(Received for publication, February 21, 1995; and in revised form, April 24, 1995)
From the
Actin depolymerizing factor (ADF) occurs naturally in two forms,
one of which contains a phosphorylated Ser and does not bind G-actin or
depolymerize F-actin. Removal of this phosphate in vitro by
alkaline phosphatase restores full F-actin depolymerizing activity. To
identify the phosphorylation site, [
Actin depolymerizing factor (ADF),
Phosphorylated forms of both ADF and
cofilin have been identified in vertebrate
cells(9, 10, 11) . We isolated phosphorylated
ADF and showed it to be inactive in depolymerizing F-actin and in
inhibiting the assembly of G-actin(11) . Phosphoamino acid
analysis identified phosphoserine as the phosphorylated amino
acid(11) . Here, we demonstrate that pADF can be completely
reactivated following phosphatase treatment. We also determine the
location of the phosphoserine and show by site-directed mutagenesis and
expression in vertebrate cells that this is the site for regulation by
phosphorylation in vivo.
Figure 1:
Reactivation of pADF by alkaline
phosphatase. Partially purified pADF, which was eluted from an HAP
column, contained no cofilin or ADF as determined by two-dimensional
gel electroblotting. It was boiled in 1% SDS to inactivate protease and
phosphatase activity. The protein was precipitated, dissolved in SDS,
and treated with 10 units of alkaline phosphatase (
Figure 2:
Purification of pADF. A,
fractions from the HAP column were subjected to SDS-PAGE and silver
stained. The pADF ran just slightly above the ADF standard (S)
with the major amounts eluting in fractions 4-6. B,
autoradiograph of the gel in A showing that
[
Figure 3:
HPLC peptide maps comparing endopeptidase
Lys-C digests of purified brain ADF and pADF. The position of the
single
Figure 4:
HPLC-electrospray mass spectrometry of the
Lys-C phosphopeptide following digestion with the endopeptidase Asp-N.
The sequence of the N-terminal Lys-C peptide from pADF is shown above with the expected Asp-N peptides underlined.
Peptides A, B, and C all arose from the
Lys-C peptide. Peptide D was a contaminating peak arising from
the large Lys-C peptide, which eluted directly after the
phosphopeptide. This peptide contained no Asp and was identified by
direct sequencing of the peak from the Lys-C peptide fractionation
shown in Fig. 3.
Figure 5:
Identification of the phosphorylation site
by tandem mass spectrometry. A, N-terminal Asp-N peptide from
pADF. B, same peptide after alkaline phosphatase treatment. C, peptide fragments expected from the N-terminal end (b) and C-terminal end (y) of the peptide. Those that
are readily identified in the mass spectra are labeled in A and B. p, parent ion. Amino acid B is
phosphoserine (serine in the dephosphorylated
peptide).
Figure 6:
Two-dimensional immunoblot analysis of ADF
species in extracts of HeLa cells transfected as follows. 1a,
nothing; 2a, wild-type chick ADF; 3a, ADF(E3) mutant; 4a and 4b, mixture of samples in 2a and 3a; 1b, ADF(A24,25) mutant; 2b, ADF(A3)
mutant; 3b, ADF(
Figure 7:
Activity of the recombinant ADF(E3) is
reduced to about 10% of that of wild-type ADF. Bacterially expressed
wild-type ADF and the ADF(E3) mutant were purified and assayed for
their ability to depolymerize F-actin in the DNase I inhibition assay.
The equivalent inhibition of DNase I was achieved with 0.4 µg of
ADF and 4 µg of ADF(E3). Each point is the average of triplicate
assays.
Regulation of proteins by phosphorylation is a well
established mechanism for controlling biological activity (for review,
see (28) ), nuclear translocation (for review, see (29) ), and protein degradation(30) . Since ADF and
cofilin contain identical nuclear translocation signal
sequences(31, 32) , and both can be transported to the
nucleus of cells under stress(33, 34) , their
phosphorylation in cells may be involved in regulating one or more of
the above events. In order to demonstrate that the reversible
phosphorylation of ADF controls its cellular activity, we needed to
show that it can be reactivated by removal of the phosphate. Partially
purified preparations of pADF contain proteases, which degrade ADF when
incubated at 37 °C(11) . However, by boiling the proteins
with SDS, these proteases are permanently inactivated. ADF can be
reactivated after denaturation and dephosphorylation.
Regulation by
phosphorylation/dephosphorylation may control the activity of ADF in
cells. ADF is normally down-regulated in developing chick skeletal
muscle in vivo but not in primary myocyte cultures of
equivalent age in vitro. Interestingly, myotubes developing in vitro compensate for their inability to down-regulate ADF
expression by converting the abundant ADF to the inactive form, pADF,
which accumulates during the process of in vitro myogenesis(11) . ADF colocalizes with actin in the
membrane ruffles of motile cells and, in developing neuronal growth
cones, areas containing highly dynamic actin filaments(35) .
Rapid cycling of actin assembly/disassembly could be regulated by ADF
phosphorylation.
Recent studies show phosphorylation of both ADF and
cofilin are altered by various signal transduction pathways that are
activated by growth factors and pharmacological agents. Treatment of
cultured rat primary astrocytes with dibutyryl-cAMP or the myosin light
chain kinase inhibitor, ML-9, causes dramatic shape changes and
reorganization of the actin filament network and increased
dephosphorylation of both the regulatory light chain of myosin and
ADF(36) . Additionally, treatment of primary cultures of dog
thyroid cells with thyrotropin causes distinct morphological changes
and a redistribution of the actin stress fiber network, as well as
rapid dephosphorylation of both ADF and cofilin(37) . Since the
thyrotropin effects could be mimicked by forskolin, it seems likely
that the dephosphorylation of ADF is regulated in both astrocytes and
thyroid cells by a cAMP-dependent process. Additionally, cofilin
dephosphorylation in human platelets could be stimulated by GTP
Dephosphorylation of ADF (37) and cofilin (9, 37) accompanies nuclear transport in many cells
placed under stress. In addition, the costimulatory signals for human
T-cell activation induce the dephosphorylation of cofilin and its
translocation to the nucleus of human T-cells(39) . Cofilin
dephosphorylation correlates with the induction of interleukin-6
responsiveness and interleukin-2 secretion. Interestingly, cofilin
dephosphorylation and translocation to the nucleus occur spontaneously
in autonomously proliferating T-lymphoma cells. However,
dephosphorylation of ADF and cofilin, which occurs in thyroid cells in
response to thyrotropin(37) , is not accompanied by nuclear
translocation, suggesting that dephosphorylation is not sufficient for
nuclear uptake.
Ser
We have identified the single
regulatory phosphorylation site within the chick brain ADF primary
sequence as Ser
Figure 8:
N-Terminal sequences for proteins in the
ADF/cofilin family from different sources. Sequences are compared with
human ADF and are aligned for maximum homology around Ser
Although the major actin binding regions of proteins in
the ADF/cofilin family have been identified as domains in the
C-terminal half of the protein(48, 49) , recent
evidence suggests that amino acids near the N terminus also play a
role.
The identification of the
regulatory phosphorylation site on ADF and the development of
site-directed mutants will enable further investigation into the
cellular role of this regulation. We are currently isolating stable,
clonal populations of various cell lines, which have been transfected
with vectors expressing the ADF(A3) and ADF(E3) mutants and will
examine the effects of these mutations on actin-based functions
including cell motility, neurite outgrowth, and nuclear rod formation.
We thank Dr. K. M. Swiderik, Department of Immunology,
Beckman Research Institute of the City of Hope, Duarte, CA for expert
technical assistance in obtaining the mass spectrometry results
presented here and Craig Miles, Macromolecular Resources, Colorado
State University, for protein sequencing. We also thank Judith Sneider
for technical assistance and Drs. Norman Curthoys, Barbara Bernstein,
and A.-Young Woody for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P]pADF was
purified and digested with endoproteinase Lys-C. The digest contained
only one
P-labeled peptide. Further digestion with
endoproteinase Asp-N and mass spectrometric analysis showed that this
peptide came from the N terminus of ADF. Alkaline phosphatase treatment
of one Asp-N peptide (mass 753) converted it to a peptide of mass 673,
demonstrating that this peptide contains the phosphate group. Tandem
mass spectrometric sequence analysis of this peptide identified the
phosphorylated Ser as the encoded Ser
(Ser
in
the processed protein). HeLa cells, transfected with either chick
wild-type ADF cDNA or a cDNA mutated to code for Ala in place of
Ser
or Thr
, express and phosphorylate the
exogenous ADF. Cells also expressed high levels of mutant ADF when
Ser
was deleted or converted to either Ala or Glu. However,
none of these mutants was phosphorylated, confirming that Ser
in the encoded ADF is the single in vivo regulatory
site.
(
)is
an 18.5-kDa protein with a pH-dependent F-actin binding/depolymerizing
activity (1, 2) very similar to that of its structural
homolog, cofilin(3, 4) . Proteins in the ADF/cofilin
family appear to be ubiquitous in eukaryotes (for review, see (5) ). Mutations that inactivate the ADF/cofilin gene in Saccharomyces cerevisiae(6) , Drosophila
melanogaster(7) , and Caenorhabditis elegans(8) are lethal.
Purification of Proteins
pADF was isolated free
from ADF and cofilin from embryonic day 10 (E10) or E11 chick brain as
described previously(11) . It was further purified for
digestion and sequence analysis as described under
``Results.'' Chick brain ADF was purified according to the
method of Giuliano et al.(12) . Skeletal muscle actin
was purified from rabbit muscle acetone powder(13) .
Dephosphorylation of pADF
The in vitro reactivation of pADF by treatment with alkaline phosphatase
(Sigma) is described under ``Results.''
ADF Activity Assay
The amount of G-actin
depolymerized from F-actin in the presence of ADF was quantified by
using the DNase I inhibition assay(10, 14) .
Sample Preparation for Electrophoresis
Cells in
10-cm culture dishes were scraped in 300 µl of ice-cold extraction
buffer (10 mM Tris, pH 7.6, 1% SDS, 15 mM NaF, 10
mM dithiothreitol (DTT), 2 mM EGTA, 0.3 mM sodium orthovanadate, 10 µl/ml protease inhibition
mixture)(11) , sonicated, and immersed 5 min in a boiling water
bath. After cooling the samples on ice, proteins were precipitated with
chloroform/methanol(15) . The pellets were dissolved in sample
preparation buffer (0.125 M Tris, pH 6.8, 1% SDS, 5% glycerol,
10% 2-mercaptoethanol, 0.01% bromphenol blue) for SDS-polyacrylamide
gel electrophoresis (PAGE) or in lysis buffer (9.5 M urea, 2%
Nonidet P-40, 2% ampholytes, pH 3-10, 10% 2-mercaptoethanol) for
two-dimensional gels (16) . Protein concentrations were
determined by the solid phase dye-binding method(17) .
Gel Electrophoresis and Immunoblotting
SDS-PAGE
was performed by the method of Laemmli (18) on 15% acrylamide
(2.7% cross-linker) isocratic mini-slab gels. Two-dimensional,
nonequilibrium pH gradient electrophoresis (19) on mini-gels,
immunoblotting onto polyvinylidene difluoride membrane (Immobilon P;
Millipore Corp., Bedford, MA) for 1 h at 0.3 A, and immunostaining for
ADF were performed as described previously(10) . Alkaline
phosphatase-conjugated secondary antibody (Amersham Corp.) was used,
and blots were developed after a quick rinse in high pH buffer (100
mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl), first with Lumiphos substrate (Boehringer
Mannheim, Indianapolis, IN) and then in 0.165 mg/ml of
5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and 0.33
mg/ml of nitro blue tetrazolium chloride (Life Technologies, Inc.)
diluted in the pH 9.5 buffer. Hyperfilm ECL (Amersham Corp.) exposures
of the Lumiphos images and the stained blots were scanned using a
Microscan 2000 image analysis system (Technology Resources, Inc.,
Knoxville, TN). Internal standards of chick brain ADF were included on
all one-dimensional immunoblots.
Metabolic Labeling of Chick Skin Fibroblasts
Chick
skin fibroblasts, prepared by trypsin dissociation of skin from the
dorsal side of 8-day embryos, were grown in 10-cm culture dishes in
Dulbecco's modified Eagle's medium (Sigma) containing 10%
fetal bovine serum. When 70% confluent, cells were transferred to a low
phosphate medium (Dulbecco's modified Eagle's medium
without phosphate) (Sigma) containing 10% fetal bovine serum that had
been exhaustively dialyzed against 0.15 M NaCl.
[P]Orthophosphate (5 Ci/mmol; 100 µCi/ml
culture medium) was added for 12 h. The cells were washed with 3
10 ml of 4 °C phosphate-buffered saline, and then harvested
in extraction buffer as described above. The cell lysate was added to a
homogenate prepared from 6 g of 10-11-day chick embryo brain in
10 ml of the same buffer, and the mixture was further homogenized with
five to six more strokes of a Teflon-glass homogenizer. The
P-labeled pool of pADF was then purified as described
under ``Results.''
Protease Digests
For endopeptidase digestion,
approximately 60 µg of precipitated ADF or pADF were solubilized in
20 µl of 8 M urea, 20 mM Tris-Cl buffer, pH 9.0.
The volume was adjusted to 100 µl with 20 mM Tris-Cl, pH
9.0. Endopeptidase Lys-C (Wako Bioproducts, Richmond, VA) was added at
an enzyme/substrate molar ratio of 1:50, and the mixture was incubated
at 30 °C for 8 h. Aliquots were removed periodically to evaluate
the progress of digestion by SDS-PAGE. The digested samples were either
immediately fractionated or frozen in liquid nitrogen and stored at
-80 °C for later use. For endopeptidase Asp-N digestion of
the N-terminal fragment from Lys-C digestion, the appropriate fractions
were lyophilized and resolubilized in 10 µl of 8 M urea,
50 mM sodium phosphate, pH 8.0. The volume was adjusted to 60
µl with 50 mM sodium phosphate, pH 8.0. Endopeptidase
Asp-N (Boehringer Mannheim) was added at an enzyme/substrate weight
ratio of 1:20. The mixture was incubated for 8 h at 36 °C and then
lyophilized.
HPLC Separation of Peptides
Peptides from Lys-C
digests of ADF or [P]pADF were fractionated by
HPLC on a column of Microsorb-MV C18, 5 µm, 300-Å pore (5.6
mm
25 cm) (Rainin Inst., Woburn, MA) eluted with a linear step
gradient (2.0-37.5% B, 60 min; 37.5-75% B, 30 min;
75-90% B, 15 min; solvent A, 0.1% trifluoroacetic acid; solvent B
80% acetonitrile, 0.1% TFA) (Millipore, Waters Chromatography, Milford,
MA). The flow rate was 0.5 ml/min, and the column effluent was
monitored at 220 nm. Fractions (0.25 ml) were collected every 30 s, and
Cerenkov radiation was measured.
Mass Spectrometry
HPLC electrospray mass
spectrometry and tandem mass spectrometry were performed at the Beckman
Institute, City of Hope, Duarte, CA on a TSQ-700 triple quadrupole mass
spectrometer (Finnigan-MAT, San Jose, California), equipped with an
electrospray ion source operating at atmospheric pressure. Mass spectra
were recorded in the positive ion mode. The solvent delivery system,
fused silica capillary columns, and UV detection system have been
described in detail(20) .
Plasmids and Site-directed Mutagenesis
A 656-base
pair EcoRI/AvrII fragment containing the full ADF
cDNA was excised from pUC 19 (21) and cloned into the EcoRI-XbaI sites of M13mp18. Oligonucleotide-directed
point mutagenesis (22) was used to mutate the Ser and Thr
codons to Ala using the mutagenic primers
5`-CCTCAGGCGCTGCGCATTTCCG-3`, 5`-CCTCAGGCGTTGCGCCATTT-3`,
5`-CCTCAGGCGCAGAGCATTTCCG-3` giving the ADF(A24,25), ADF(A24), and
ADF(A25) mutant cDNAs, respectively. NcoI/HindIII
fragments encoding ADF(A24,25), ADF(A24), or ADF(A25) were subcloned
into the modified pBR 322 bacterial expression vector (pET) as
described previously(21, 23) . BamHI/AvrII fragments (560 base pairs) from pET
containing the full ADF or ADF(A24,25) cDNAs were cloned into the
multi-cloning site of the eukaryotic expression vector pcDNA3
(Invitrogen, San Diego, CA), giving the vectors pcDNA3ADF and
pcDNA3ADF(A24,25). Site-directed mutagenesis was performed on pcDNA3ADF
using the Transformer Mutagenesis Kit (Clontech, Palo Alto,
CA)(24) . Three mutagenic primers were used, encoding mutations
in the Ser
codon of the ADF cDNA (Ser
Ala,
5`-CTTGTACTCCAGCTGCCATGGATC-3`; Ser
Glu,
5`-CGGCAACTTGTACTCCTTCTGCCATGGATCCGAGC-3`; Ser deletion,
5`-CGGCAACTTGTACTCCTGCCATGGATCCGAGC-3`). A selection primer,
eliminating a unique pcDNA3 HindIII restriction site, was also
synthesized (5`-GGTACCAAGCAAGGGTCTCCC-3`). All mutant cDNAs were
confirmed by DNA sequencing (Sequenase Version 2.0 DNA Sequencing Kit,
U. S. Biochemical Corp., Cleveland, OH). All oligonucleotides were
synthesized by Macromolecular Resources, Colorado State University,
Fort Collins, CO.
Bacterial Expression and Isolation of Recombinant
Proteins
Bacterial expression of the ADF(A24,25) and ADF(E3)
mutants was performed as described previously for the expression of ADF (21) . The postinduction bacterial pellets were resuspended in
50 mM Tris, pH 8.0, 2.0 mM EGTA, 2.4 mM phenylmethylsulfonyl fluoride, 20 µl/ml protease inhibition
mixture(11) , and then lysed in 2.0 mg/ml lysozyme on ice for
15 min, followed by addition of 15 mM MgCl, 0.2
mM MnCl
, and 8 µg/ml DNase I. After incubation
at room temperature for 10 min, 20 mM DTT and 10 µl/ml
protease inhibition mixture were added. After centrifugation at 60,000
g
for 30 min at 4 °C, the supernatant
was passed through a column of DEAE-cellulose (DE-52; Whatman), and the
flow-through was applied to a column of Green A-agarose (Amicon Corp.,
Danvers, MA). The ADF was eluted with 175 mM NaCl in 10 mM Tris, pH 7.6, 5 mM DTT, concentrated on a Centricon-10
(Amicon Corp.), frozen in liquid nitrogen, and stored at -80
°C.
Cell Transfections
HeLa cells were cultured in
minimal essential medium (Sigma) containing 10% fetal bovine serum, 50
units/ml penicillin, and 50 µg/ml streptomycin. Cells at
30-50% confluency on 10-cm dishes were transfected by the calcium
phosphate method (25) with 10 µg of pcDNA3 vector
containing ADF cDNA or one of the site-directed mutants of ADF cDNA.
The precipitate was removed after 12 h by washing 3 times with
phosphate-buffered saline, and extracts of cells were prepared for
SDS-PAGE 48 h after transfection as described above.
pADF Is Reactivated by Alkaline Phosphatase
We
previously demonstrated that the phosphorylated form of ADF, partially
purified from chick embryo brain, had no actin depolymerizing
activity(11) . Partially purified preparations of pADF contain
proteases that degrade ADF when incubated at 37 °C(11) .
While the SDS-denatured pADF could be dephosphorylated by alkaline
phosphatase, we did not demonstrate reactivation. We found that brain
ADF, denatured by boiling in 1% SDS, could be completely reactivated
after removal of SDS and renaturation. We used these conditions to
renature ADF produced from denatured pADF following alkaline
phosphatase treatment. Complete ADF activity was regained after
dephosphorylation, whereas no ADF activity was found in the pADF
control (Fig. 1). Electrophoresis of both samples confirmed that
dephosphorylation occurred in the alkaline phosphatase-treated sample,
as previously reported(11) .
) in the
presence of a 5-fold excess of Triton X-100 to bind excess SDS. After
60 min at 37 °C, the protein was reprecipitated, solubilized in 8 M urea, and dialyzed against 10 mM Tris, pH 7.5
containing 1 mM DTT. A control sample of pADF (
) was
treated identically, except no alkaline phosphatase was added. Activity
was assayed by the DNase I inhibition assay(10, 14) .
Actual ADF content of the partially purified sample was determined by
quantitative immunoblotting. The theoretical curve for 100% activity (dashedline) and points obtained with renatured
recombinant ADF (
) are shown for
comparison.
Purification of pADF
The combined homogenate from
the P-labeled chick skin fibroblasts and chick embryo
brain was centrifuged at 100,000
g. pADF was partially
purified from the supernatant by chromatography on DEAE-cellulose,
Green A-agarose, and hydroxylapatite (HAP). SDS-PAGE of HAP fractions (Fig. 2A) and the corresponding autoradiograph (Fig. 2B) demonstrate that
[
P]pADF eluted in the position expected for
pADF, as previously identified by immunoblotting(11) . The
pADF-containing fractions were boiled in 0.1% SDS, combined, and
concentrated to less than 100 µl in a Centricon-10
microconcentrator. The volume was adjusted to 1 ml with 0.1 M Tris-Cl, pH 7.6, and the protein was reduced, and alkylated with
4-vinylpyridine(26) . Proteins were precipitated (15) and redissolved in sample preparation buffer for
separation on a preparative SDS-PAGE mini-slab gel (0.5 mm). After
electrophoresis, proteins were visualized by KCl
precipitation(27) , and the pADF band was excised from the gel.
pADF was electroeluted into a Centricon-10 chamber in 0.5
SDS-PAGE running buffer (18) and concentrated to about 0.1 ml
by centrifugation. An aliquot of this material was rerun on SDS-PAGE to
check for homogeneity (Fig. 2C).
P]orthophosphate was incorporated into the
pADF. C, Coomassie Blue-stained gel of the pooled HAP
fractions (4, 5, 6) before (lane1) and after (lane2) reduction and
alkylation. The pADF has a slightly slower mobility than the ADF
standard (compare lanesS and 1), which is
amplified following alkylation with 4-vinylpyridine (lane2). The purified electroeluted pADF is shown in lane3.
pADF Contains Only a Single
Phosphopeptide
Electroeluted ADF and pADF were
precipitated(15) , dissolved in buffer, and digested with
endopeptidase Lys-C. The resulting peptides were separated by HPLC and
Cerenkov radioactivity in each fraction determined (Fig. 3). A
single phosphopeptide, unique to the pADF digest, was identified (Fig. 3, arrow). This single peptide contained about
60% of the total radioactivity loaded onto the column, the remainder
being accounted for in the unbound fraction and a few small peaks
visible in Fig. 3. No sequence could be obtained by direct
sequence analysis of the single phosphopeptide, whereas neighboring
peptides from the same digest were readily sequenced. This suggested
that the labeled peptide contained a blocked N terminus and might arise
from the N terminus of ADF, a peptide containing only a single Ser
residue.
P-labeled phosphorylated peptide is shown by the arrowhead with the CPM in each fraction shown
below.
Identification of the Phosphorylation Site by Mass
Spectrometry
The N-terminal Lys-C peptide from pADF contains
only a single Ser and has the sequence Ac-ABGVQVADEVJRIFYDMK,
where B is phosphoserine, and J is pyridylethylcysteine. This sequence
suggests that the N-terminal Met is removed and that the penultimate
Ala is acetylated. This sequence was confirmed by mass spectrometric
analysis as follows. The isolated Lys-C phosphopeptide (containing some
of the contaminating trailing peak) was digested with Asp-N and rerun
on HPLC. The undigested contaminating peptide and two major peptide
fragments of the phosphopeptide were obtained (Fig. 4). The
parent ion masses of the two major Asp-N peptides are 753.6 and 1150.3,
as determined by electrospray mass spectrometric analysis. These
correspond to the masses expected for peptides derived from the
sequence shown above: A, Ac-ABGVQVA (mass 753); B,
DEVJRIFY (mass 1150). In addition, the C-terminal peptide (C)
DMK was identified by its mass of 393. Analysis of the N-terminal
peptide by tandem mass spectrometry produced the spectrum in Fig. 5A. The parent ion (P) at m/z 753 matches the value predicted for that of the N-terminal
peptide. The predicted fragment ions, type b and y are shown in Fig. 5C, and those ions observed are labeled in Fig. 5A. Fragment ions b through b
yield the C-terminal sequence -VQVA, confirming the origin of the
peptide. The b
ion m/z corresponds to the
N-terminal fragment Ac-ABG-. Also apparent in the spectrum are ion
species representing the loss of phosphate as phosphoric acid (98 mass
units) due to collision. The N-terminal peptide (A) in Fig. 4was isolated by HPLC and treated with alkaline
phosphatase. Analysis of the resulting peptide by tandem mass
spectrometry yielded the spectrum in Fig. 5B. The
parent ion at m/z 673 is that of the dephosphorylated peptide
(-80 mass units). Fragment ions b
through b
yield an identical C-terminal sequence but are decreased by 80
mass units due to the loss of phosphate. Additionally, there are no
peaks representing the loss of phosphoric acid due to collision. The
b
fragment (AcASG-) contains only one phosphorylatable
residue, Ser, and therefore, the phosphorylation site on pADF is
Ser
(Ser
in the encoded protein).
Confirmation of Ser
Extracts from
HeLa cells, transiently transfected with pcDNA3 carrying wild-type ADF,
ADF(A24,25), ADF(A3), ADF(E3), or ADF(as the in Vivo
Regulatory Site by Site-directed Mutagenesis
3) expressed the transfected
forms of ADF as shown on two-dimensional immunoblots (Fig. 6).
Endogenous HeLa cell ADF only weakly cross-reacts with the chick ADF
antiserum (Fig. 6, 1a), eliminating the need to epitope
tag the exogenously expressed protein. Two-dimensional immunoblots of
extracts from cells expressing wild-type ADF (Fig. 6, 2a) or the ADF(A24,25) mutant (Fig. 6, 1b)
typically show two species, the more acidic one arising from the in
vivo phosphorylation of ADF. Extracts of cells transfected with
ADF(A3) (Fig. 6, 2b), ADF(E3) (Fig. 6, 3a), or ADF(
3) (Fig. 6, 3b) do not
contain the phosphorylated forms of these proteins, confirming that
Ser
of the encoded protein is the regulatory site for
phosphorylation in vivo.
3) mutant.
Bacterial Expression and Characterization of Mutants of
ADF
Bacterially expressed forms of wild-type ADF, ADF(A24),
ADF(A25), and ADF(E3) were purified from supernatants of bacterial
lysates. Proteins were homogeneous on SDS-PAGE (not shown). Wild-type
ADF, ADF(A24) and ADF(A25) were identical to brain ADF in
depolymerizing F-actin. The ADF(E3) mutant, a structural homolog of the
phosphorylated ADF, had F-actin depolymerizing activity that was about
10% of the wild-type (Fig. 7).
S
or by free calcium but not by activation of protein kinase
C(38) . Again, the GTP
S effects could very well be
mediated through increases in cAMP via the protein G
.
Together, this evidence suggests that ADF is a major regulator of actin
filament dynamics in vivo and that the regulation of ADF by
phosphorylation may provide a rapid, localized, and reversible control
mechanism for regulating actin filament dynamics in areas of high
filament turnover.
in both ADF and cofilin exists
within a conserved CaM kinase II consensus sequence. This seemed a
likely site for phosphorylation as it is situated next to a nuclear
translocation sequence, conserved between ADF and cofilin, both of
which have been shown to translocate to the nucleus. It was suggested
that the dephosphorylation of cofilin at this site may be involved in
its nuclear translocation(9) . However, ADF was not
phosphorylated in vitro to any significant extent with several
common kinases including CaM kinase II, protein kinase C, protein
kinase A, and myosin light chain kinase(11) . The studies
reported here in which the ADF(A24,25) mutant is expressed and
phosphorylated in HeLa cells demonstrates convincingly that Ser
is not the regulatory site.
of the encoded protein. This is a conserved
residue within the ADF/cofilin family, present in mammals, birds,
amphibians, and insects (Fig. 8). Echinoderms (40) and
yeast (6) have an extra amino acid between this Ser and the
initiating Met, while plants (41) have four extra amino acids,
two of which are Ser. In the amoeba protein, actophorin(42) ,
this Ser is adjacent to the initiating Met. In mammals, birds, and
amphibians, the only classes so far in which phosphorylation of
ADF/cofilin proteins have been demonstrated, a consensus sequence for
the phosphorylation site appears to be
N-ASGVXVXD. Such a phosphorylation consensus
sequence has not been reported(43, 44) . Although
autophosphorylation of the N-terminal penultimate Ser residue in
c-mos has been characterized, this Ser is surrounded by two
Pro residues, which are likely to be involved in determining the
phosphorylation specificity of this meiotic cell cycle regulatory
protein(30) .
.
-, gap in sequence for alignment; :, identical amino acid with
the human ADF. Reference numbers refer to references at end of
manuscript. XenopusADF was as described in Footnote
3.
The small GTP-binding protein Rac1 is a key
regulator of the rapid actin reorganization accompanying membrane
ruffling during the early phases of growth factor-induced signal
transduction (for review, see (45) ). ADF is co-localized with
actin in regions where Rac1 induces dramatic alterations in actin
dynamics. Overexpression of a constitutively active mutant of Rac1 in Drosophila neurons inhibits nerve growth and increases the
rhodamine-phalloidin staining of growth cones(46) . This
finding suggests that growth inhibition may arise from a decrease in
F-actin turnover. Conversely, overexpression of a dominant negative
form of Rac1 also led to inhibition of nerve growth, but in this case
growth cones did not stain with rhodamine phalloidin, suggesting that
they contained little or no F-actin(46) . One downstream
effector of Rac1 is the serine/threonine kinase,
p65(47) , which we believe could be the kinase
that is directly or indirectly responsible for the phosphorylation of
ADF. Interestingly, Drosophila ADF has the phosphorylation
consensus sequence found in ADF/cofilin from mammals, birds, and
amphibians (Fig. 8). These results are at least consistent with
a model in which ADF plays a prominent role in growth cone actin
dynamics.
(
)Introduction of a phosphate group nearby
an actin binding domain could directly affect the interactions, but
until the structure of the entire molecule is resolved, we can only
speculate on how the phosphorylation of the penultimate serine brings
about the changes in the actin binding properties of ADF. It is
interesting to note, however, that extensions to the N terminus (three
amino acids in recombinant ADF (21) or the addition of a 26-kDa
glutathione S-transferase fusion protein)
(
)do not inhibit the actin depolymerizing or
pH-sensitive F-actin binding of ADF.
S, guanosine
5`-3-O-(thio)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.