(Received for publication, November 6, 1995; and in revised form, March 13, 1996)
From the
Human fascin is an actin-bundling protein that is thought to be
involved in the assembly of actin filament bundles present in
microspikes as well as in membrane ruffles and stress fibers. We have
found that human fascin is phosphorylated in vivo upon
treatment with 12-O-tetradecanoylphorbol-13-acetate, a tumor
promoter. The in vivo phosphorylation is gradually increased
from 0.13 to 0.30 mol/mol during 2 h of treatment, concomitant with the
disappearance of human fascin from stress fibers, membrane ruffles, and
microspikes. Human fascin can also be phosphorylated in vitro by protein kinase C at the same sites as observed in vivo as judged by phosphopeptide mapping. The extent of phosphorylation
depends on pH: the stoichiometries are 0.05, 0.38, and 0.6 mol of
phosphate/mol of protein at pH 7.0, 6.0, and 5.0, respectively.
Phosphorylation greatly reduces actin binding of human fascin, while
lowering the pH to 6.0 alone does not affect fascin-actin binding. With
the incorporation of 0.25 mol of phosphate/mol of protein, the actin
binding affinity is reduced from 6.7 10
to 1.5
10
M
. The actin
bundling activity is also decreased. These results suggest that
phosphorylation of fascin plays a role in actin reorganization after
treatment with 12-O-tetradecanoylphorbol-13-acetate.
HeLa 55-kDa actin-binding protein and sea urchin fascin are similar in that they both cause aggregation of F-actin into bundles and that their molecular masses are similar. Sea urchin fascin is a 58-kDa protein, first isolated from sea urchin eggs(1) . Fascin is thought to act in the formation of microvillar cores at fertilization (2) and is involved in the formation of filopodia by coelomocytes, phagocytotic defense cells of echinoderms(2) . We have purified a 55-kDa actin-bundling protein from HeLa cells(3) . Immunofluorescent studies have revealed that this human protein is localized in filopodia and membrane ruffles as well as in stress fibers(4) , suggesting the involvement of this protein in the organization of these structures.
The molecular cloning of sea urchin fascin by Bryan et al.(5) showed that fascin has 35% identity at the amino acid level to the product of the Drosophila singed gene, a 55-kDa protein of heretofore unknown function. The derived amino acid sequence of sea urchin fascin shows homology to three peptide sequences of HeLa 55-kDa protein, which suggested that HeLa 55-kDa protein is a human homologue of sea urchin fascin. Two groups have independently cloned a human cDNA homologous to Drosophila singed. They have demonstrated, in collaboration with us, that HeLa 55-kDa protein is a homologue of Drosophila singed protein and sea urchin fascin (6, 7) . Subsequently, mouse and Xenopus fascins have also been cloned(8) . The amino acid sequences of these fascins share no apparent homology with other actin-bundling proteins (including villin and fimbrin), indicating that the fascins represent a new family of actin-binding proteins.
It appears that each of these three proteins functions as an actin-bundling protein in microspikes and stress fibers of fibroblasts (4, 9) , in filopodia of coelomocytes(2) , and in bristles and nurse cells of Drosophila(10) . Many Drosophila singed mutants exhibit two phenotypes: gnarled bristle development and female sterility(11) . A common feature shared between the two phenotypes involves the association of the singed protein with actin fibers(10) . In bristles, actin-containing filament bundles that are oriented along the length of the shaft surround microtubules in the center of the bristle. In singed mutants, a decreased number of microfilament bundles are observed, resulting in a short, curved bristle shaft(10, 12) . In Drosophila oogenesis, each developing oocyte is surrounded by and connected by intercellular bridges to 15 nurse cells(13) . Nurse cell cytoplasmic contents flow into the oocyte along actin filaments traversing these cytoplasmic bridges. A singed allele associated with female sterility has been reported to affect the microfilament structure required for this nurse cell cytoplasmic flow(10) .
It is not clear what
regulates the actin binding and bundling activities of human fascin.
This report shows that human fascin is phosphorylated in vivo up to 0.30 mol/mol of protein by TPA ()treatment. Human
fascin can also be phosphorylated in vitro by PKC when the pH
is lowered to 6.0. In vitro phosphorylation occurs at the same
sites as observed in vivo as judged by phosphopeptide mapping.
Phosphorylation greatly reduces the actin binding ability of human
fascin, although lowering the pH to 6.0 alone does not affect
fascin-actin binding. While it remains to be established whether PKC is
indeed responsible for in vivo phosphorylation of human
fascin, these results suggest that phosphorylation of human fascin
regulates its actin binding and bundling activities, thereby
controlling the organization of microspikes as well as stress fibers.
For S labeling, cells
(one 100-mm dish, 70-80% confluency) were incubated in
methionine-free Dulbecco's modified Eagle's medium
containing 55 µCi/ml Tran
S-label (ICN) and 10%
dialyzed newborn calf serum. Cells were treated with TPA in the same
way as described above.
Immunoprecipitation was performed with two
monoclonal antibodies (clone 55k-2 and 55k-14), which have been
characterized previously(4) . After being thawed quickly, the
samples lysed in the SDS sample buffer were first diluted 10 times with
phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl,
1.5 mM KHPO
, 8 mM Na
HPO
(pH 7.3)) containing 50 mM NaF, homogenized by several passages through a 25-gauge needle,
and then centrifuged at 100,000
g for 30 min. To the
supernatants containing the same total radioactivities was added 0.025
volume of ascites fluids of the monoclonal antibodies. After incubation
at 4 °C for 1.5 h, rabbit antibodies against mouse IgG that had
been conjugated to protein A-Sepharose were added and further incubated
for 1 h. After washing, one-half of the immune complexes were loaded on
one- or two-dimensional gels and analyzed by autoradiography. Human
fascin is focused as multiple spots on two-dimensional
gels(3) . To unambiguously identify fascin spots on
two-dimensional gels, the other half of the immunoprecipitate samples
were mixed with purified, nonradioactive HeLa fascin and separated on
two-dimensional gels, and fascin spots were identified by comparison of
the patterns detected by Coomassie Blue staining and by
autoradiography. The amounts of radioactivity in
S- or
P-labeled protein spots separated on one- or
two-dimensional gels were quantitated with a PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA).
Figure 1:
In vivo phosphorylation of
human fascin upon treatment with TPA. Human neuroblastoma cells were
labeled with [P]P
for 2.5 h and then
treated with TPA for 2 h. TPA was not added to control cells. Human
fascin (indicated by 55K) was immunoprecipitated and analyzed
by SDS-polyacrylamide gel electrophoresis. Lane 1, fascin from
control cells; lane 2, fascin from TPA-treated cells.
Molecular mass markers are indicated to the left (from top to bottom: 200, 116, 96, 68, 42, 30, and 21
kDa).
We examined
the time course of fascin phosphorylation after TPA treatment. As Fig. 2shows, fascin phosphorylation is not a rapid reaction,
gradually increasing over 2 h after TPA treatment. To correlate fascin
phosphorylation with changes in the localization of fascin, we have
immunolocalized fascin during the time course of fascin phosphorylation (Fig. 3). Human neuroblastoma cells are heterogeneous,
consisting of two kinds of cells: spindle-shaped cells with few stress
fibers but with long neurite-like extensions (Fig. 3A)
and well spread cells with stress fibers and short microspikes (Fig. 3B). In a short time (10 min) after TPA
treatment, the neurite-like extensions disappear. However, numerous
membrane ruffles that show strong staining with fascin antibody are
induced in both types of cells (indicated by arrowheads in Fig. 3, C and D), as reported previously with
the other type of cells(16) . In addition, short microspikes
are often associated with membrane ruffles, which are also stained with
fascin antibody. In contrast, stress fibers are more resistant to TPA:
fascin staining of stress fibers persists at this time (Fig. 3D), when the phosphorylation level is low.
Fascin staining of stress fibers disappears 1-2 h after TPA
treatment (Fig. 3F), when the phosphorylation level is
reaching a near maximum level. Concomitantly, the fascin staining in
membrane ruffles also becomes diffuse, with the appearance of
perinuclear cytoplasmic staining of fascin in many cells (Fig. 3E). Cell shape becomes more rounded. These
observations suggest that in vivo phosphorylation of fascin
appears to be correlated with the disappearance of fascin in stress
fibers and membrane ruffles at later stages of TPA treatment.
Figure 2:
Time
course of in vivo phosphorylation of human fascin after TPA
treatment. Fascin was immunoprecipitated from P-labeled
cells at the indicated times after TPA treatment and separated on a
one-dimensional gel, and phosphate incorporation was quantitated by a
PhosphorImager.
Figure 3: Immunolocalization of fascin before and after TPA treatment. Cells at different times of TPA treatment were stained with the monoclonal antibody against human fascin. A and B, before TPA treatment. Human neuroblastoma cells consist of two types of cells: spindle-shaped cells with long neurite-like extensions (A) and well spread cells with stress fibers and short microspikes (B). Both structures are stained with the fascin monoclonal antibody. C and D, 10 min after TPA treatment. Long neurite-like extensions rapidly disappear; instead, numerous membrane ruffles (indicated by arrowheads) stained with the fascin antibody are induced (C). Stress fibers, which are stained with the fascin antibody, are more resistant to TPA (D). E and F, 1 h after TPA treatment. Fascin staining of membrane ruffles disappears, and cells become more rounded (E). Stress fibers also disappear (F).
We
next determined the stoichiometry of in vivo phosphorylation
in the following way. Because human fascin is known to be focused as
multiple spots on two-dimensional gels(3) , we first
identified, by in vivoP labeling, which spots
represent phosphorylated isoforms of human fascin. Then, by labeling in vivo with [
S]methionine followed by
two-dimensional gel separation, the level of both phosphorylated and
unphosphorylated isoforms of fascin can be determined by PhosphorImager
analysis. Fig. 4A shows a Coomassie Blue-stained
two-dimensional gel of purified, nonradioactive HeLa fascin for
reference, which exhibits two major basic and two minor acidic spots
(designated as spots a-d from basic to acidic). Fascin was
immunoprecipitated from
P-labeled cells after 2 h of TPA
treatment. Fig. 4B shows an autoradiograph of a
two-dimensional gel of the immunoprecipitates where five spots are
observed (spots c-g). Co-electrophoresis of the
P-labeled immunoprecipitates with unlabeled human fascin
(data not shown) revealed that two (Fig. 4B, spots
c and d) of the five phosphorylated spots comigrate with
the two minor acidic forms (Fig. 4A, spots c and d), but not with the two major basic forms (spots
a and b), of purified HeLa fascin. The three most acidic
spots (Fig. 4B, spots e-g) are too minor
to be detected on the Coomassie Blue-stained two-dimensional gel
pattern of the purified protein (Fig. 4A). These are,
however, isoforms of human fascin because Western blotting using
enhanced chemiluminescence (Amersham Corp.) detected these minor spots
on two-dimensional gels (data not shown).
Figure 4:
Two-dimensional gel analyses of
TPA-induced phosphorylation of human fascin. Only relevant parts of the
two-dimensional gels are shown. For reference, human fascin purified
from HeLa cells was also analyzed in A, and protein spots were
visualized by staining with Coomassie Blue. For in vivo labeling, human neuroblastoma cells were labeled with either
[P]P
or Tran
S-label in
the presence or absence of TPA; human fascin was then
immunoprecipitated and analyzed by two-dimensional gel electrophoresis. B, TPA-induced,
P-labeled human fascin. C,
S-labeled human fascin in the absence of TPA. D,
S-labeled human fascin in the presence of
TPA.
With the identification of
phosphorylated isoforms of human fascin on two-dimensional gels, we
determined the stoichiometry of in vivo phosphorylation of
human fascin after a 2-h treatment of human neuroblastoma cells with
TPA. Cells were labeled with [S]methionine in
the presence or absence of TPA, and fascin was immunoprecipitated and
separated on two-dimensional gels. Fig. 4(C and D) shows the two-dimensional gel patterns of human fascin in
the absence and presence of TPA, respectively. Again, the
identification of these spots as fascin was confirmed by
co-electrophoresis of radioactive spots with unlabeled purified HeLa
fascin. Due to the lower sensitivity of
S in comparison
with
P, we cannot see the very minor spots from d to g in the control (Fig. 4C) and from e to g in the TPA-treated sample (Fig. 4D) on these autoradiographs. However, we can
detect spots a-d with a PhosphorImager and thus
determined the levels of the spots from a to d. We
found that the level of phosphorylated fascin (sum of spots
c-d divided by sum of spots a-d) is increased
from 13 to 30% after a 2-h TPA treatment of human neuroblastoma cells.
Figure 5: In vitro phosphorylation of human fascin by PKC. Human fascin (indicated by 55K) purified from HeLa cells was incubated with PKC under the conditions described under ``Materials and Methods.'' A, one-dimensional gel analyses. Lanes 1 and 2, Coomassie Blue staining; lanes 3 and 4, corresponding autoradiography. Lanes 1 and 3, with PKC; lanes 2 and 4, without PKC. Molecular mass markers are indicated by the arrowheads to the left and are the same as described for Fig. 1. B, two-dimensional gel analyses of in vitro phosphorylated human fascin. Panel 1, Coomassie Blue staining; panel 2, corresponding autoradiography.
In vitro phosphorylation of
fascin by PKC depends greatly on pH (Fig. 6). At pH 7.0 and
above, only 5% of fascin is phosphorylated, which initially made us
think that fascin is a poor substrate of PKC. However, the
stoichiometry of fascin phosphorylation is greatly improved as the pH
is lowered, while the activity of PKC itself is decreased. At pH 6.0,
38% of fascin is phosphorylated within 20 min, and 60% of fascin
is phosphorylated at pH 5.0. In contrast, phosphorylation of myosin
light chain, a good substrate of PKC, is decreased by 30% when the pH
is decreased from 7.0 to 5.0. The increase in phosphorylation of fascin
at lower pH values is probably not due to the activation of contaminant
kinases because a PKC peptide inhibitor (Sigma) inhibits
phosphorylation reactions of both fascin and myosin light chain to the
same extent at pH 6.0.
Figure 6:
pH dependence of in vitro phosphorylation of human fascin by PKC. HeLa human fascin was
phosphorylated by PKC at the indicated pH for 20 min under the
conditions described under ``Materials and Methods.''
Phosphate incorporation was determined by counting radioactivities of
fascin bands separated on SDS gels. As a control, myosin light chain
was also phosphorylated under the same conditions. , human fascin;
, myosin light chain.
To determine whether in vitro phosphorylation of fascin with PKC occurs at the same sites as observed in vivo, two-dimensional phosphopeptide mapping was performed. Fig. 7(A and B) shows phosphopeptide maps of in vitro phosphorylated fascin and in vivo phosphorylated fascin, respectively. These two maps appear to be identical, which is confirmed by peptide mapping of a mixture of in vivo and in vitro phosphorylated fascin (Fig. 7C). While the extent of in vitro phosphorylation greatly depends on pH, the patterns of two-dimensional peptide maps are found to be the same (data not shown). We also analyzed the phosphoamino acids in these spots and found that the two major and one minor phosphopeptide spots (indicated by arrowheads 1, 2, and 4) contain phosphoserine, while the other minor spot (indicated by arrowhead 3) contains phosphothreonine (data not shown).
Figure 7: Two-dimensional tryptic phosphopeptide mapping of in vitro and in vivo phosphorylated human fascin. A, in vitro phosphorylated fascin; B, in vivo phosphorylated fascin; C, a mixture of in vivo and in vitro phosphorylated fascin. O, origin. Note that the phosphopeptide spots of in vitro phosphorylated fascin match those of in vivo phosphorylated fascin.
Fig. 8shows actin binding of unphosphorylated (lanes 1-4) and phosphorylated (lanes 5-8) fascin at two different fascin concentrations (1 µM total fascin for lanes 1, 2, 5, and 6; 1.5 µM total fascin for lanes 3, 4, 7, and 8). Coomassie Blue staining (lanes 1-8) revealed that the levels of free fascin in the supernatants with phosphorylated fascin (lanes 5 and 7) are higher than those with control unphosphorylated fascin (lanes 1 and 3) (compare lane 5 with lane 1 and lane 7 with lane 3), indicating that phosphorylated fascin shows a lower actin affinity. Furthermore, the autoradiographs (lanes 9-12) corresponding to the Coomassie Blue-stained gels (lanes 5-8) show that most of the phosphorylated fascin remains in the supernatants (lanes 9 and 11).
Figure 8: Actin binding of phosphorylated human fascin. Two different concentrations (1 µM for lanes 5, 6, 9, and 10; 1.5 µM for lanes 7, 8, 11, and 12) of phosphorylated fascin (indicated by 55 K; 0.25 mol of phosphate incorporation/mol of protein) were incubated with 12 µM F-actin and centrifuged in a Beckman Airfuge (lanes 5-8 for Coomassie Blue staining and lanes 9-12 for the corresponding autoradiography). As a control, the same concentrations (1 µM for lanes 1 and 2; 1.5 µM for lanes 3 and 4) of unphosphorylated fascin were incubated under the same conditions but without PKC (Coomassie Blue staining; autoradiography is not shown because of lack of phosphate incorporation). Lanes 1, 3, 5, 7, 9, and 11, supernatants; lanes 2, 4, 6, 8, 10, and 12, pellets. Note that more fascin remains in the supernatants (lanes 5 and 7) when phosphorylated fascin is used compared with the control (lanes 1 and 3). Also note that most of the radioactivity remains in the supernatants (lanes 9 and 11), but not in the pellets (lanes 10 and 12). Molecular mass markers are indicated by the arrowheads to the left and are the same as described for Fig. 1.
We determined the level of radioactivity of fascin in the pellets and supernatants after actin binding. The phosphate incorporation of the original phosphorylated sample is 0.25 mol/mol of protein. After actin binding, the phosphate incorporation of fascin in the supernatant (Fig. 8, lanes 5 and 9) is increased to 0.71 mol of phosphate/mol of protein, while that in the pellet (lanes 6 and 10) is decreased to 0.07 mol/mol of protein. These results indicate that phosphorylated fascin has a greatly reduced actin binding activity.
We used 25% phosphorylated fascin to estimate its actin binding constant. Fig. 9shows a comparison of actin binding between unphosphorylated and phosphorylated fascin. Phosphorylated fascin shows reduced actin binding. The reduction in actin binding is not due to denaturation caused by pH 6.0 as judged from the following results. First, the actin binding affinity of fascin is not affected by the pH range from 7.5 to 6.0. Second, dephosphorylation of phosphorylated fascin by treatment with calcineurin (protein phosphatase 2B) restores actin binding. Although fascin is not completely dephosphorylated by this treatment, phosphate incorporation is decreased from 0.25 to 0.1 mol of phosphate/mol of protein. As Fig. 9shows (open square marked with an arrowhead), fascin treated with phosphatase shows a higher affinity than does untreated phosphorylated fascin, indicating that phosphorylation indeed regulates actin binding of fascin.
Figure 9:
Effects of phosphorylation by PKC on actin
binding of human fascin. Fascin was phosphorylated at pH 6.0 to a ratio
of 0.25 mol/mol of protein, and then actin binding of phosphorylated
fascin () was assayed at pH 7.0 as described under
``Materials and Methods.'' As a control, fascin was incubated
in the same way except for the addition of PKC and assayed for actin
binding (
). To examine the effect of dephosphorylation, 1
µM phosphorylated fascin was dephosphorylated by treatment
with calcineurin and examined for actin binding (indicated by the open square marked with an arrowhead). Note that
dephosphorylation restores actin binding of
fascin.
Fig. 10shows Scatchard plots of actin binding,
which yield a bell-shaped curve, suggesting that actin binding of
either phosphorylated or unphosphorylated human fascin is cooperative.
Actin binding constants calculated from 25-75% saturation points
give 6.7 10
M
for
control unphosphorylated fascin and 1.5
10
M
for 25% phosphorylated fascin.
These plots also indicate that both control and phosphorylated fascin
give a similar saturation of actin binding: one fascin molecule to four
to five actin molecules, which is similar to the value reported
previously (3) .
Figure 10:
Scatchard plots of actin binding of
phosphorylated () and control (
) fascin. Concave-down
shapes for the curves indicate that actin binding of both
phosphorylated and unphosphorylated fascin is
cooperative.
Finally, we examined the effects on actin bundling activities. As Fig. 11shows, the actin bundling activity of phosphorylated fascin is considerably lower than that of the control. The concentrations required for the half-maximal bundling activity are 1.1 µM for unphosphorylated fascin and 1.6 µM for phosphorylated fascin (0.25 mol of phosphate/mol of protein).
Figure 11:
Effects of phosphorylation by PKC on
actin bundling of human fascin. Fascin phosphorylated at a ratio of
0.25 mol/mol of protein was assayed for its actin bundling activity by
centrifugation in an Eppendorf centrifuge as described under
``Materials and Methods.'' , phosphorylated fascin;
, control unphosphorylated fascin.
We have shown in this paper that 1) fascin is phosphorylated in vivo up to 30% after TPA treatment; 2) PKC is able to phosphorylate fascin in vitro at the same sites as observed in vivo; and 3) in vitro phosphorylation greatly reduces fascin-actin binding. TPA treatment is known to induce movement of membrane ruffles and to cause disassembly of stress fibers in many cells(16) . Human neuroblastoma cells also show similar but complicated effects upon TPA treatment (Fig. 3). In a short time (10 min) after TPA treatment, when the phosphorylation level is still low, long neurite-like extensions are rapidly retracted; instead, numerous membrane ruffles are induced, which can be strongly stained with fascin antibody. Stress fibers are disassembled gradually over 2 h of incubation, which apparently causes rounding of most cells. Concomitantly, fascin staining of stress fibers as well as membrane ruffles disappears, resulting in more diffuse staining in the cytoplasm. The maximum level of phosphorylation roughly corresponds to these alterations of fascin distribution. These observations suggest that fascin phosphorylation may play a role in the dissociation of fascin from stress fibers and membrane ruffles, but does not appear to be involved in the initial rapid retraction of neurite-like extension upon TPA treatment.
In vivo phosphorylation of fascin is
substoichiometric (30%). However, the actin binding experiments
with partially phosphorylated fascin (Fig. 9) have shown that
the actin binding affinity is reduced to one-fourth of its normal
level, suggesting that phosphorylation at this level can have a
considerable effect on fascin-actin interactions. Furthermore, there
have been some reports that substoichiometric phosphorylation produces
biological effects. For example, phosphorylation of only 5 or 20%
(depending on the source of smooth muscle) of myosin light chain is
sufficient for tension development in smooth
muscle(18, 19) . We have also reported (22) that 15-30% of myosin light chain is phosphorylated
at Ser-1/Ser-2 during mitosis, which is suggested to inhibit myosin
from premature activation before cytokinesis.
It is interesting to note that phosphorylation of human fascin with PKC depends greatly on pH. The optimal pH of PKC is known to be pH 7.5, and indeed, myosin light chain is better phosphorylated by PKC at pH 7.0 or above. Thus, the pH dependence of fascin phosphorylation should not be attributed to the pH-dependent activity of PKC itself. Rather, it is likely that fascin phosphorylation sites become more accessible to PKC at a lower pH. Lowering the pH to 6.0 alone does not affect the actin binding ability, suggesting that fascin is not denatured at this pH. The observation that actin binding ability is recovered by dephosphorylation with calcineurin (protein phosphatase 2B) treatment confirms that phosphorylation actually regulates actin binding of fascin.
At present, we cannot conclude that PKC is responsible for phosphorylation of fascin in vivo. It is possible that local changes in pH may occur where both fascin and PKC are present. Indeed, some isoforms of PKC are known to be localized in microfilament structures(20) . However, it is not known whether pH drops upon TPA treatment. Furthermore, the time course of PKC activation upon TPA treatment does not appear to coincide with the time course of fascin phosphorylation. Further studies are required to identify whether PKC or another kinase (or kinases) is involved in fascin phosphorylation in vivo.
Phosphorylation appears to have a drastic effect
on actin binding of fascin. The actin binding assay (Fig. 11)
shows that the actin binding constant was decreased 4-fold with the
incorporation of 0.25 mol of phosphate/mol of human fascin. However,
this actin binding constant was obtained with a mixture of 75%
unphosphorylated and 25% phosphorylated human fascin. Densitometry of
the Coomassie Blue-stained gel shown in lanes 5 and 6 of Fig. 8revealed that 72% of fascin binds to actin.
On the other hand, densitometry of the autoradiograph of the same lanes
showed that only 20% of radioactive fascin binds to actin. Thus, if we
were able to use 100% phosphorylated fascin, we would find a much
reduced actin binding constant, perhaps >10 times lower than that of
unphosphorylated fascin.
We have recently identified one of the
sites of human fascin phosphorylation as Ser-39. ()This site
is well conserved among many fascins, including human, mouse, Xenopus, and Drosophila (sea urchin fascin has a Thr
residue instead). Furthermore, the sequence around this site is one of
the most conserved domains of these fascins, suggesting its importance
in the functions of fascin. We are in the process of generating fascin
mutants with the Ser residue replaced by Ala or Asp. It will be very
interesting to see whether the expression of such mutants changes the
organization of filopodia, membrane ruffles, and stress fibers. Such
studies will facilitate the understanding of the physiological
significance of phosphorylation of fascin in microfilament organization
and cell motility.