33) . This correlates with our observation
that the mutants Y6F and K25Q, both with a substitution in the region
of the PLP-binding site, exhibited diminished binding to PLP.
We
found that mutating residue Arg-88 on the opposite side of profilin
caused a decrease in profilin's inhibition of PLC1,
suggesting that the binding site actually exists on the opposite side
of the protein. This result is consistent with the presence of a
positive electrostatic potential over the analogous region of Acanthamoeba profilin-II(15) . Mutagenesis applied to
yeast profilin has shown that substituting Arg-72 decreased PIP
binding(35) , providing further evidence that the binding
site for PIP
is localized to this area. We also showed that
a decapeptide comprised of the sequence around residue 88 was able to
inhibit PLC
1 activity just as well as the entire molecule of
wild-type profilin, thereby providing strong evidence that a binding
site for PIP
on human profilin is located near the loop
between
-strands 5 and 6.
If the loop between -strands 5
and 6 represents a binding site for PIP
, then the binding
sites for PIP
and actin would overlap since biochemical
studies, x-ray crystallography, and mutational analysis of yeast
profilin have implicated the region spanning
-helix 3,
-strands 4-6, and the first portion of
-helix 4 as the
binding site for
actin(9, 16, 34, 35) . Such overlap
has already been demonstrated for cofilin (45) and yeast
profilin(35) , and this could account for the ability of
PIP
to dissociate profilin-actin complexes, although
PIP
-induced changes in conformation may also be important
in precluding actin binding(13) . Overlap of the two binding
sites is consistent with our finding that R88L exhibited markedly
diminished interactions with actin, an effect not simply explained by
large-scale denaturation since R88L demonstrated unaltered protease
sensitivity and exhibited normal binding to PLP. Smaller scale effects
on conformation, however, are more difficult to exclude. In fact, just
as PIP
binding induces a conformation that may disfavor
actin binding, the mutation R88L may be stabilizing an intermediate
conformation that favors neither PIP
or actin binding.
Crystallographic analysis is underway and should provide us with
specific structural information concerning this mutant.
Why the
charge differences between Acanthamoeba profilin-I and -II,
two isoforms displaying markedly different affinities for
PIP, cluster to the side of the molecule that does not bind
PIP
remains unclear. It may be that the overall positive
charge of profilin is important in facilitating an interaction with
PIP
, as would be consistent with the increased affinity of
D8A for PIP
, but that a separate non-electrostatic
interaction is crucial for the cooperative binding of multiple
PIP
molecules. This is supported by the fact that the
relative abilities of our three synthetic decapeptides to inhibit
PLC
1 activity did not correlate with the net charge of each
peptide, a result also seen with PIP
-binding peptides
derived from gelsolin(46) . Examination of the loop at the
proposed site shows that it protrudes from the molecule and could
potentially serve as a core for the clustering of PIP
molecules, an arrangement perhaps stabilized by a hydrophobic
interaction between the proximal aspects of the acyl chains of
PIP
and the aliphatic residues at the tip of the loop
(Gly-93, Gly-94, and Ala-95). This may explain why profilin does not
bind to IP
(11) , the acidic head group of PIP
cleaved by PLC
1 from the remainder of the molecule.
Recently, the PIP
-binding region of the N-terminal homology
domain of pleckstrin was found to include a loop contained in the
sequence KKGSVFNTWK(47) . This bears a striking resemblance to
the loop between
-strands 5 and 6 of Acanthamoeba profilin-II contained in the sequence KKGSAGVITVK. Admittedly, the
corresponding sequence for human profilin is not so similar, but it is
interesting to note that human profilin has a 10-fold greater affinity
than Acanthamoeba profilin-II for PIP
(11) and that the single major difference between the two
profilin tertiary structures is in this same loop, which is much larger
and more protrusive in human profilin. (
)Furthermore, the
affinity of pleckstrin for PIP
(K
30 µM) (47) is much closer to that
of Acanthamoeba profilin-II than to that of human profilin for
PIP
.
The details of how profilin binds PIP is certainly complicated and will require additional research to
establish more clearly the structures and mechanisms involved.
Mutational studies directed to other residues in the vicinity of
residue 88 are currently underway, and three-dimensional studies such
as nuclear magnetic resonance spectroscopy or x-ray crystallography of
the profilin-PIP
complex will be needed to confirm our
proposed region of human profilin as the true PIP
-binding
site. Meanwhile, the generation of profilin mutants, which are
deficient for the profilin-PIP
interaction but not for the
actin interactions, should help us to determine through their
overexpression in mammalian cells the physiologic importance of the
PIP
interaction in vivo. As for R88L, its intact
PLP-binding activity with diminished PIP
and actin
interactions may be useful in determining the importance of the
recently reported interaction between profilin and
vasodilator-stimulated phosphoprotein, an interaction mediated by
proline-rich domains on vasodilator-stimulated
phosphoprotein(48) , by acting as a competitive inhibitor of
wild-type profilin. As such, site-directed mutagenesis provides us with
a powerful tool for deciphering the molecular interactions between this
multifunctional protein and its many ligands.