From the Institute of Clinical Biochemistry and
Pathobiochemistry, University of Würzburg,
Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany and the
¶ Medical Proteome Center, Ruhr University of Bochum,
Universitätsstrasse 150, D-44780 Bochum, Germany
Received for publication, September 4, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Various drugs that elevate cGMP levels and
activate cGMP-dependent protein kinase (cGK) inhibit
agonist-induced platelet activation. In the present study we identified
the LIM and SH3 domain protein (LASP) that was recently cloned from
human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C.,
Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK
in human platelets. Recombinant human LASP was phosphorylated by
cGMP- and cAMP-dependent protein kinase (cAK) in
vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed
phosphorylation of LASP in vivo. Studies with human LASP
mutants identified serine 146 as a specific phosphorylation site for
cGK and cAK in vivo. LASP is an actin-binding protein, and
the phospho-LASP-mimicking mutant S146D showed reduced binding affinity
for F-actin in cosedimentation experiments. Immunofluorescence of
transfected PtK2 cells demonstrated the localization of LASP in the
tips of cell membrane extensions and at cell-cell contacts. Expression
of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the
cells. Taken together, these data suggest that phosphorylation
of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.
The activation process of human platelets and vessel wall-platelet
interactions is tightly regulated under physiological conditions and is
often impaired in thrombosis, arteriosclerosis, hypertension, and
diabetes. Platelet activation can be inhibited by a variety of agents,
including aspirin and Ca2+ antagonists as well as cGMP- and
cAMP-elevating agents such as NO and prostaglandin I2 (for
review see Ref. 1). The inhibitory effects of cGMP and cAMP are
mediated by cAMP-dependent protein kinases types I- and
II The molecular mechanisms of platelet inhibition by cGMP signaling
downstream of cGK activation are only partially understood. In
cGK-deficient mice cGMP-mediated inhibition of platelet aggregation is
impaired (4). To date, only a few substrates for cAK and cGK have been
identified and characterized in human platelets. The 22-kDa small
GTP-binding protein rap 1b is phosphorylated by cAK and cGK in intact
platelets (Ref. 5 and this study). Phosphorylation of rap 1b is
associated with translocation of the protein from the membrane to the
cytosol (6). The vasodilator-stimulated phosphoprotein VASP is another
major substrate of cAK and cGK in human platelets (7). Its three
phosphorylation sites are phosphorylated with different specificities
by these two kinases (8). VASP phosphorylation is thought to be
involved in the negative regulation of integrin
Several other proteins have been reported to be phosphorylated in
response to cGK activation either in vitro or in intact cells, including cGMP-specific phosphodiesterase (2), myosin light
chain kinase (17), an inositol 1,4,5-trisphosphate (IP3) receptor-associated cGMP kinase substrate (18),
Na+/K+-ATPase (19), cysteine-rich protein 2 (20), MEKK1 (21), and endothelial NO synthase (22). None of these
proteins, however, has been established as a downstream target of cGK
in platelets.
Here we report the identification of a specific substrate for cAK and
cGK in intact human platelets using differential phosphoproteomic display of radiolabeled human platelets. The protein was identified as
the LIM and SH3 domain protein (LASP) (27) by nano-liquid chromatography-electrospray ionization mass spectrometry.
Phosphorylation of LASP at Ser-146 leads to a redistribution of the
actin-bound protein from the tips of the cell membrane to the cytosol,
accompanied with a reduced cell migration.
Materials--
Urea (ultrapure), [
cGK I Isolation of Platelets--
Freshly donated blood from healthy
volunteers (50 ml) was collected in acid-citrate dextrose and
centrifuged for 10 min at 300 × g to yield
platelet-rich plasma (PRP). PRP was centrifuged for 20 min at 500 × g, and the platelet pellet was resuspended and washed
once in an isotonic buffer containing 10 mM Hepes (pH 7.4),
137 mM NaCl, 2.7 mM KCl, 5.5 mM
glucose and 1 mM EDTA at a density of 1 × 109 cells/ml. After resuspension, platelets were allowed to
rest at 37 °C for 15 min.
32P-Labeling of Platelets Two-dimensional Gel Electrophoresis--
Isoelectric focusing
for two-dimensional gel electrophoresis was performed using the Protean
IEF cell from Bio-Rad according to the instructions of the
manufacturer. The platelet pellet (about 200 µg of protein) was
solubilized for 15 min by sonication in 320 µl of lysis buffer
containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 15 mM dithiothreitol (electrophoresis grade), 0.5%
carrier ampholytes, pH 3-10. Pellet homogenate was loaded on a 17-cm
immobilized IPG strip, pH 3-10, and reswollen overnight at 50 V. Focusing was carried out for 1 h at 250 V, 1 h at 500 V,
and 15 h at 7000 V.
After equilibration in 50 mM Tris, pH 8.9, 6 M
urea, 30% (w/v) glycerol, 2% (w/v) SDS, gels were immediately applied
to a vertical 10% SDS gel without a stacking gel. Electrophoresis was carried out at 8 °C with a constant current of 40 mA per gel. The
gels of radioactively labeled platelet proteins were fixed in 30%
ethanol, 10% acetic acid and exposed. Radioactive spots visualized by
autoradiography were excised.
Mass Spectrometry--
Gel pieces were washed sequentially for
10 min in tryptic digestion buffer (10 mM
NH4HCO3) and digestion buffer: acetonitrile (1:1). These steps were repeated three times and led to a shrinking of
the gel. It was reswollen with 2 µl of protease solution (trypsin at
0.05 µg/µl) in digestion buffer and incubated overnight at 37 °C.
Analysis of the resulting peptides was carried out using a nano-HPLC
system coupled directly to an electrospray ionization-ion trap mass
spectrometer equipped with a custom-built nano-electrospray ion source
(LCQTM Classic, Thermo Finnigan, San Jose, CA). Fifteen µl of
5% (v/v) formic acid were added to the gel pieces and the peptides
extracted by sonication for 15 min. The extraction step was repeated
once. The supernatants were transferred to glass tubes, and the
peptides were automatically flushed and preconcentrated on a
µC18-precolumn (Nano-PrecolumnTM, 0.3-mm inside diameter × 1 mm, C18 PepMapTM, LC Packings Dionex, Idstein, Germany) for 10 min
with 0.1% (v/v) trifluoroacetic acid and a flow of 40 µl/min. Tryptic peptides were injected automatically on the
reversed-phase C18-column (75 µm inside diameter × 250 mm, C18 PepMapTM, 5-µm particle size, LC Packings Dionex) using the
SwitchosTM system (LC Packings Dionex). Separation of the peptides was
carried out in a gradient consisting of 0.5% (v/v) formic acid
(solvent A) and 0.5% formic acid/84% acetonitrile (solvent B) with
5-15% B in 10 min, 15-20% B in 10 min, 20-50% B in 70 min, and
50-100% B in 5 min. The flow rate was adjusted from 200 µl/min to
160 nl/min using a precolumn split. Eluting peptides were transferred online to a heated capillary (Pico TipTM, FS360-20-10, New Objective Inc., Cambridge, MA) of an ion trap mass spectrometer (LCQTM
Classic, Thermo Finnigan). The following electrospray ionization
parameters were used: spray voltage, 1.8 kV-2.15 kV; capillary
temperature, 200 °C; capillary voltage, 42 V; tube lens offset
voltage, 30 V; and the electron multiplier at Molecular Cloning of LASP and LASP Mutants--
Gene-specific
primers used for PCR amplification of LASP and LASP mutants were
designed based on the published human cDNA sequence
(GenBankTM Access. No. X82456). Oligonucleotides used to
generate wild-type LASP:
AATGGATCCATGAACCCCAACTGCGCCCGGTGCGGCAAG (sense, starting at
position 76) and CGGGAATTCTCAGATGGCCTCCACGTAGTTGGCCGGCA
(antisense, starting at position 862) with BamHI and
EcoRI restriction sites (underlined). Full-length human LASP
cDNA was cloned into the BamHI/EcoR1sites of pGEX4T1 to
generate a glutathione S-transferase (GST)-LASP fusion
protein. The single and double mutants were made by performing a second
round of PCR using the wild type and single mutant as a template,
respectively, and the appropriate pairs of oligonucleotides. For
eukaryotic expression, LASP was cloned into pcDNA3. All constructs
were confirmed by DNA sequence analysis.
Expression of GST·LASP Fusion Proteins--
Recombinant LASP
and LASP mutants S61D, S146D, and S61/146D were expressed in
Escherichia coli as GST fusion proteins using pGEX-4T1. Expression and purification of the GST fusion proteins were
performed according to the manufacturer's protocol. Removal of GST
from LASP was achieved by digestion with thrombin overnight at 4 °C.
Purity was analyzed by examination of Coomassie-stained SDS-polyacrylamide gels.
LASP Polyclonal Antibody Generation--
To generate a
polyclonal antibody specific for LASP, recombinant human GST·LASP
that had been expressed and affinity-purified from bacteria was
injected into New Zealand rabbits (Immunoglobe, Himmelstadt, Germany).
Immunoreactive serum was affinity-purified against LASP protein coupled
to a HiTrap-NHS-activated affinity column according to the
manufacturer's instructions.
Western Blot Analysis of LASP--
Cell extracts were resolved
by 10% SDS-PAGE. After blotting on nitrocellulose membrane and
blocking with 3% nonfat dry milk in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% (w/v) Tween 20, the membrane was first
incubated with the polyclonal antibody raised against LASP (1:16,000)
followed by incubation with horseradish peroxidase-coupled goat
anti-rabbit IgG (1:5000) and detection by ECL.
In Vitro Phosphorylation of LASP--
LASP and its mutants S61A,
S146A, and S61/146A (0.5 µM each) were incubated at
30 °C in a total volume of 20 µl with 10 mM Hepes, pH
7.4, 5 mM MgCl2, 1 mM EDTA, 0.2 mM dithiothreitol, and the C subunit of cAK or cGK I Eukaryotic Expression and in Vivo Phosphorylation--
PTK2
cells were grown in Dulbecco's modified Eagle's medium in 6-well
plates to about 70% confluency and then transiently transfected with
one of the LASP constructs cloned into a pcDNA3 vector alone or
together with pCMV-cGKI Immunofluorescence--
For immunofluorescence microscopy,
transfected PtK2 cells grown on glass chamber slides were fixed in 4%
(w/v) paraformaldehyde in phosphate-buffered saline, permeabilized with
0.1% (w/v) Triton X-100 in phosphate-buffered saline, and then stained
with affinity-purified LASP antibody (1:1000) followed by secondary
Cy3-labeled anti-rabbit antibody.
F-actin Cosedimentation Assay--
F-actin cosedimentation
assays were performed essentially as described by the actin
manufacturer (Cytoskeleton). Briefly, purified recombinant human LASP
(40 µg/ml) was incubated with 400 µg/ml freshly polymerized actin
(F-actin) for 30 min at room temperature. Following incubation, the
LASP/F-actin solution was subjected to centrifugation at 160,000 × g to pellet F-Actin and LASP bound to F-actin. After
solubilization of the pellet fraction in a volume equal to the initial
incubation volume, 20 µl of the pellet and supernatant fractions were
analyzed by SDS-PAGE.
Migration Experiments--
PtK2 cells were grown in Dulbecco's
modified Eagle's medium in 5-cm dishes to about 80% confluency and
then transiently transfected with pcDNA3 vector containing WT-LASP
or S146D-LASP using LipofectAMINE. After 24 h, 1 × 105 cells per 100 µl incubation medium (Dulbecco's
modified Eagle's medium with 1 mM MgCl2) were
seeded in the upper chamber of bovine serum albumin-coated transwells.
PtK2 cells were allowed to migrate through the porous membrane for
4 h at 37 °C. Cells remaining at the upper surface were
completely removed using a cotton carrier. The lower surfaces of the
membranes were then stained in a solution of 1% (w/v) crystal violet
in 2% ethanol for 30 s and rinsed afterward in distilled water.
Cell-associated crystal violet was extracted by incubation in 10%
acetic acid for 20 min and measured at 595 nm.
Phosphorylation of LASP in Intact Human Platelets Treated with the
cGK-specific Stimulus 8pCPT-cGMP--
To identify substrates of cGK in
intact human platelets, cells were labeled with
[32P]orthophosphate, stimulated with 500 µM
of the specific cGMP-dependent protein kinase activator
8pCPT-cGMP, and proteins of the resulting platelet lysate were
separated by two-dimensional gel electrophoresis. The two-dimensional
phosphoproteomes resulting from this experiment (Fig.
1) demonstrate the
phosphorylation/dephosphorylation of several proteins after stimulation
with 8pCPT-cGMP. For identification of these proteins, the spots were
excised from the gel, digested with trypsin, and the resulting peptides
were analyzed by electrospray ionization mass spectrometry/mass
spectrometry. Spot 2a-c was identified as VASP, a well known
substrate of cGK in human platelets (8) with multiple phosphorylation
isoforms (26). Spot 3 was characterized as rap 1b, a low molecular
weight GTP-binding protein. Phosphorylation of rap 1b has been observed
in nitric oxide-stimulated human platelets through stimulation of
guanylyl cyclase and activation of the cGMP-dependent
protein kinase (5). Spot 1 was identified as LASP, a novel type of
actin binding protein (27). Spots 4, 5, and 8 have not been identified
yet because of their low amounts of protein in the gel.
Recombinant LASP Is Phosphorylated by cGK and cAK in
Vitro--
The specificity of the phosphorylation observed in intact
platelets was further investigated by studying the role of LASP as a
substrate for cGK in vitro. For this purpose, purified
recombinant human LASP was incubated with cGK I Identification of LASP Phosphorylation Sites in Vitro--
Human
LASP contains two cAK consensus motifs for serine phosphorylation,
Ser-99 (KGFS) and Ser-146 (RRDS) (28). In the
present study, phosphorylation of amino acids was determined by mass
spectrometry. A complete trypsin digest of phosphorylated LASP was
fractionated on a nano-HPLC connected online to an ion trap mass
spectrometer. The resulting MS/MS spectrum of the phosphopeptide with
the sequence QpSFTMox-VADTPENLR is presented in Fig.
3. The b-ion series shows many signals
with a loss of In Vitro Phosphorylation of LASP and LASP Mutants--
To study
the specificity of phosphorylation, the identified serine
phosphorylation sites Ser-61 and -146 were mutated to alanine (S61A,
S146A, and S61/146A). The purified, recombinant proteins (wild-type,
single mutants, and double mutant) were incubated with the cGK isoforms
I In Vivo Phosphorylation of LASP by cGK I Expression of LASP in Various Cell Lines and Tissues--
The
expression of human LASP in different cell types was studied using a
rabbit polyclonal antibody raised against GST-tagged human LASP.
Western blot analyses of cell extracts from several human tissues and
from different cell lines showed LASP expression in human platelets,
brain, heart, kidney, lung, liver, fibroblasts, smooth muscle cells,
human umbilical vein endothelial cells (HUVEC), and various cell lines
(Fig. 6, A and B).
Phosphorylation of Ser-146 Reduces Binding of LASP to
F-actin--
In earlier studies by Schreiber et al.
(27) as well as in our experiments, the filamentous expression pattern
of LASP suggested that the protein is colocalized with F-actin. To test
whether this association might be affected through phosphorylation of LASP, we performed F-actin/LASP cosedimentation experiments with wild-type LASP and the phosphorylation-mimicking mutant S146D, because
in vitro phosphorylation by cAK and cGK would also
phosphorylate Ser-61. In the absence of F-actin, LASP was exclusively
located in the soluble fraction, whereas in the presence of F-actin,
about half of the LASP protein was found in the pellet (Fig.
7). However, using LASP S146D (mimicking
the phosphorylation by cAK and cGK in vivo), two thirds of
the protein remained in the supernatant (Fig. 7, left
panel). In control experiments, the actin binding protein
Phosphorylation-dependent Redistribution of LASP in
PtK2 Cells--
In view of these results, we investigated whether the
intracellular localization of LASP is directly affected by
phosphorylation. PtK2 cells, which express no detectable amount of
endogenous LASP (Fig. 6B), were transiently transfected with
expression vectors encoding either wild-type LASP, LASP mutant S146A,
or LASP mutant S146D. Forty-eight hours after transfection, cells were
prepared for immunofluorescence. Wild-type LASP and LASP S146A were
predominantly present in the tips of cell membrane extensions and at
cell-cell contacts where it colocalizes with F-actin (Fig.
8, A, B,
D, and E). However, double staining
analysis with the LASP antibody and Oregon green phalloidin for F-actin
staining revealed no colocalization with actin stress fibers (Fig. 8,
D and E). In contrast, the
phosphorylation-mimicking mutant LASP S146D was found predominantly in
the cytosol (Fig. 8C). The specificity of the staining was
controlled with preadsorbed LASP antibody, which showed no
immunofluorescence (data not shown).
Redistribution of LASP to the Cytosol Reduces Cell
Motility--
Because LASP is prominently present within focal
contacts and the leading edges of the cell membrane, we wondered
whether the protein might be involved in cell motility. Therefore we
tested migration of PtK2 cells transiently expressing wild-type LASP or
LASP mutant S146D in a modified Boyden chamber system. Cells were
seeded in the upper chamber of a transwell polycarbonate membrane, and
after 4 h cells that had migrated through the porous membrane were
counted. Cells transfected with LASP S146D, mimicking the
phosphorylation and exhibiting cytosolic localization (Fig. 8C), showed a 25% reduced motility compared with
untransfected cells or WT-LASP-expressing cells displaying membrane
localization (Fig. 9).
As an approach to identify novel substrates of
cGMP-dependent protein kinase, we analyzed cGK-mediated
protein phosphorylation in intact human platelets using two-dimensional
gel electrophoresis. In addition to the previously known substrates
VASP and rap 1b, we identified LASP as a novel substrate for cGK and
cAK. LASP consists of an N-terminal zinc-binding LIM domain, followed
by two actin binding sites and a Src homology region 3 at the
C-terminal end (30). The human LASP gene was previously cloned and
identified from a human breast cancer cDNA library (31). It
was mapped to human chromosome 17 q12-q21 and was shown to be
amplified and overexpressed in breast tumors (32).
Theoretical sequence analysis of human LASP revealed two potential
phosphorylation sites at Ser-99 (KGFSF) and Ser-146 (RRDSQ), with Ser-146 having an additional basic amino acid
on either side of the consensus sequence. These sites agree well with
the minimal motif for efficient cGK phosphorylation (RKXS/T) (33). The data presented here show that human LASP Ser-146 is directly phosphorylated by cAK and cGK in vivo. Using site-directed mutagenesis of all serine residues, we
excluded phosphorylation at Ser-99 and at Ser-61 in vivo,
although Ser-61 was phosphorylated in vitro. Interestingly,
serine in position 146 is only found in human and rabbit, whereas the
corresponding amino acid in mouse and rat is an alanine. In contrast,
Ser-99 and Ser-61 are conserved in all four species. Just recently,
Chew et al. (34) identified Ser-146 and Ser-99 as the major
in vitro and in vivo phosphorylation sites of
rabbit LASP by cAK. Phosphorylation of rabbit LASP at Ser-146 induced a
Mr band shift that is absent in human LASP
phosphorylated by cAK and cGK, indicating differences in the structure
of the two proteins. Further studies are underway to explore
phosphorylation of LASP in the different species and its possible
physiological role.
LASP is expressed in all human tissues tested, including platelets,
brain, heart, kidney, lung, liver, endothelial cells, smooth muscle
cells, and fibroblasts. Northern blot analysis of murine LASP revealed
a constant expression of the protein during embryogenesis from day 7.5 to day 18.5 with various levels in all adult tissues, which is
consistent with an essential role for LASP in basic cellular
function (32).
Immunofluorescence analysis of LASP subcellular distribution showed
that the protein colocalizes with F-actin at focal adhesion plaques and
membrane edges in mouse cardiac fibroblasts and rat mesangial
cells.2 These results
confirmed earlier observations by Schreiber et al.
(27), who found LASP at peripheral cell extensions in individual epithelial cancer cells. Experiments performed using PtK2 cells transfected with wild-type LASP also demonstrated that the protein is
colocalized with F-actin at membrane extensions, although not along
intracellular stress fibers. In PtK2 cells, however, the LASP mutant
S146D, which simulates phosphorylation at Ser-146, accumulates in the
cytoplasm when transiently expressed, suggesting that phosphorylation
of human LASP by cAK and cGK regulates the intracellular localization
of the protein.
Recently, it was shown that the cAK-dependent acid
secretory agonists histamine and forskolin induce a rapid sustained
rise in LASP phosphorylation in rabbit gastric parietal cells, and this
increase is closely correlated with the acid secretory response (28).
In parallel, LASP redistributes from a predominantly cortical location
to a region surrounding the intracellular canaliculus, which is the
site of active HCl secretion (35). Mutation of the two major cAK
phosphorylation sites in rabbit LASP, Ser-99 and Ser-146, to alanine
appears to block this recruitment (34).
The function of LASP in living cells seems to be complex and cell
type-specific. The localization of LASP to the part of active membrane
extension in addition to our observations of a reduced cell migration
after phosphorylation and relocalization to the cytosol indicates a
prominent role for LASP in cell movement, either by interacting
directly with actin and promoting actin polymerization or by acting as
a scaffolding molecule recruiting other motility proteins to the tips
of the cells involved in the organization of the cytoskeleton. In
gastric parietal cells, LASP was identified to bind to dynamin, a large
GTPase involved in vesicular fission and control of membrane
trafficking in the H+/K+-ATPase pathways at the
apical membrane (36).
Apart from LASP, cGK phosphorylates VASP (8), a protein that has been
implicated in the regulation of actin dynamics and associated processes
such as cell adhesion and motility by its ability to associate with
F-actin, profilin, zyxin, and vinculin (37). In platelets, VASP
phosphorylation seems to be involved in the negative regulation of the
integrin As a newly identified signaling protein within the cGMP and cAMP
pathway, the specific function of LASP is still under investigation. Future experiments will address the question of whether there are
platelet-specific binding partners for LASP and determine whether LASP
might be a phosphorylation-dependent molecular switch that
regulates the interaction of other proteins with F-actin-rich compartments, thereby modulating platelet function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and by cGMP-dependent protein kinase I
(cAK
I,1 cAK II
, and cGK I
,
respectively), representing the major forms of cyclic
nucleotide-dependent protein kinases in human platelets (2,
3).
IIbbIII (9). Experiments in vitro
revealed reduced F-actin binding and actin polymerization of
phosphorylated VASP (10). Two recent studies investigated RhoA-mediated
myosin light chain (MLC) activation and its contribution to platelet
aggregation and secretion, showing that cGK phosphorylates RhoA and
counteracts the phosphorylation of MLC through activation of MLC
phosphatase (11, 12). We previously identified heat shock protein 27 (Hsp27) as a substrate for cGK in intact platelets (13).
Phosphorylation of Hsp27 by cGK reduced the stimulatory effect of
mitogen-activated protein kinase-activated protein kinase 2-phosphorylated Hsp27 on actin polymerization. There is also evidence that at least part of the inhibitory response mediated by cGK
depends on phosphorylation of the thromboxane receptor (14, 15) and the
inositol 1,4,5-trisphosphate-receptor (16).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]ATP,
[32P]orthophosphate (HCl-free), Protein A-Sepharose
beads, thrombin,
isopropyl-1-thio-
-D-galactopyranoside, glutathione,
HiTrap-NHS-activated HP column, pGEX4T1, and the ECL detection kit were
purchased from Amersham Biosciences. Actin was obtained from
Cytoskeleton (Denver, CO), CalyculinA was from Calbiochem, trypsin was
from Promega, 8pCPT-cGMP was from BioLog (Bremen, Germany), IPG
strips, goat anti-rabbit IgG, and nonfat dry milk were from
Bio-Rad, FuGENE, the rapid ligation kit, and Complete
MiniTM were from Roche, and nitrocellulose membrane
was obtained from Schleicher und Schuell (Kassel, Germany). Primers
were ordered from MWG Biotech (Ebersberg, Germany), restriction enzymes
were from New England Biolabs, the first strand cDNA synthesis kit was from MBI Fermentas (St. Leon-Rot, Germany), LipofectAMINE, TA-vector, and pcDNA3 were from Invitrogen, xL1-blue competent cells were from Stratagene, Cy3 was from Dianova (Hamburg, Germany), Dulbecco's modified Eagle's medium was from Invitrogen, and Boyden chambers were from Corning Costar (Cambridge, MA). All other
chemicals, reagents, and solvents of the highest purity available were
purchased from Sigma.
and the catalytic subunit of cAK type II were purified from
bovine lung and bovine heart, respectively (23). cGK I
and cGK II
were expressed in and purified from the baculovirus-Sf9 cell
system (24).
Platelet preparation
was carried out essentially as described above. After washing, 1 ml of
platelets at a concentration of 1 × 109/ml was
incubated with 500 µCi of [32P]orthophosphate
(HCl-free) for 1.5 h at 37 °C. Platelets were then centrifuged
at 500 × g for 7 min and resuspended in 1 ml of
isotonic buffer. Aliquots of 100 µl (corresponding to 200 µg of
protein) were treated with 500 µM 8pCPT-cGMP for 30 min
at 37 °C. After stimulation, platelets were briefly centrifuged
(500 × g for 3 min) to yield a pellet.
950 V. The collision
energy was set automatically depending on the mass of the parent ion. Gain control was set to 107. The data were collected in the
centroid mode using one MS experiment (Full-MS) followed by three MS/MS
experiments of the three most intensive ions (intensity at least 3 × 105). "Dynamic exclusion" was used for data
acquisition with an exclusion duration of 5 min and an exclusion mass
width of ± 1.5 Da.
,
I
, or II (0.05 µM each) and 5 µM cGMP.
Reactions were started by the addition of 50 µM ATP
containing 0.5 µCi of [
-32P]ATP and terminated after
30 min or at the times indicated in the figures by the addition of 10 µl of Laemmli SDS stop solution. Proteins were separated by SDS- PAGE
on 10% gels. Incorporation of 32P was visualized by autoradiography.
(25) using FuGENE. After 48 h, cells
were washed once with phosphate-free Dulbecco's modified Eagle's
medium and labeled with [32P]orthophosphate for 1 h
at 37 °C. Then cells were exposed to buffer alone and either 10 µM forskolin or 50 µM 8pCPT-cGMP for 20 min. After washing with ice-cold phosphate-buffered saline, the cells
were scraped into RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton x-100, 0.1%
SDS, 10% sodium pyrophosphate (all w/v %), 10 mM EDTA, 10 mM NaF, 100 units/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM benzamidine, 10 nM
Calyculin A, and 0.5 mM phenylmethylsulfonyl difluoride). Immunoprecipitation was performed with 0.3 mg of proteins. Lysates were
incubated with 1 µl of antibody (1.6 µg) for 2 h at 4 °C, then 1.5 h with pre-equilibrated protein A-Sepharose. Bound immune complex was washed three times with ice-cold phosphate-buffered saline.
The pellets were resuspended in 30 µl of Laemmli SDS stop solution.
Proteins were separated by SDS-PAGE on 10% gels. Incorporation of
32P was visualized by autoradiography.
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phosphorylation of LASP in intact human
platelets. Human platelets were incubated in the presence of
[32P]orthophosphate and treated with buffer alone
(Control) or with 500 µM cGK activator
8pCPT-cGMP for 30 min (Stimulation). Platelet homogenate was
separated by two-dimensional gel electrophoresis, and an autoradiogram
was obtained. Spot 1, LASP; spots 2a,
-b, and -c, VASP; spot 3, rap 1b;
spots 4, 5, and 8 have not yet been
identified. The pH gradient is indicated at the top of the
gel. The gels shown are representative of three separate
experiments.
and with the
catalytic subunit of cAMP-dependent protein kinase in the
presence of [
-32P]ATP. As shown in Fig.
2, LASP is clearly a substrate of cGK I
and cAK, the two isoforms known to be present in human
platelets.
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Fig. 2.
In vitro phosphorylation of
LASP. Purified, recombinant human LASP (1 µM) was
phosphorylated by cGK I and cAK (0.05 µM each) in a
total volume of 20 µl for 30 min as described under "Experimental
Procedures." In a control experiment (Co) no kinase was
added to the mixture. Proteins were resolved by SDS-PAGE and the
phosphorylated LASP visualized by autoradiography. Similar results were
obtained in four separate experiments.
98 Da typical for phosphoserine- and
phosphothreonine-containing peptides (29), leading to the unequivocal
identification of Ser-61 as phosphorylation site for cGK. In addition,
the predicted Ser-146 was identified in a second phosphopeptide (data
not shown). However, no phosphorylation of Ser-99 was observed. The
same results were obtained with LASP phosphorylated by cAK.
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Fig. 3.
Phosphoamino acid analysis of human LASP
phosphorylated by cGK. MS/MS spectrum of the phosphorylated
peptide QpSFTMoxVADTPENLR. Nearly the complete b- and y-ion
series are visible. The typical generation of dehydroalanine from
phosphoserine resulting in the loss of 98 Da occurs from
y13, indicating unequivocally Ser-61 as the phosphorylated
amino acid. Similar spectra were obtained with LASP phosphorylated by
cAK.
, I
, and II and the catalytic subunit of
cAMP-dependent protein kinase in the presence of
[
-32P]ATP. Incorporation of phosphate was observed
after 30 min with each of the four kinases, albeit at different levels
(Fig. 4). cGK I
and cAK, the two
isoforms present in human platelets, caused the highest phosphate
incorporation. As expected, no phosphorylation was observed for the
double mutant S61/146A. In a control experiment, VASP, a well known
substrate for cAK and cGK (8), was equally phosphorylated by each of
the four kinases (Fig. 4).
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Fig. 4.
In vitro phosphorylation of LASP
by cGMP- and cAMP-dependent protein kinases. Purified,
recombinant human LASP or its mutants S61A, S146A, and S61/146A or VASP
(each 1 µM) were phosphorylated by either cGK types I ,
I
, and II or the C subunit of cAK (each 0.05 µM) in a
total volume of 20 µl for 30 min as described under "Experimental
Procedures." Proteins were resolved by SDS-PAGE and the
phosphorylated proteins visualized by autoradiography. The
autoradiogram shown is representative of three separate
experiments.
and cAK at
Ser-146--
To evaluate the role of LASP as an in vivo
substrate of cGK I
and cAK (the two isoforms present in human
platelets), LASP-deficient PtK-2 cells were transfected with wild-type
LASP or the mutants S61A and S146A and cGK I
simultaneously or with
either protein alone. The cells express cAK endogenously.
Phosphorylation of the proteins by cGK I
or cAK was analyzed after
stimulation of the cells with 8pCPT-cGMP or forskolin, respectively,
followed by immunoprecipitation. Wild-type LASP and the mutant S61A
showed identical in vivo phosphorylation (Fig.
5). In contrast, no phosphorylation was
observed with the S146A mutant, indicating that in vivo
Ser-146 is the only cGK I
phosphorylation site in LASP. In the
absence of cGK I
no phosphorylation was detected. Similar results
were obtained for cAK phosphorylation of LASP (data not shown).
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Fig. 5.
In vivo phosphorylation of LASP
expressed in PtK-2 cells. LASP-deficient PtK-2 cells were
transfected with wild-type LASP or the mutants S61A and S146A and cGK
I simultaneously or with either protein alone. Cells were stimulated
20 min with 50 µM 8pCPT-cGMP for cGK activation or 20 min
with 5 µM forskolin for cAK activation; this was followed
by immunoprecipitation of LASP. Immunoprecipitates were analyzed by
SDS-Page (total LASP) and then subjected to autoradiography
to reveal [32P] incorporation (upper panels).
The autoradiograms shown are representative of two separate
experiments.
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Fig. 6.
Expression of LASP in human tissues and cell
lines. A rabbit polyclonal LASP antibody was used to detect LASP
protein by Western blot analysis of crude cell lysates from various
human tissues and different cell lines. A, human platelets,
brain, heart, kidney, lung, and liver. B, human embryonic
kidney (HEK 293), human umbilical vein endothelial cells
(HUVEC), PtK-2 cells, human fibroblasts, COS-7 cells, and
human smooth muscle cells (SMC).
-actinin (positive control) cosedimented almost completely with
F-actin, whereas >95% of bovine serum albumin (negative control) remained in the soluble fraction (Fig. 7, right panel).
These results suggested that upon phosphorylation of Ser-146, LASP
loses its ability to bind to F-actin.
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Fig. 7.
Binding of LASP to F-actin.
Cosedimentation analysis of 2 µM pre-polymerized actin
with 1 µM wild-type LASP (WT) or the
phosphorylation-mimicking mutant S146D. Densitometric analysis of two
separate experiments determined the amount of LASP remaining in the
supernatant (S) versus the amount of LASP in the
pellet (P). The positive control with actinin and the
negative control with bovine serum albumin (BSA) are shown
in the right panel.
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Fig. 8.
Subcellular localization of LASP.
Immunofluorescence microscopy of the subcellular distribution of
wild-type LASP and the mutants S146A and S146D in PtK-2 cells. Cells
were fixed with paraformaldehyde and permeabilized, and LASP was
immunostained with the LASP polyclonal antibody and Cy3-labeled goat
anti-rabbit secondary antibody. Actin was stained with oregon green
phalloidin. Wild-type LASP (A, D) and LASP S146A
(B) are predominantly present at the leading edges and focal
contacts of cells (indicated by arrows), whereas the LASP
mutant S146D is mainly localized in the cytosol (C). The
colocalization of LASP and F-actin is demonstrated by double-staining
in D and E (arrows), whereas no LASP
binding to actin stress fibers (E, arrowheads) is
observed.
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Fig. 9.
Migration. Relative migration of
uninfected PtK2 cells (Co) and PtK2 cells expressing
wild-type LASP (WT) or mutant LASP S146D (S146D).
Cell migration was measured over 4 h in a Transwell® cell
culture chamber. At least four chambers from two separate experiments
were analyzed (* value significant different from that of Co and WT by
t test; p < 0.001). Western blot of
untransfected control cells and LASP-expressing cells probed with
anti-LASP polyclonal antibody are shown in the lower
panel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIbbIII (9). Actually, in
vitro phosphorylation of VASP reduces F-actin binding (10); however, in contrast to LASP, phosphorylation of VASP plays no obvious
role in subcellular targeting (38).
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ACKNOWLEDGEMENTS |
---|
We thank U. Walter and M. Zimmer for helpful suggestions and comments.
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FOOTNOTES |
---|
* This work was supported by Grants Bu 740 and ME 765 from the Deutsche Forschungsgemeinschaft (DFG).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Institute of Clinical Biochemistry and Pathobiochemistry, Medical University Clinic, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany. Tel.: 49-931- 201-45771; Fax: 49-931-201-45153; E-mail: butt@klin-biochem.uni-wuerzburg.de.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M209009200
2 E. Butt, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: cAK, cAMP-dependent protein kinase; LASP, LIM and SH3 domain protein; C, catalytic subunit; cGK, cGMP-dependent protein kinase; VASP, vasodilator-stimulated phosphoprotein; 8pCPT-cGMP, 8-para-chlorophenylthio-cGMP; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase.
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