expression is usually constitutive, levels can also be tightly
regulated during development in a tissue- and isofom-
Summary
Despite its small size, profilin is an amazingly diverse and
sophisticatedprotein whose precise role in cells continues
to elude the understanding of researchers 15 years after
its discovery. Its ubiquity, abundance and necessity for
life in more evolved organisms certainly speaks for its
extreme importance in cell function. So far, three ligands
for profilin have been well-characterized in vitro: actin
monomers, membrane polyphosphoinositides and poly-
L-proline. In the years following its discovery, profilin's
role in vivo progressed from that of a simple actin-bind-
ing protein which inhibits actin polymerization, to one
which, as an important regulator of the cytoskeleton, can
even promote actin polymerization under the appropri-
ate circumstances. In addition, interactions with compo-
nents of the phosphatidylinositolcycle and the RAS path-
way in yeast implicate profilin as an important link
through which the actin cytoskeleton is able to communi-
cate with major signaling pathways.
Introduction
In 1976, while purifying calf spleen actin for crystallization
studies, Carlsson discovered the 12-15 kDa protein which
co-purifies with actin monomers in a stable 1:l complex(').
That same year, Tilney used the term 'profilamentous actin'
to describe the large amounts of unpolymerized actin stored
in the periacrosomal region of Thyone briareus sperm, main-
tained in this state by some as yet unknown complex of pro-
teind2). Carlsson's small protein subsequently became
known as profilin.
Profilin has since been found in all of the eukaryotic sys-
tems examined to date including plants, amoebae, plas-
modia, slime molds, yeast, flies, sea urchins, frogs and mam-
mals. Additionally, vaccinia virus carries a gene for
expressing its own profilin in infected animal host celld3).Of
interest to the 15% of us afflicted with hay fever is the
hypothesis that endogenous profilin may be keeping us sensi-
tized to plant profilin, now identified as a major allergen in
pollen(4).
Although every cell in an organism expresses profilin,
they do so in tissue-specific amounts, with intracellular con-
centrations ranging from 4 yM in pig smooth muscle, to 50
yM in human platelets, to 100 yM in amoebae(5).While the
specific manner. In the amoeba Acanthamoeba(6)and the
slime mold Dicty~stelium(~), for example, two separate genes
encode different isoforms; in flies, a single gene provides two
different transcripts, one of which is expressed throughout
the organism while the other is only expressed in ovaries dur-
ing oogenesis(*). In sea urchins, a particular isoform is
expressed only in response to injury(9);and in human gastric
carcinoma, transcript levels for profilin, along with those for
an assortment of other proteins associated with embryonic
development, is increased by several-fold in comparison to
normal mucosa cells(I0).Recently, researchers identified a
human gene which encodes a protein with high sequence
similarity to human profilin I, previously thought to be our
only isoform("). This gene for human profilin I1 provides
transcripts of three different lengths which display tissue
expression patterns distinct from that of human profilin I.
Hence, there are many varieties of profilin within and
between species, and as might be expected, functional differ-
ences between them have been demonstrated in vitro, partic-
ularly in their relative ability to bind their major substrates,
actin and PIP2 (phosphatidylinosito14,5-bipho~phate)(~).
The amino acid sequences for the twenty or so different
profilins cloned thus far show that they are poorly conserved
across species, since just one residue remains strictly invari-
ant. At the same time, these same sequences show enough
conservative substitutions and similarities between them,
especially at the C- and N-terminal a-helices, to allow for
their easy identification as profilins. As a result, the sec-
ondary and tertiary structures are well-preserved between
those of calf profilin(12)(Fig. 1) and Acanthamoeba profilin-
I(' 3). The common feature revealed in their three-dimen-
sional structures is a large, multi-stranded antiparallel p-
pleated sheet bisecting the protein, with both terminal
a-helices packed side-by-side on the same surface.
Profilin inhibits actin polymerization
Cells are not Aoppy bags. They maintain resilient forms and
shapes through the rigid support of an intricate endoskeleton
composed of microtubules, intermediate filaments and actin
filaments. In addition, many of these cells respond to external
stimuli by exhibiting a diverse repertoire of motile activities
such as crawling, protruding, secreting, retracting, engulfing
and ruffling. These activities result from the rapid, coordi-
nated remodeling of the same endoskeleton that provides
these cells with their shapes. Thus, our visual perceptions of
these movements simply reflect the changing shape of the
underlying cytoskeletal network.
Rapid remodeling of the actin cytoskeleton, a major com-
ponent of this network, is usually associated with a markedly
increased rate of actin filament turnover, characterized by the
selective polymerization of actin into specific regions con-
current with the selective depolymerization of actin from
other regions. Additionally, whole filaments can be translo-
cated from one cell region to another. Clearly, such processes
need to be strictly regulated, and indeed, at least 100 actin-
binding proteins, belonging to more than 30 families, have
been identified that affect the dynamics of actin polymeriza-
tion.
Noting that profilin forms a 1: 1 stable complex with G-
actin (short for Globular-actin, the monomeric form of actin),
but does not interact significantly with F-actin (short for Fila-
mentous-actin), Carlsson cleverly postulated profilin to be an
actin monomer sequestering protein whose effect is to pro-
tect pools of G-actin from po1ymerizing(l4).
Sequestration must play an important role in regulating the
actin cytoskeleton since cells keep astonishingly high con-
centrations of unpolymerized actin, between 50-200 pM.
The very same actin concentrations in vitro would polymer-
ize rapidly until the G-actin concentration fell to approx. 0.1
pM, the so-called critical concentration for actin. This con-
centration is determined in a test tube as the G-actin concen-
tration at which polymerization is exactly balanced by
depolymerization at steady state. These opposing processes
provide a feedback system which, in response to any sudden
fluctuation in the G-actin concentration, compensates imme-
diately to pull G-actin back to its critical Concentration. This
being so, the only way cells can maintain G-actin concentra-
tions a thousand times higher than the critical concentration
is through strict barriers to prevent polymerization, such as
by blocking filament ends, and by hiding monomers with
sequestering proteins so as to prevent the formation of new
filaments (a process known as nucleation). By inhibiting
nucleation in vitro, profilin can significantly retard actin
polymerization, since nucleation represents the rate-limiting
step in the formation of F-actin from pure G-actin(15).Inter-
estingly, profilin overexpression protects actin-overexpress-
ing mutant yeast cells from lethality(16),but it is not clear
whether this can be ascribed to profilin’s role as a
sequesterer.
Still, profilin’s role as an important actin monomer
sequesterer in vivo is dubious. Over the years, profilin lost its
position as the major sequesterer in favor of an even smaller
(5 kDa) actin-binding protein known as thymosinp4 which,
Fig. 1. Topology diagram for the three-dimensional
structure of bovine profilin as determined by X-ray
crystallography(12).The entire polypeptide chain con- NH2
sists of 139 amino acids. The central antiparallel, p-
pleated sheet is comprised of P-strands 1, 2, 5-7, and
the carboxy-terminal portion of P-strand 4.
like profilin, is ubiquitous and binds to G-actin and not to F-
actin(17).As compared to profilin, thymosinp4 in platelets is
more abundant (560 pM versus 50 pM for profilin), and
binds actin more tightly (Kd -0.4-0.7 pM versus -0.5-10 pM
for profilin). Furthermore, the decrease in thymosinp6actin
complexes in platelets upon activation with thrombin corre-
lates with the observed increase in F-actin(18), while the
decrease in profilin-actin complexes in neutrophils stimu-
lated with chemoattractants cannot account for the observed
increase in F-actin(19).Thus, it appears that sequestration is,
at most, a limited component of profilin’s interaction with the
actin cytoskeleton.
Profilin promotes actin polymerization
The polar appearance of an actin filament complexed with
myosin head fragments, when viewed by electron
microscopy, has led to the opposite ends being called the
barbed end and the pointed end. As it turns out, the two ends
behave very differently when in solution with G-actid20).
Since the barbed end exhibits faster kinetics, and has a criti-
cal concentration which is nearly one-tenth that of the
pointed end, it is at this end that productive polymerization
usually occurs. Conversely, the pointed end tends to depoly-
merize. The relationship between the net rates at the two ends
determines whether a filament elongates, shortens, or tread-
mills (disregarding for now the effects of proteins which cap
and sever filaments).
In the actin cycle, G-actin binds tightly to ATP to become
G-actin-ATP, which adds onto the filament’s barbed end to
become a subunit of F-actin. The ATP is hydrolyzed, with
release of the inorganic phosphate group, and the subunit
eventually falls off the filament as G-actin-ADP. Why F-actin
functions as an ATPase, able to consume a large fraction of
the ATP supply in quiescent cells, is still unclear. Interest-
ingly, the much lower ATPase activity of G-actin, as com-
pared to F-actin, is completely blocked by profi1id2l), sug-
a4
Cl
gesting that profilin may serve to increase the efficiency of
some as yet unidentified ATP-dependent function of F-actin
by reducing background hydrolysis. To complete the actin
cycle, G-actin must release its ADP so that ATP can take its
place. Unless this happens, G-actin-ADP will accumulate and
become detrimental, since G-actin-ADP polymerizp several
times slower than G-actin-ATP(20).Additionally,?G-actin-
ADP forms filaments which, under some conditions, are not
as stifff22),although the physiological significance of this is
unknown. As luck would have it, this step represents the rate-
limiting step of the entire cycle (off-rate, approx. 0.01 s-l).
Profilin is the only actin-binding protein known to increase
the rate of nucleotide exchange on G - a ~ t i n ( ~It~ accom-
).
plishes this by boosting the off-rate of the bound nucleotide
by more than lOOO-f~ld(~~). It becomes self-evident that in a
state of high actin filament turnover when G-actin-ADP
would tend to accumulate, profilin can profoundly increase
the rate of polymerization by ensuring a constant supply of
G-actin-ATP. In fact, profilin is so efficient in catalyzing the
release of bound-ADP that even if most of the G-actin is
sequestered by thymosinp4, profilin is still able to recharge
effectively and quickly a large pool of G-actin-ADP with
ATP(25).The selective affinity of Thymosinp4 for G-actin-
ATP may actually facilitate this process, by making G-actin-
ADP relatively more available for profilid2@.
An additional fascinating mechanism by which profilin
promotes actin polymerization has recently emerged. Careful
analysis shows that in the presence of thymosinp4 and excess
ATP, profilin actually desequesters G-actin from
thymosinp4 and, by lowering the critical concentration for
actin, increases filament assembly(27).The mechanism for
this effect remains to be elucidated, but it is suggested that
profilin couples the ATPase activity of F-actin to the addition
of profilin-actin complexes onto barbed ends with subse-
quent release of profilin, such that this process is actually
favored over the addition of actin alone. Support for this
model may come, unexpectedly, from crystallographic data
of the profilin-a-actin complex(12). In this complex, actin
apparently adopts a ribbon-like structure which, although
thought to be incompatible with bulk filament formation(28),
may represent a conformation occurring only at the barbed
end of filaments. Such a conformation may allow profilin,
when complexed with actin subunits, to function as a pump
for polymerization.
In the light of these proposed mechanisms, there is
increasing in vivo evidence to support profilin’s role as a pro-
moter of actin polymerization. For example, in CHO (Chi-
nese hamster ovary) cells which overexpress profilin, F-actin
is more abundant than in control cells. In addition, the fila-
ments formed are more stable and are more resistant to, and
recover faster from, cytochalasin D treatment(29).Cytocha-
lasin D caps barbed ends and produces G-actin-ADP. At least
in this case, the beneficial effects of profilin on actin poly-
mers probably derive from its effect on nucleotide exchange.
The bacteria Listeria rnonocytogenes provides a more
spectacular example of profilin’s role as a promoter of actin
polymerization. These intracellular pathogens, which cause
disease in humans, especially in infants and in those who are
immunocompromised, require profilin to propel themselves
through the interior of cells via an actin-based motility sys-
As they move, they leave behind long comet-like
tails of actin filaments which continue to polymerize rapidly
at their barbed ends interfaced with the rear ends of the bacte-
ria. Host profilin localizes to this interface and is necessary
for Listeria motility, since cell extracts depleted of profilin
are unable to support Listeria motion.
Profilin as a regulator of actin polymerization
When profilin is deleted from cells, the effects are severe, if
not lethal. Yeast cells without profilin exhibit slow growth,
temperature sensitivity, cell multinucleation and distorted
cell shape and size. In addition, they contain unusual actin
structures and lack actin cabled3’). Flies without profilin die
during late embryonic development and display abnormal
regulation of mitosis, cell binucleation, stalled cell migra-
tion, and aberrant actin structures including the absence of
actin filament bundles(32).Finally, mice without profilin die
at approximately the 100-cell stage of embryonic develop-
ment (D. J. Kwiatkowski and W. Witki, personal communi-
cation).
While it is tempting to pin down profilin’s role either as an
inhibitor or as a promoter of actin polymerization, it is likely
that the combination of these two effects permits definition of
its role more accurately as a reguZator of actin polymeriza-
tion. Indeed, profilin consistently localizes to areas where the
actin cytoskeleton undergoes rapid change. For example, it
concentrates in the lamellipodia of fibroblasts(33),and along
the cleavage furrow of dividing protozoa(34).In the fission
yeast Schizosaccharomyces pombe, normal expression of
profilin is necessary for the formation of the F-actin contrac-
tile ring. Either deleting or overexpressing profilin results in
the absence of ring formation, with subsequent arrest at
cytokinesi~(~~). Microinjecting rat kidney cells with profilin
results in a drop of F-actin content, while microinjecting pro-
filin-actin complexes results in a rise of F-actin content and
an increase in membrane ruffling(36).Thus, profilin seems to
act either as an inhibitor or as a promoter of actin polymeriza-
tion, depending on its intracellular level relative to actin.
Profilin’s effect on actin polymerization is highly con-
dition-dependent. This characteristic suits profilin’s role as a
regulator of the actin cytoskeleton particularly well, since
conditions in vivo can vary dramatically between different
regions of the same cell. In this way, profilin’s role in signal
transduction, which will henceforth serve as the focus of this
review, provides an appropriate link to a system which
demonstrates the very essence of regulation.
Profilin binds polyphosphoinositides
In 1985, nearly a decade after discovering profilin, the same
group of researchers found that profilin will bind the mem-
brane phospholipid PIP2 (phosphatidylinositol 4,5-bisphos-
phate), and to a lesser degree its precursor PIP (phos-
phatidylinositol 4-monophosphate), with a stochiometry of
I :4-5(37).In addition, PIP2, unlike poly-L-proline which is
able to bind profilin as well as profilin-actin complexes(38),
dissociates profilin-actin complexes rapidly and efficiently
in vitro, leading under certain conditions to actin polymeriza-
tion (a result consistent with a role for profilin as an actin
monomer sequesterer). This result was exciting since it pro-
vided a new link between the actin cytoskeleton and the sig-
nal transduction pathway known as the PI cycle (phos-
phatidylinositol cycle).
Why PIP2 dissociates profilin from actin so effectively is
unclear, since its affinity for profilin is nearly equal to that of
actin for the same molecule. Furthermore, the putative bind-
ing sites on profilin for actin and PIP2 do not overlap(’3).
Recent spectroscopic evidence sheds light on this effect by
suggesting that profilin may undergo a significant structural
change upon binding to PIP2(39),perhaps enough to disrupt
the binding site on profilin for G-actin.
In trying to determine whether any of these results corre-
late with the situation in vivo, researchers examined the sub-
cellular distribution of profilin. In resting platelets and leuko-
cytes, profilin does localize to the periphery of cells near the
plasma membrane(40), although in quiescent fibroblasts, its
distribution is largely cytoplasmic(33).Nevertheless, in acti-
vated platelets and moving fibroblasts, there is a significant
increase in profilin’s association with the periphery. While
these findings suggest that profilin may bind PIP2 in cells,
there is still no conclusive evidence for this since it is not
clear how much of the profilin is actually associating with the
membranes or simply localizing to the region underneath it.
Assuming for now that profilin does bind PIP2, we may
wonder how this interaction could account for the movement
of profilin to the periphery in activated cells. One hypothesis
is that PIP2 localization is altered when cells are activated,
and profilin re-localizes accordingly. However, there are no
good data yet to support such a change in PIP2 distribution.
Another hypothesis is that some profilin is bound to PIP2 in
resting cells, and that this binding increases when the cells
are activated. However, profilin at the periphery of activated
cells has been found to co-localize with actin f i l a m e n t ~ ( ~ ~ 3 ~ ~ ) .
Perhaps the most attractive hypothesis is that profilin is
released from PIP2 during activation concomitant with PIP2
hydrolysis, and is hence able to interact with actin. But why
then does profilin accumulate there? It may be that as actin
monomers diffuse toward membranes to replace monomers
consumed there by the rapid formation of cortical actin fila-
ments, they carry profilin along with them while they
exchange their ADP molecules for ATP. Profilin accumu-
lates at the periphery because unlike G-actin, which gets
incorporated into filaments, it has nowhere to go. Such piling
up of profilin may actually be useful in providing a negative
control for otherwise disordered rapid polymerization near
membranes, by invoking profilin’s ability to inhibit sponta-
neous nucleation.
Profilin and the receptor tyrosine kinase pathway
RTKs (receptor tyrosine kinases) are transmembrane recep-
tors which, as a consequence of binding extracellular growth
factors such as EGF (epidermal growth factor) and PDGF
(platelet-derived growth factor), dimerize and phosphorylate
themselves on cytoplasm-exposed tyrosine residues. These
phosphorylated residues subsequently serve as targets for
specific signaling proteins that contain and bind via SH2 (Src
homology region 2) domains. Once bound to RTKs, these
proteins are activated by the phosphorylation of their own
tyrosine residues. Thus, growth factors outside cells use
RTKs to activate signaling pathways inside cells. Examples
of signaling proteins activated in this way include PLCyl
(phospholipase Cyl) and P13-K (phosphatidylinositol 3-
kinase), two enzymes regulating the PI cycle, and both
GTPase-activating proteins and Ash, a docking protein for
guanine nucleotide-releasing factors of the Ras pathway
(Fig. 2).
Cells anchor themselves to the extracellular matrix using
actin bundles, which span the cytoplasm and attach to focal
adhesions composed of aggregates of transmembrane inte-
grins and associated cytoplasmic protein-ligands. When
CHO cells are starved in 0.5% serum, they become quiescent
and contain many actin bundles. Upon exposure to 10%
serum rich in growth factors, they ruffle, become motile, and
show a dramatic reduction in the number of actin bundles.
While tyrosine kinase inhibitors of the tyrphostin family
have little effect on starved cells, they completely block the
response of these cells to 10% serum (unpublished observa-
tions). These data suggest the following: first, starved cells
have little, if any, inhibitor-sensitive tyrosine kinase activity
Response
F’
II ,9
AA
\ \
Fig. 2. How growth factors use the tyrosine kinase pathway to pro-
duce changes in the actin cytoskeleton of mammalian cells. Abbre-
viations: arachidonic acid, AA; GTPase-activating protein, GAP;
leukotriene, LT; mitogen-activated protein kinase, MAPK; mito-
gen-activated protein kinase kinase, MAPKK; phosphatidylinositol
4,5-bisphosphate, PIP2; phosphatidylinositol 3-kinase, PI3-K;
phosphatidylinositol 3,4,5-trisphosphate, PIP3; phospholipase A2,
PLA2; phospholipase C, PLC; phosphorylated tyrosine residues, P;
prostaglandin, PG; receptor tyrosine kinase, RTK; Son of Sevenless
(a guanine nucleotide-releasing factor which is linked to RTKs
through a docking protein, Ash, not shown for clarity), Sos.
targeted towards the regulation of the actin cytoskeleton; and
second, the changes in the actin cytoskeleton of cells exposed
to 10% serum depend on an increase in tyrosine kinase
activity. Interestingly, CHO cells overexpressing profilin
contain nearly as many cell-spanning actin bundles as control
cells when starved, but show an exaggerated reduction in the
number of actin bundles when exposed to 10%
response which, as in control cells, is completely blocked by
tyrphostin. These results suggest that profilin is probably act-
ing downstream of the RTK pathway.
As alluded to earlier, one possible connection between
profilin and the RTK pathway is through PLCyl and the PI
cycle. Profilin protects PIP2 from hydrolysis by the unphos-
phorylated form of P L C Y ~ ( ~an ’ ) ,effect demonstrated even
with plant profilin and plant PLC-II(42). However, when
PLCyl is phosphorylated by RTKs bound to growth factors
such as EGF, it is able to associate with the plasma mem-
brane(43),overcome the protective effects of profilin and
hydrolyze PIP2 as efficiently as uninhibited PLCyl. In this
way, profilin serves to reduce the background hydrolysis of
PIP2 in unstimulated cells.
IP3 (inositol 1,4,5-trisphosphate), one of the products of
PIP2 hydrolysis, finds its way through the cytoplasm to
receptors on intracellular Ca+2 stores and triggers the Ca+2
response associated with PIP2 hydrolysis. The other product,
DAG (1,2-diacylglycerol), remains embedded in the plasma
membrane and activates PKC (protein kinase C). Both prod-
ucts eventually recycle back to PIP2. Interestingly, PKC can
phosphorylate profilin in vitro, an effect potentiated by
PIP2(44),but the physiological relevance of this remains with-
out proof.
Since profilin binds neither IP3 nor DAG(45),it is presum-
ably released in an unbound form into the cytoplasm, where
it may then interact with G-actin. Thus, not only could pro-
filin cause the PI cycle to be regulated by the phosphorylation
status of PLCyl, it could also, by the very same mechanism,
serve as a second messenger to the actin cytoskeleton. This
hypothesis is certainly attractive, and plausible too, since
most of the profilin in cells could be bound to
However, numerous studies have down-played the impor-
tance of the PI cycle in producing an actin response. For
example, blocking the PI cycle in human foreskin fibroblasts
with neomycin does not diminish the PDGF-induced actin
response. Strangely, neomycin by itself will induce an actin
response similar to that of PDGF(46).Given that neomycin
blocks the PI cycle by binding PIP and PIP2, thus preventing
their hydrolysis by PLC, it may be that neomycin triggers its
actin response by displacing actin regulatory proteins from
complexes with PIP and PIP2.
Even if profilin proves to be an important link between the
PI cycle and the actin cytoskeleton, it will probably represent
one of many molecules which do. Other actin-binding pro-
teins which potentially interact with the PI cycle include a-
actinin, gelsolin, protein 4. 1(47),c ~ f i l i n ( ~g~C) a, ~ 3 9 (and
~~)
MARKS (myristoylated, alanine-rich C kinase substrate)(50).
In addition, DAG has been shown to increase the nucleation
of actin filaments at plasma membranes(51).
Other links between the actin cytoskeleton and RTKs
include proteins other than PLCyl which contain SH2
domains. Tensin, an actin-binding protein involved in mem-
brane-associated cytoskeletal structures such as focal con-
tacts, contains an SH2 dornaiG). The EGF receptor itself is
an actin-binding protein(53).PI3-K, which catalyzes the pro-
duction of PIP3 (phosphatidylinositol 3,4,5-trisphosphate),
a complexes with a non-catalytic docking subunit containing
an SH2 domain, and is activated by RTKs bound to growth
factors. Interestingly, while PIP3 is nearly absent in quiescent
neutrophils, its level rises quickly upon stimulation of the
neutrophils with chemoattractants, and follows a time course
which closely matches that of actin polymerization(54).
Finally, the arachidonic acid metabolites prostaglandins and
leukotrienes, which are normally produced by EGF stimula-
tion, will by themselves reproduce the EGF-induced actin
response(55).
Profilin and small GTP-binding proteins
Another potential link between the actin cytoskeleton and
RTKs involves small G-proteins (GTP-binding proteins).
Small G-proteins are essential for the proper regulation of
diverse cellular processes involving growth and differen-
tiation. The more than 30 small G-proteins known to date are
structurally related to Ras, a proto-oncogene product found
in normal mammalian cells. Ras captured the attention of
researchers approximately ten years ago when the rus genes
encoding it were found to be carried by cancer-causing retro-
viruses. It is now appreciated that nearly one-third of human
tumors contain abnormally activated rus gene products.
A G-protein, in a manner analogous to actin, functions in a
catalytic cycle. It binds GTP to become G-protein-GTP,
which represents the ‘on’ state. It is turned ‘off‘ by hydroly-
sis of the GTP to produce G-protein-GDP. To turn itself back
‘on’ and thus complete the cycle, the G-protein must release
its GDP so that GTP can take its place. Just as F-actin cat-
alyzes the hydrolysis of ATP on actin subunits, specialized
proteins known as GAPs (GTPase-activating proteins) accel-
erate the hydrolysis of GTP on G-proteins; just as profilin
facilitates the recharging of G-actin with ATP by causing the
release of ADP, certain proteins known as GRFs (guanine
nucleotide-releasing factors) accelerate the recharging of G-
proteins with GTP by causing the release of GDP. Thus,
GAPs turn ‘off‘ G-proteins while GRFs turn ‘on’ G-proteins.
In mammalian cells, both GAPs and GRFs are activated by
the RTK pathway, and both proteins are regulated in an
opposite manner by membrane phospholipids(56).Potentially
important in the regulation of these proteins by phospho-
lipids is the fact that in reconstituted membranes, a rise in
Ca+2concentration, as would occur in cells upon activation
of the PI cycle, induces the aggregation of phospholipids into
discrete patches from an otherwise diffuse distribution(57).
In yeast, the small G-protein known as RAS, when turned
‘on’, activates adenylate cyclase to produce CAMP (adeno-
sine 3’,5’-cyclic monophosphate), an important effector of
diverse biological processes. For this response to occur, the
catalytic subunit of adenylate cyclase must be complexed
with the protein known as CAP (cyclase-associated protein).
Mutational analysis shows that the N-terminal domain of
CAP by itself mediates the responsiveness of adenylate
cyclase to RAS. Deleting the C-terminal domain of CAP
results in a number of abnormalities unrelated to adenylate
cyclase function including temperature and nutritional sensi-
tivities, and morphologic enlargement and rounding(58).
Since the effects of deleting the two domains are dissociable,
CAP appears to be truly bifunctional.
An exciting link between CAP and profilin was made
when researchers showed that overexpressing profilin saves
cells from the detrimental effects of deleting the C-terminal
domain of CAP, but has no rescue effect on the N-terminal
deleted mutants(59).The fact that cells lacking this domain
look and behave much like cells without either profilin or
PLC 1(60)may provide insights into the mechanism involved,
but little else is known about this interaction. Mutational
analysis of yeast profilin has shown that only profilins which
have both actin- and PIP2-binding abilities are able to save
the CAP C-terminal-deficient mutants(61).It may be that
CAP interacts directly with actin, especially since ASP-56,
its homolog in pig platelets, is an actin-binding protein(62).
CAP may also bind profilin. Interestingly, CAP contains pro-
line-rich domains which, as to be discussed below, may serve
as binding sites for profilin. Finally, CAP may bind mem-
brane lipids, in particular those of the PI cycle. This would
not surprise us since RAS, actin and profilin all seem to inter-
act with components of the PI cycle.
In mammalian cells, small G-proteins do not activate
adenylate cyclase, and hence would not require CAP for this
function. However, it is possible that a separate interaction
between small G-proteins, lipids and profilin is conserved.
Rac and Rho, two small G-proteins of the Ras family that are
important in controlling actin network reorganization, may
be important in such an interaction. Rac promotes the accu-
mulation of actin filaments in the periphery of cells as seen in
membrane ruffling, while Rho induces the formation of actin
stress fibers and focal adhesions(63).
Does profilin contain an SH3 domain?
In addition to binding G-actin and PIP2, profilin also binds to
PLP (p~ly-L-proline)(~~). This has led PLP-Sepharose affin-
ity chromatography to become the most commonly used
method for purifying profilin from cell extracts(@).Varying
conditions reveal that at least eight consecutive prolines are
required for this interaction(65),although fluorescence and
nuclear magnetic resonance spectroscopy suggest that only
six consecutive prolines are necessary@@.Interestingly,
binding of profilin to PLP does not occur as a high affinity
complex in vitro. However, transitions between the cis- and
trans-conformations of a proline stretch within a protein, in
response to the changing hydrophobicity of the surrounding
medium, may provide a regulatory mechanism for this inter-
action.
Thus far, researchers have failed to identify any cellular
ligands which use PLP domains to bind profilin. However,
candidates exist. As alluded to above, CAP contains a stretch
of six consecutive prolines and a separate proline-rich
domain between the N- and C-terminal domains. Since pro-
filin is able to bind either a ~ t i n (or
~ PIP2
~ )(@') simultaneously
with PLP, profilin may serve to target CAP to the actin
cytoskeleton or the plasma membrane.
Listeria monocytogenes expresses a surface protein, ActA,
which contains the amino acid sequence motif DFPPPPT-
DEEL repeated four times with slight variations, and is
another candidate for this interaction. ActA co-localizes with
host profilin to the rear ends of bacteria in infected cells.
However, profilin can only bind ActA when inside infected
cells or in cell-free extracts, suggesting that another host fac-
tor is required for its binding(3o).This other factor may regu-
late transitions between the cis- and trans-conformations of
the proline stretch.
Ten-amino acid motifs which are proline-rich have
recently been identified as binding sites for SH3 (Src homol-
ogy region 3) domains(67).SH3 domains work downstream
of RTK pathways where they target signaling proteins, par-
ticularly those regulating the actin cytoskeleton, to their
active sites. Since profilin has associations with both signal-
ing pathways and the actin cytoskeleton, it is conceivable for
profilin to contain an SH3 domain. Indeed, pig profilin shows
a 54% sequence similarity and 24% identity with the SH3
domain of PLCy1(68).
SH3 domains consist structurally of multiple anti-parallel
P-sheets forming a solvent-exposed hydrophobic patch of
conserved aromatic residues(69). Profilin has an SH3-like
fold with its anti-parallel P-sheets, as well as a solvent-
exposed hydrophobic patch of aromatic residues between the
N- and C-terminal a-helices(12).However, the two features
are not associated as they are in SH3 domains, making simi-
larities difficult to delineate.
Recent evidence implicates the hydrophobic patch
between the two terminal a-helices as the binding site for
PLP. For example, mutagenesis of aromatic residues in the
hydrophobic patch(68)or truncation of more than three C-ter-
minal residue@) abolishes profilin's binding to PLP.
Admittedly, conformational effects are difficult to rule out
since this same area is important for the stability of the mole-
cule@'). However, fluorescence and nuclear magnetic reso-
nance spectroscopies reveal that residues localized to this
hydrophobic patch are those which are most perturbed by
PLP binding@@.Thus, the SH3-like fold on profilin is dis-
tinct from the PLP binding site, and as yet there is no evi-
dence to suggest that this fold actually represents a functional
SH3 domain.
Conclusion
Profilin is remarkable as a small protein with diverse proper-
ties and effects. lnitially thought to be just an actin sequester-
ing protein, profilin is now known to regulate the dynamics
of actin polymerization in a sophisticated manner which
makes it difficult to categorize by any general effects. Its
interactions with signaling pathway components adds further
depth by placing profilin at the crossroads of transmembrane
signal transduction and the actin cytoskeleton. Adding to the
already intricate system of communication between signal-
ing pathways, profilin is poised to reinforce the innumerable
checks and balances existing between RTK pathways, mem-
brane lipids, actin-binding proteins and the actin cytoskele-
ton. As for the future, continued thoughtful and meticulous
research will surely provide us with even more fascinating
roles for this elegant molecule.
Acknowledgments
This research was supported in part by grants from Syntex,
the Bernard A. and Rebecca S. Bernard Foundation, and the
American Heart Association (G-I-A, Maryland Affiliate,
Inc.). P.J.G.-C. was selected as a Syntex Scholar in 1992.
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Richard H. Sohn and Pascal J. Goldschmidt-Clermont* are
at the Bernard Laboratory for Fundamental Research in Preven-
tive Cardiology, Division of Cardiology, Department of Medi-
cine, and the Department of Cell Biology and Anatomy, The
Johns Hopkins University School of Medicine, Baltimore,
Maryland 21287, USA.
*Author for correspondence at Ross Research Building, Room
1023,720 Rutland Avenue, Baltimore, MD 21287, USA.