Institute of Molecular Medicine and Genetics, Medical College of Georgia,
Augusta, GA 30912-3175, USA
Present address: Genzyme Corporation, One Mountain Road, Framingham, MA 01701,
USA
Present address: Department of Pharmaceutical Sciences, University of Southern
California, Los Angeles, CA 90089, USA
* Author for correspondence (e-mail: cchew{at}immagene.mcg.edu)
Accepted 15 September 2002
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Summary |
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In this study, we show that lasp-1 binds to non-muscle filamentous (F)
actin in vitro in a phosphorylation-dependent manner. In addition, we provide
evidence that lasp-1 is concentrated within focal complexes as well as in the
leading edges of lamellipodia and the tips of filopodia in non-transformed
gastric fibroblasts. In actin pull-down assays, the apparent
Kd of bacterially expressed his-tagged lasp-1 binding to
F-actin was 2 µM with a saturation stoichiometry of 1:7.
Phosphorylation of recombinant lasp-1 with recombinant PKA increased the
Kd and decreased the Bmax for lasp-1 binding to
F-actin. Microsequencing and site-directed mutagenesis localized the major in
vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to
S99 and S146. BLAST searches confirmed that both sites
are conserved in human and chicken homologues. Transfection of lasp-1 cDNA
encoding for alanine substitutions at S99 and S146, into
parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1
to the intracellular canalicular region. In gastric fibroblasts, exposure to
the protein kinase C activator, PMA, was correlated with the translocation of
lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent
focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC,
these data suggest that other mechanisms in addition to cAMP-dependent
phosphorylation can mediate the translocation of lasp-1 to regions of dynamic
actin turnover. The localization of lasp-1 to these subcellular regions under
a range of experimental conditions and the phosphorylation-dependent
regulation of this protein in F-actin rich epithelial cells suggests an
integral and possibly cell-specific role in modulating
cytoskeletal/membrane-based cellular activities.
Key words: Gastric parietal cell, Stomach, Rabbit, Protein phosphorylation, Cytoskeleton, cAMP-dependent protein kinase
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Introduction |
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Lasp-1 is a recently identified cAMP-dependent signaling protein that may
also be involved in tyrosine kinase signaling
(Chew et al., 1998;
Schreiber et al., 1998
). It is
widely expressed, but differentially distributed, in normal epithelial tissues
and in brain (Chew et al.,
2000
; Chew et al.,
1998
; Schreiber et al.,
1998
). There is a particularly prominent expression of this
protein in the gastric parietal cell and in other F-actin rich
ion-transporting cells including pancreatic and salivary duct cells as well as
certain distal tubule and collecting duct cells in the kidney
(Chew et al., 2000
). In the
parietal cell, elevation of intracellular cAMP ([cAMP]i) induces a partial
translocation of lasp-1 to the apically directed F-actin rich intracellular
canaliculus, which is the site of active HCl secretion. This
stimulus-associated phosphorylation and translocation of lasp-1 to the
canalicular region suggests that lasp-1 may play role in the regulation of
actin cytoskeleton plasticity and, possibly vesicle trafficking
(Chew et al., 2000
).
Lasp-1 was initially identified as pp40, a phosphoprotein that migrated on
SDS-PAGE gels with an apparent molecular mass of 40 kDa
(Chew and Brown, 1987
).
Phosphorylation of pp40 was increased in gastric parietal cells following
elevation of [cAMP]i and was correlated with histamine H2-receptor-activation
of HCl secretion. Subsequently, pp40 was isolated, sequenced and cloned
(Chew et al., 1998
) and shown
to be identical to lasp-1 (LIM and SH3 domain-containing protein), a product
of the human gene, MLN 50, which is amplified in some cancers
(Tomasetto et al., 1995
). In
addition to an N-terminal LIM domain and a carboxyl terminal SH3 domain,
lasp-1 contains two nebulin repeats. Although the specific cellular functions
of lasp-1 have not been defined, the presence of several major
protein-interacting motifs predicts multiple binding partners. Sequence
homology comparisons as well as analyses of physical characteristics further
suggest that one or more these interacting proteins is likely to be
cytoskeletal (Chew et al.,
1998
; Schreiber et al.,
1998
). In this regard, actin is a strong candidate because lasp-1
has been localized to non-stress fiber, actin-rich subcellular regions
(Chew et al., 2000
;
Schreiber et al., 1998
) and
also reportedly associates with actin on blot overlays and in GST pull down
assays (Schreiber et al.,
1998
).
The initial goals of this study were to define the actin binding properties of lasp-1 and to determine if phosphorylation can modulate the interaction. Our results demonstrate that lasp-1 binds to filamentous (F) actin and that cAMP-dependent phosphorylation modifies this interaction in vitro. In the course of these experiments, lasp-1 was found to be highly expressed not only in the gastric parietal cell but also to be present in focal adhesions and focal complexes as well in the extreme tips of lamellipodia and filopodia in gastric mucosal fibroblasts. Since these subcellular regions are rich in F-actin and are associated with a range of activities, including cell migration and membrane trafficking, our results suggest that lasp-1 may play an important signaling-dependent role in the regulation of one or more of these processes.
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Materials and Methods |
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DNA constructs and bacterial protein expression
Polyhistidine (his)-tagged lasp-1 protein was generated using the pET15b
expression vector (Novagen, Madison, WI) by transformation into BL21(DE3)pLysS
bacteria (Promega, Madison, WI) and isopropylthio-(-D-galactoside) induction.
His-tagged protein was purified on a Hi Trap chelating column
(Amersham-Pharmacia Biotech, Piscataway, NJ) followed by Mono Q purification
as previously described (Chew et al.,
1998). Immediately after purification, proteins were dialyzed,
aliquoted and lyophilized. Glutathione-S-transferase (GST)-lasp-1 fusion
protein was generated by inserting cDNA encoding for the rabbit lasp-1 open
reading frame downstream of the GST fragment in the pGEX4T-3 vector (Pharmacia
Biotech, Piscataway, NJ) with BamHI and EcoRI restriction
sites at the 5' and 3' ends, respectively. Bacterial
transformations and inductions were performed with lasp-1-pGEX4T-3 and empty
pGEX4T-3 plasmid (to generate GST protein). A similar procedure was used to
generate mutated lasp-1 GST fusion proteins (below). GST-tagged proteins were
purified using glutathione-sepharose 4B beads (Pharmacia Biotech).
For expression in MDCK cells, constructs containing an N-terminal
hemagglutinin (HA) tag upstream of the coding region for lasp-1 were subcloned
into the pcDNA3 vector (Invitrogen, Carlsbad, CA). Constructs were generated
by PCR-amplification using an Advantage®-HF 2 PCR kit (Clontech, Palo
Alto, CA). The pET15b plasmid containing lasp-1 cDNA served as the template
for primers that generated BamHI and EcoRI restriction sites
at the 5' and 3' ends, respectively. Primers based on the rabbit
lasp-1 cDNA sequence (GenBank accession # AF017438) were designed with OLIGO
Primer Analysis software, version 6.6 for MacIntosh (National Biosciences,
Plymouth, MN) and synthesized by Gibco BRL Life Sciences, PCR conditions were
as follows: Sense primer: 5' GCC GGA TCC ACC ATG GGC TAC CCA TAC GAT GTT
CCA GAT TAC GCT AAC CCC AAC TGC GCC; anti-sense primer: 5' GGC CGA ATT
CTC AGA TGG CTT CCA CGT AGT T; Initial denaturation, 94°C, 30 seconds
followed by 35 cycles of 94°C, 30 seconds; 60°C, 30 seconds; 72°C,
45 seconds and a final 10 minute extension at 72°C. PCR products were gel
isolated and ligated into EcoRI and BamHI-digested pcDNA3
vector. After transformation into Escherichia coli JM109 bacteria,
plasmids containing lasp-1 cDNA were isolated (Qiagen Miniprep kit, Valencia,
CA). Positive clones were identified by PCR. The sequences of all clones were
confirmed prior to use (Medical College of GA Core DNA Sequencing Facility;
ABI Prism 377 automated DNA sequencer; ABI Prism Cycle Sequencing Dye
Terminator Ready Reaction kits). For transfections, plasmid DNA was isolated
and purified with Qiagen Maxiprep Endo-Free kits as previously described
(Parente et al., 1999).
Site-directed mutagenesis was performed with the Stratagene Quick-Change
mutagenesis kit using pcDNA3 vector containing rabbit lasp-1 cDNA as a
template. Primers for single serine to alanine substitutions were as follows:
S146 (RRDA): sense, 5' CGA GCG CCG GGA CGC CCA GGA
CAG CAG C; antisense, 5' GCT GCT GTC CTG GGC GTC CCG GCG CTC G;
S99 (RGFA): sense, 5' GGG CAG AGG CTT CGC CGT GGT
GGC AGA C; antisense, 5' GTC TGC CAC CAC GGC GAA GCC TCT GCC C. PCR
reactions were performed with Pfu taq using the following conditions:
95°C, 1 minute then 16 cycles; 95°C, 30 seconds, 55°C, 1 minute,
68°C, 12 minutes. After 16 cycles, 1 µl (10 U/µl) DPNI restriction
enzyme was added to the reaction and, after a brief centrifugation, samples
incubated at 37°C for 2 hours to digest supercoiled dsDNA. Vectors
were transformed into Epicurean Col:XL1 Blue Super Competent cells as per
manufacturer's instructions. Plasmid DNA was isolated and sequences of all
constructs confirmed after mutagenesis as described above. For double
S99/S146 (RRDA/RGFA) mutants, the
same strategy was employed using the RRDA mutant in pcDNA3 vector as the
starting material. For in vitro experiments with his-tagged lasp-1 mutants,
pcDNA3 plasmids containing the appropriately mutated inserts were transferred
to the pET15b plasmid using a PCR-based approach (Advantage®-HF 2 PCR kit)
as described above with oligodeoxynucleotide primers containing NdeI
(5') and BamHI (3') restriction sites respectively as
follows: 5' GGG AAT TCA TAT GAA CCC CAA CTGC GCC CGG TG and 5' CCG
GAT CCT TCA GAT GGC TTC CAC GTA GTT GGC A.
GST-based assays
GST `pull down' assays were performed at 4°C using parietal cell
extracts that were prepared by incubating freshly isolated cells for 15
minutes in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM
EGTA, 1% NP-40, 0.1% SDS, 10 mM NaF containing the following inhibitors: 0.2
mM AEBSF, 5 mM benzamidine, 10 µg/ml each of leupeptin, pepstatin).
Cellular debris was removed by centrifugation (10 minutes, 10,000
g) and supernatants (0.5-1 mg protein in 0.5 ml) precleared by
incubating with 150 µl of a PBS-washed, 50% GST-sepharose bead slurry for 1
hour on a nutator. Precleared supernatants were collected by centrifugation
(500 g, 5 minutes) then incubated (90 minutes, nutator) with
lasp-1-GST fusion protein (30 µg) bound to washed glutathione-sepharose
beads (7.5 µl bed volume) as per manufacturer's instructions. All samples
were run in duplicate with the following controls: GST protein + precleared
cell lysate + sepharose beads; precleared cell lysate + sepharose beads; GST
lasp-1 + sepharose beads. Beads were harvested by centrifugation (1500
g, 15 seconds), washed three times with cell lysis buffer then
solubilized with 40 µl of 2x SDS stop buffer. Supernatants were
analyzed on SDS-PAGE gels (8-12%), which were either silver stained
(InvestigatorTM Silver stain kit, ESA, Chemsford, MA) or with a modified
Coomassie Blue colloidal staining protocol
(Chew et al., 1998), and by
western blot with enhanced chemiluminescent (ECL) detection as previously
described (Chew et al., 2000
).
Replicate western blots were probed for lasp-1 (anti-lasp-1 monoclonal
antibody (mab, clone 3H8, diluted 1:1000)
(Chew et al., 2000
) and actin
(anti-non-muscle actin mab, clone AC40, Sigma Aldrich, ST Louis, MO, diluted
1:5000). ECL detection was performed with HRP-conjugated sheep anti-mouse Ig
(1:5000 dilution, Amersham Pharmacia, Piscataway, NJ) as the secondary
antibody.
Actin interaction assays
Actin co-sedimentation assays were performed with bacterially-expressed
lasp-1. Monomeric (G)-actin was generated by incubating human platelet actin
(99% purity, 5:1 ß/
isoforms, Cytoskeleton, Denver, CO) at a
concentration of 1 µg/ml in a buffer containing 5 mM Tris-HCl, pH 8.0, 0.2
mM CaCl2, 0.2 mM ATP, 0.5 mM DTT for 1-2 hours, 4°C.
Lyophilized his-tagged or GST-tagged lasp-1 was dissolved in the same buffer
at a concentration of 1-2 µg/ml then centrifuged (100,000
g, 1 hour, Airfuge (Beckman Instruments) to remove protein
aggregates. Lasp-1 (0.5-16 µM) in the resulting supernatants was incubated
with G-actin (14-23 µM, 40 minutes, 4°C) then polymerized by addition
actin polymerization buffer (2 mM Tris, pH 8, 50 mM KCl, 2 mM
MgCl2, 1 mM ATP). After incubation for 30 minutes, room temperature
to allow actin polymerization to reach a steady state, samples were
centrifuged (100,000 g, 1 hour) to pellet F-actin.
Supernatants (which contained G-actin) and pellets were dissolved in SDS-PAGE
buffer and resolved on 8% SDS-PAGE gels. Gels were stained with Coomassie®
Brilliant Blue R250. Images of destained gels were digitized with a Syngene
Gene Genius system and bands quantitated with Gene Tools software (Synoptics,
UK) using BSA as a standard. BSA (Sigma) and
-actinin (Cytoskeleton)
were used respectively as negative and positive controls for F-actin
co-sedimentation. In preliminary experiments in which actin was polymerized
prior to lasp-1 addition, similar amounts of lasp-1 were found to co-sediment
with F-actin as compared to experiments in which actin was polymerized after
lasp-1 addition. This latter approach was used in all subsequent experiments
to avoid quantitation problems associated with the transfer of small
quantities of F-actin.
To assess the association of endogenous lasp-1 with F-actin, a modification
of previously described methods was used
(Weed et al., 2000). Parietal
cells were temperature equilibrated then rapidly pelleted, rinsed in cold PBS
then lysed by sonicating cells (3x10 seconds, 4°C) in a lysis buffer
containing 10 mM imidazole, pH 7.2, 75 mM KCl, 5 mM MgCl2, 1 mM
EDTA, 0.5 mM DTT plus proteolytic inhibitors (mini EDTA-free tablet, Roche
Diagnostics, Mannheim, Germany). After centrifugation (30 minutes, 100,000
g, 4°C), the resulting supernatants were preincubated with
platelet-derived actin (5-8 µM). Samples were sedimented as for the
recombinant protein following actin polymerization. Lasp-1 associated with
F-actin was detected by western blot using the 3H8 monoclonal antibody and ECL
detection as described above. Chemiluminescent signals on western blots were
quantitated with a Syngene GeneGnome 16 bit CCD-based chemiluminescent
detection system and Gene Tools software (Synoptics).
In vitro and in vivo phosphorylation site analyses
To generate phosphorylated lasp-1 for actin co-sedimentation assays,
lyophilized his- or GST-tagged lasp-1 (0.5-1 µg/µl) was dissolved in
1x cAMP-dependent protein kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2). Following addition of recombinant cAMP-dependent protein
kinase (catalytic subunit, New England Biolabs, Beverly, MA; 1-2 U/µg
lasp-1), reactions were initiated with 1 mM ATP and continued for 10 minutes,
30°C. For controls, 1 µg synthetic rabbit protein kinase inhibitor
(PKI, Sigma Chemicals, St Louis, MO) was included in the reaction mixture.
32P-labeling using [-32P]ATP as a substrate (0.2
mM, specific activity 400 cpm/pmol) was used to confirm that this
concentration of PKI completely blocked the phosphorylation of lasp-1 by
cAMP-dependent protein kinase. At the end of the incubation period, samples
were immediately dialyzed (20x volume of 5 mM Tris-HCl, pH 8.0) and
concentrated by centrifugation (2500 g, 4°C) in Centricons
(Amicon, Beverly, MA). To prevent any further phosphorylation in the
ATP-containing actin polymerization buffer, PKI was added to samples in which
it was not added initially. Phosphorylated lasp-1 was incubated with actin and
polymerization performed as described above. Because recombinant protein
kinase migrates close to the phosphorylated form of his-tagged lasp-1 on
SDS-PAGE gels, western blot analyses were also performed using the lasp-1 mab
in conjunction with ECL detection as above. Quantitation was performed using
multiple film exposures to ensure film linearity as previously described
(Chew et al., 2000
) or with
the Syngene GeneGnome system as above.
To identify in vitro cAMP-dependent protein kinase phosphorylation sites,
20 µg of his-tagged lasp-1 was phosphorylated with 15 U recombinant
cAMP-dependent protein kinase catalytic subunit (New England Biolabs) for 5
minutes, 30°C in kinase buffer (25 mM HEPES, pH 7.4, 10 mM Mg (C2H3O2)2,
0.33 mM DTT) containing 0.1 mM [-32P]ATP (specific activity,
36,000 cpm/pmol). Phosphorylated protein was resolved on an 8% SDS-PAGE gel
and subjected to `in gel' tryptic digestion as previously described
(Chew et al., 1998
;
Parente et al., 1996
).
Peptides in digests were resolved on a µRPC C2/C18 column (0-40% linear
acetonitrile gradient, 100 µl/minutes) using a Pharamacia SMART system then
re-purified on the same column at a flow rate of 50 µl/minutes. Peaks
containing radiolabeled peptides were identified by Cerenkov counting of each
fraction (Parente et al.,
1996
). Sequencing by Edman degradation was performed at the Emory
University Microchemical Core Facility, Atlanta, GA. This facility also
performed mass spectrum analyses comparing signals in V8 digests of His-tagged
lasp-1 before and after phosphorylation with recombinant cAMP-dependent
protein kinase catalytic subunit.
Time course experiments were performed by adding
[-32P]ATP (final concentration 0.2 mM, SA, 400 cpm/pmol) to
temperature-equilibrated (5 minutes, 30°C) assay tubes containing 10 µg
histagged lasp-1 plus recombinant cAMP-dependent protein kinase catalytic
subunit (20 U) in kinase buffer. Aliquots (
1 µg lasp-1) were withdrawn
at different time points, placed in an equal volume of 2x SDS stop
solution, boiled for 3 minutes and subjected to SDS-PAGE. Radiolabeled bands
were located by autoradiography, excised and quantitated by Cerenkov counting
(Parente et al., 1996
).
In vivo phosphorylation site analyses were performed with MDCK cells.
Exponentially growing cells were transfected with the pcDNA3 vector containing
HA-tagged wild-type lasp-1 cDNA and cDNA from phosphorylation site mutants
using Effectene (Qiagen) as previously described
(Parente et al., 1999).
Forty-eight hours later, cells were incubated with forskolin (10 µM, 15
minutes) or an equal volume of DMSO vehicle. For one dimensional (1D)
Mr band shift analyses, cells were rinsed in cold PBS and
immediately lysed in 1x SDS stop buffer. Lysates were fractionated on
SDS-PAGE gels and lasp-1 detected by western blot with ECL detection as
previously described (Chew et al.,
2000
). For two dimensional (2D) analyses, cells were lysed with
hot 0.3% SDS-1% ßME, 10 mM Tris, pH 7.4. Lysates were precipitated at
room temperature with 4x volumes of acetone and redissolved in
rehydration buffer (8 M urea, 2% CHAPS, 18 mM DTT, 0.5% IPG buffer, pH 3-10
(Amersham-Pharmacia), 0.001% bromphenol blue). First dimension IEF was
performed on IPG strips (pH 3-10 NL or L) with an IPGPhor (Amersham-Pharmacia)
as follows: 12 hour rehydration; 500 V, 1 hour; 1000 V, 1 hour; 8000
V
28,000 volt hours. For second dimension SDS-PAGE, strips were incubated
for 15 minutes, room temperature in SDS equilibration buffer (50 mM Tris, pH
8.8, 6 M urea, 30% glycerol, 2% SDS, 65 mM DTT, 0.001% bromphenol blue).
Resolved proteins were transferred to nitrocellulose for western blot analyses
of lasp-1 using the lasp-1 mab and ECL detection. Lasp-1 phosphorylation was
defined as an acidic shift resulting from an addition of negatively charged
phosphate residue(s) to the protein (the predicted acidic shifts were
confirmed in metabolic 32P labeling experiments as previously
described (Chew et al., 1998
).
Phosphorylation site analyses were performed with the Phosphobase program on
the Center for Biological Sequence Analysis (CBS) web site
(Kreegipuu et al., 1999
).
Indirect immunofluorescence microscopy
Endogenous lasp-1 was localized by indirect immunofluorescence (primary
antibody, anti-lasp-1 mab 3H8); secondary antibody, cyanine (Cy)-5-labeled
goat anti-mouse IgG (Jackson Immunoresearch Labs, Westgrove, PA) with primary
and secondary antibody controls as previously described
(Chew et al., 2000). In brief,
gastric cells grown on glass coverslips were fixed with 4% paraformaldehyde,
permeabilized with 0.2% Triton X-100, blocked with 5% non-fat milk (BioRad) in
PBS and sequentially incubated with the lasp-1 antibody (diluted 1:50 in 1%
milk/PBS) followed by the Cy-5-labeled secondary antibody (diluted 1:100 in
0.1% milk/PBS). PBS rinses (3-6x5 minutes) were performed after each
incubation step. In most experiments, cells were dual labeled for F-actin by
adding Oregon Green phalloidin (1:400 dilution, Molecular Probes, Eugene, OR)
simultaneously with the secondary antibody. Transfected lasp-1 and mutants
were immunolocalized using a similar protocol with monoclonal anti-HA antibody
(BabCo/Covance, 1:1,000), Alexa 488 chicken anti-mouse secondary antibody
(Molecular Probes, 1:100). Dual labeling for F-actin was accomplished using
Alexa 647-labeled phalloidin Molecular Probes, 1:400). Fluorescently labeled
cells were optically sectioned using a Molecular Dynamics 2010 confocal
microscope equipped with a krypton/argon laser
(Chew et al., 2000
).
Statistical analyses
Where appropriate, values are expressed as means±s.e.m. with
n representing the number of independent experiments. For paired
samples, data was analyzed for statistical significance using the Student's
t-test for paired comparisons. Analysis of variance and Dunnett's
tests were used to analyze multiple comparisons
(Chew and Brown, 1987).
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Results |
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To characterize further the interactions between lasp-1 and actin, actin
co-sedimentation assays were performed using highly purified, platelet-derived
non-muscle actin in order to mimic epithelial cell physiology as closely as
possible. In initial experiments we confirmed that both endogenous and
bacterially expressed lasp-1 cosediment with F-actin
(Fig. 1A,B). The
well-characterized actin binding protein, -actinin, also co-sedimented
with F-actin in these assays but BSA, which does not bind actin, did not
(Fig. 1A). Cumulative data from
several independent experiments demonstrated reproducible and saturable
lasp-1-F-actin co-sedimentation that was significantly greater than controls
(Fig. 1C).
|
To determine if lasp-1 also binds to monomeric (G)-actin, a range of assay conditions were tested including GST pull down assays with GST-lasp vs GST alone, blot overlay assays with 32P-labeled lasp-1, and far western blots using his-tagged lasp-1 in conjunction with the anti-lasp-1 monoclonal antibody. No actin binding was detected in blot overlays or far westerns (not shown). Although actin was present in GST pull downs, there was no significant difference between samples containing GST-lasp vs GST alone (Fig. 1D). Thus, a variety of approaches indicated that lasp-1 does not bind to monomeric actin; however, additional studies are required to establish this point unequivocally.
Identification of major cAMP-dependent phosphorylation sites in vitro
and in vivo by microsequencing, mass spectrometry and site-directed
mutagenesis
Before testing the effects of phosphorylation on the interaction between
recombinant lasp-1 and F-actin, it was essential to define the pattern of in
vitro phosphorylation and to determine if the phosphorylation sites targeted
by PKA in vitro were the same as those regulated by cAMP in vivo. In intact
cells, lasp-1 is phosphorylated on serine residues following elevation of
[cAMP]i (Chew et al., 1998).
This could be the result or either a direct or an indirect involvement of
cAMP-dependent protein kinase (PKA). Previous work predicted that lasp-1 is a
direct substrate for PKA because the recombinant his-tagged protein was
strongly phosphorylated by recombinant cAMP-dependent protein kinase catalytic
subunit, but not by several other serine/threonine kinases including protein
kinase C (Chew et al., 1998
).
As shown in Fig. 2A, the
kinetics of lasp-1 phosphorylation with PKA (recombinant catalytic subunit) is
also rapid and monophasic, reaching a maximum within
30 minutes.
|
Two cAMP-dependent protein kinase phosphorylation consensus sites, both of
which contain serine residues, have been identified in the rabbit lasp-1
protein (Chew et al., 1998;
Chew et al., 2002
). Blast
searches of the GenBank have confirmed that these sites are conserved in the
human (NM_006148) and chicken (#BI394039) homologues but only the
K/RGFS99 site is conserved in rat (NM_032613) and mouse (NM_010688)
(C.S.C., unpublished). As shown in the diagram in
Fig. 2A, the first consensus
phosphorylation site falls between the two nebulin repeats
(RGFS99) and the second is immediately downstream of
this region (RRDS146). To confirm that these sites are,
indeed, targeted by PKA, his-tagged lasp-1 was incubated with recombinant
cAMP-dependent protein kinase catalytic subunit (using
[
-32P]ATP as a substrate) then subjected to tryptic
digestion followed by micro-HPLC purification and microsequencing (as
described in Materials and Methods). Both PKA consensus sites were tentatively
identified within the three major peaks containing phosphorylated peptides
(Fig. 2B). Mass spectrum
analyses were also performed on the same preparation of his-tagged lasp-1
before and after phosphorylation with recombinant PKA. Nanocolumn eluates were
analyzed using a nanospray device and positive, negative and phosphate ion
scans. Positive and negative scans revealed that proteins were digested and
fragments produced. Analysis of his-tagged lasp-1 revealed only background
signal in the phosphate ion scans. Significant phosphate ion peaks were
identified in the 50% methanol eluate of phosphorylated lasp-1. Based on the
predicted proteolytic map, two proteolytic fragments were identified with a
high degree of certainty (GFS99VADTPELQR) and
(MGPSGGEGAEPERRDS146QDSSNYR).
Because other serine residues were present in the sequenced peptides and in one of the two mass spectrum-based phosphate analysis products, neither sequencing nor mass spectrum analyses unequivocally identified Ser99 and Ser146 as specific phosphorylation sites. Therefore, additional analyses were performed with mutated his-tagged lasp-1 proteins in which one or both of these putative cAMP-dependent protein kinase phosphorylation sites were mutated to alanines. As shown in Fig. 3A, cAMP-dependent protein kinase catalyzed 32P incorporation into both RRDA146 and RGFA99 mutants (65% and 26% of total 32P incorporation into wild-type lasp-1, respectively). There was also a lesser degree of 32P incorporation into the double (R/R) mutant. The combined data supported the conclusion that the most significant in vitro phosphorylation catalyzed by cAMP-dependent protein kinase occurs at the Ser99 and Ser146 residues with Ser99 being the most prominent site.
|
Mutation of Ser146, but not Ser99, to alanine blocked
the phosphorylation-induced Mr band shift
(Fig. 3A). Thus, this band
shift, which has been observed both in vivo and in vitro
(Chew et al., 2000;
Chew et al., 1998
), appeared
to result from the phosphorylation of Ser146. To test this
hypothesis more directly, MDCK cells were transiently transfected with pcDNA3
plasmids containing HA-tagged wild-type lasp-1 cDNA or lasp-1 cDNA containing
S
A substitutions for Ser99 and Ser146
respectively. Transfected cells were incubated with forskolin (10 µM, 15
minutes) to elevate endogenous cAMP and to activate endogenous cAMP-dependent
protein kinase. Cell lysates were analyzed for lasp-1 phosphorylation by
western blot. [The lasp-1 mab (clone 3H8) was used for direct analysis of the
expressed rabbit lasp-1 protein because this antibody does not recognize the
endogenous canine protein (see mock transfection lanes in
Fig. 3B).] In these
experiments, S
A mutation of the RRDS146, but not the
RGFS99 site, did indeed block the Mr band shift
of lasp-1 (Fig. 3B). This
experimental approach confirmed that Ser146 is an in vivo target
for cAMP-dependent protein kinase and that phosphorylation of this site
induces a Mr band shift.
To determine if the RGFS99 site (Fig. 3A) is also targeted by cAMP-dependent protein kinase in vivo, pcDNA3 plasmids containing either wild-type lasp-1, the RRDA146 construct, or the double mutant (R/R) construct were transfected into MDCK cells. Cells were stimulated with forskolin (as above), lysed and subjected to 2D gel electrophoresis followed by western blot analysis with the anti-lasp-1 mab. Fig. 3C demonstrates that forskolin stimulation of cells transfected with wild-type lasp-1 led to the expected shift in apparent Mr which was accompanied by an acidic shift (presumably reflecting the increased negative charge of the phosphate groups). With the RRDA146 mutant, there was an acidic shift, but no shift in apparent Mr. Both the acidic and Mr shifts were abolished in the double mutant. The combined results from microsequencing, mass spectrum and mutational analysis studies, therefore, demonstrate that Ser99 and Ser146 are the major in vitro targets of cAMP-dependent protein kinase and are consistent with direct PKA phosphorylation of these sites in vivo.
Phosphorylation of lasp-1 inhibits cosedimentation with F-actin in
vitro
To determine if phosphorylation of Ser99 and Ser146
by cAMP-dependent protein kinase modifies the interaction between lasp-1 and
F-actin, his-tagged lasp-1 was phosphorylated with recombinant PKA prior to
actin cosedimentation assay. As shown in
Fig. 4, phosphorylation
suppressed the interaction (as detected by both Coomassie blue staining
(Fig. 4A) and western blot (not
shown)). In more detailed kinetic analyses, his-tagged lasp-1 was found to
bind to F-actin in a saturable, concentration-dependent manner with a
Kd of 2.2±0.3 µM (n=4) and
stoichiometry of 1 mol lasp-1:7 mol actin
(Fig. 4B). PKA-dependent
phosphorylation of his-tagged lasp-1 increased the Kd by
more than three times (Fig.
4B).
|
Lasp-1 is concentrated within focal contacts, the edges of
lamellipodial extensions, the tips of microfilaments and in orthogonal
filament junctions
Lasp-1 has been detected in cell membrane extensions in some breast cancer
cell lines and in the cortical region in several normal epithelial cell types
(Schreiber et al., 1998;
Chew et al., 1998
;
Chew et al., 2000
). In
contrast, lasp-1 does not appear to be present along stress fibers
(Chew et al., 2000
;
Schreiber et al., 1998
) or in
focal contacts in the cancer cell lines in which it has been characterized
(Schreiber et al., 1998
). As
shown in Fig. 5 (a-c,
arrowheads), however, lasp-1 is prominently present within focal contacts in
non-transformed gastric mucosal fibroblasts, where it co-localizes with
F-actin. Lasp-1 is also present within the extreme tips of F-actin enriched
filopodia (Fig. 5d-f) and
lamellipodial membrane extensions (Fig.
5a-c, arrows). Finally, lasp-1 can be detected within the
orthogonal actin filament junctions that abut lamellipodial extensions
(Fig. 5a'-c', arrowheads).
|
Elevation of cAMP leads to disruption of focal adhesions and stress
fibers and translocation of lasp-1 from the cortex to the cell interior
Elevation of [cAMP]i in some cultured cells is known to cause a reduction
in stress fibers and to disrupt focal adhesions
(Schoenwaelder and Burridge,
1999). To determine if such cytoskeletal alterations could (1) be
induced in cultured gastric fibroblasts and (2) alter lasp-1 distribution,
gastric mucosal cells were incubated with forskolin then fixed and processed
for immunofluorescent localization of lasp-1 and F-actin as described in
Materials and Methods. Fibroblasts were identified based on the presence of
stress fibers and focal contacts which were not found to be present in
differentiated mucosal cells. As shown if
Fig. 6, the addition of
forskolin (10 µM), which elevates [cAMP]i through activation of adenylyl
cyclase, promotes this response in gastric fibroblasts whereas unstimulated
cells that were cultured from the same cellular isolates retained
well-developed stress fibers and focal contacts
(Fig. 6a). After a 30 minutes
exposure to forskolin, stress fibers were disrupted and focal contacts had
disappeared in most fibroblasts (Fig.
6b,c). In agreement with data shown in
Fig. 5, lasp-1 was clearly
localized within focal contacts in controls but, after forskolin stimulation,
was translocated to intracellular regions that frequently contained complexes
of F-actin.
|
Elevation of [cAMP]i in cultured parietal cells induces the
recruitment of lasp-1 to the canalicular membrane region and to the leading
edge of lamellopodia
In gastric parietal cells in the same primary cultures, lasp-1 was
predominately localized to the cortical membrane region along with F-actin
(Fig. 7a,b). Following
forskolin stimulation, the lasp-1 signal in the expanded F-actin rich
canalicular membrane region is increased
(Fig. 7c-f, arrows). A similar
change in lasp-1 distribution was previously observed in freshly isolated
parietal cells in gastric glands (Chew et
al., 2000). In addition to canalicular expansion, parietal cells
in culture generate cell extensions following stimulation with forskolin
(Ammar et al., 2001
). As shown
in Fig. 7g,h, these newly
formed membranes extensions contain lasp-1 and F-actin at their leading edges.
Thus, elevation of [cAMP]i induces lasp-1 recruitment to F-actin rich cellular
compartments not only in gastric fibroblasts but also in two different
intracellular compartments in cultured gastric parietal cells.
|
To determine if PKA-dependent phosphorylation is necessary for lasp-1
translocation following elevation of [cAMP]i, experiments were designed to
compare the distribution profile of wild-type HA-tagged lasp-1 and the
HA-tagged phosphorylation site mutants following transfection into cultured
parietal cells. The general outcome of these experiments supported the
conclusion that cAMP-dependent phosphorylation is required for lasp-1
translocation and suggested that lasp-1 may be be involved in the initiation
of the acid secretory process. Thus, in parietal cells transfected with the
wild-type lasp-1 construct, the expressed HA-tagged protein was targeted
mainly to the cortical cell membrane. In transfected cells that responded to
forskolin stimulation (based on gross morphological changes
(Chew et al., 1989), HA-tagged
lasp-1 was recruited to the F-actin rich canalicular membrane region
(Fig. 8a-d). In cells
expressing the mutant protein, however, no similar redistribution of the
HA-tagged RRDA146/RGFA99 protein could be detected. In
addition, no gross morphological changes in cells expressing the HA-tagged
RRDA146/RGFA99 protein were identified
(Fig. 8e-h).
|
Lasp-1 is recruited into some regions of dynamic actin assembly
independent of [cAMP]i elevation
Since other signaling pathways may also be involved in regulating lasp-1
interactions with the actin cytoskeleton, we sought to determine if
cAMP-independent manipulations that induce the formation of lamellipodial
extensions would alter the distribution of lasp-1. A well recognized strategy
for inducing the formation of cellular extensions is to expose fibroblasts
deprived of growth factors to the phorbol ester, PMA. Growth factor
deprivation reduces the formation of lamellipodial extensions and, under these
conditions, actin incorporation occurs only at the sites of protrusive
activity (Chan et al., 1998).
The addition of PMA induces lamellipodial formation and actin assembly
(Downey et al., 1992
;
Schliwa et al., 1984
). Thus,
sites of protrusive activity can be identified under growth factor-free
conditions and newly formed membrane extensions can be identified following
the addition of PMA (Kellie et al.,
1985
). Data depicted in Fig.
9 support this prediction. Twenty-four hours after the removal of
growth factors, lasp-1 was prominently localized within focal contacts and
microspikes present at the tips of plasma membrane extensions in gastric
fibroblasts (Fig. 9a-c). Within
two minutes after addition of PMA (100 nM), lamellipodial extensions began to
form and weak lasp-1 and F-actin signals could be detected within these newly
generated structures (Fig.
9d-f). After 5 minutes, an uninterrupted band of co-localized
lasp-1 and F-actin became obvious at the membrane-actin interface of growing
extensions (Fig. 9g-h). At 10
minutes there was some broadening of this band and focal contacts in most
cells appeared as punctate regions in which lasp-1 and F-actin signals
persisted (Fig. 9j-l). Taken
together, these results demonstrate the recruitment of lasp-1 to F-actin rich
regions that are associated with active membrane protrusion in gastric
fibroblasts. Since lasp-1 does not appear to be a direct target of PKC
(Chew et al., 1998
), the
recruitment mechanism is probably not dependent on direct phosphorylation by
PKC but may be initiated by the phosphorylation of associated protein(s).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two major serine phosphorylation sites have been identified in lasp-1 and
several lines of evidence indicate that this protein is a direct downstream
substrate for PKA in vivo. Based on the parietal cell transfection studies it
appears that cAMP-dependent phosphorylation of lasp-1 is required for the
recruitment of this protein to the intracellular canalicular region of
parietal cells. Although these findings were consistent, it should be noted
that there are several potential problems in their interpretation. First, the
mutated proteins displayed reduced affinity for lasp-1 in vitro. Thus, it is
possible that vivo functions were similarly compromised. Also, the
transfection efficiency with parietal cells is low and not all parietal cells
in primary culture respond to acid secretory agonists with obvious changes in
cytoskeletal morphology (Parente et al.,
1999). Clearly, new experimental approaches will be required to
confirm or discount a direct role for cAMP-dependent phosphorylation in lasp-1
translocation and to define the specific cellular activities that are
modulated during this translocation process.
In gastric fibroblasts, it appears that lasp-1 can also recruited to
regions of dynamic actin/membrane turnover by cAMP-independent mechanisms. In
addition, lasp-1 appears to retain its association with F-actin in
forskolin-stimulated fibroblasts in which stress fibers are disrupted. Thus,
this novel signaling protein may also be regulated either directly or
indirectly by several mechanisms, with the predominant mechanism being
dependent on the specific cell type. Tyrosine phosphorylation might also play
a regulatory role in some cell types. Although we have not been able to detect
tyrosine phosphorylation of lasp-1 in the terminally differentiated parietal
cell (C.S.C., X.C. and H.-Y.Q., unpublished), there are two conserved tyrosine
phosphorylation consensus sites in lasp-1 and this protein can be
phosphorylated on tyrosine residues in cell lines overexpressing Src kinase
(Schreiber et al., 1998). The
finding that a cytoskeletal-associated protein is differentially
phosphorylated in normal epithelial cells has precedence in that
cytoskeletal-membrane linking protein, ezrin, is phosphorylated on tyrosine
residues in A431 cells (Bretscher,
1989
) but on serine/threonine residues in the parietal cell
(Urushidani et al., 1989
).
These differences in phosphorylation patterns may reflect differences in
cellular protein kinase expression profiles and/or alterations in the coupling
of signaling pathways to the cytoskeleton.
The observation that PKA-dependent phosphorylation reduces the affinity of
lasp-1 for F-actin in vitro raises interesting questions regarding in vivo
mechanisms. If PKA-dependent phosphorylation also reduces the affinity of
lasp-1 for F-actin in vivo, why does lasp-1 translocate to the F-actin rich
canalicular membrane region in the parietal cell? There are a several possible
explanations. In the gastric parietal cell, there are at least two distinct
pools of actin. At the cell cortex, -actin predominates whereas
ß-actin predominates in the canalicular region
(Yao et al., 1995
) The
cortical actin pool is exquisitely sensitive to latrunculin B, which binds
monomeric actin, whereas the formation of microvillar filaments appear to be
highly resistant to this inhibitor as well as to cytochalasin D, which severs
actin filaments (Ammar et al.,
2001
). Thus, at the cell cortex, a reduced affinity for actin
could assist in the rapid remodeling of the actin cytoskeleton
(Pollard, 1999
). The source of
lasp-1 that translocates to the canalicular region is not yet established.
However, both detergent-soluble and -insoluble pools of lasp-1 have been
identified (Chew et al., 2002
;
Chew et al., 1998
) so it is
likely that lasp-1 is distributed within several subcellular compartments,
possibly with stronger and weaker linkages to the actin cytoskeleton. Since
lasp-1 contains both LIM and SH3 protein association domains, it is also
likely that other interacting proteins play a direct and/or indirect role in
regulating the affinity of lasp-1 for F-actin in vivo. The phosphorylation of
lasp-1 by other protein kinase(s) may also regulate this interaction. Finally,
the behavior of actin itself is extensively modified by cell membranes as well
as by cytosolic extracts (Jahraus et al.,
2001
). Thus, the local intracellular environment may also
differentially affect the association between lasp-1 and F-actin depending on
the specific pool of actin that is present.
The presence of lasp-1 in both classical and nascent focal contacts or
adhesions in gastric fibroblasts is intriguing and it will be important to
determine if this association is regulated by vesicular trafficking and/or
phosphorylation-dependent mechanisms and to determine whether F-actin and/or
other proteins within these complexes interact directly with lasp-1. Although
classical focal contacts are viewed as relatively stable structures, nascent
focal contacts and the cortical actin cytoskeleton appear to contain critical
sites for the initiation of actin polymerization
(Beningo et al., 2001;
Small et al., 1998
;
Wang, 1985
). To date, more
than 50 proteins have been detected in focal contacts and it is becoming
increasing evident that there is molecular diversity as well as complex
signaling activity and vesicular trafficking of proteins into and out of these
regions (Sastry and Burridge,
2000
). Since no systematic or comprehensive search for focal
contact proteins has been conducted, the extent of cell-type specific
distribution has not yet been established
(Zamir and Geiger, 2001
).
However, because lasp-1 does not appear to be present in focal contacts of the
human BT-474 breast cancer cell line
(Schreiber et al., 1998
), the
distribution of this signaling protein may indeed be cell-type specific.
Defining the distribution pattern and functions of lasp-1 in a range
transformed and non-transformed cell lines as well as normal epithelial cells
may provide useful insights into the functions of this recently identified
signaling molecule.
The specific localization of lasp-1 to proximal junctional branch points
within the cortical actin filament network as well as in lamellipodial tips,
where the Arp 2/3 complex proteins have also been localized
(Svitkina and Borisy, 1999),
suggests the possibility that lasp-1 might interact either directly or
indirectly with this important actin-nucleating complex. The actin-binding
protein, cortactin (Schuuring et al.,
1993
), which is also known as amplaxin and oncogene EMS-1 gene,
has recently been found to bind to the Arp 2/3 complex thereby regulating
actin polymerization (Uruno et al.,
2001
; Weaver et al.,
2001
). Lasp-1 shares a relatively high degree of homology with
cortactin in the SH3 domain and proline-rich regions as well as in their actin
binding regions (Sparks et al.,
1996
) (C.S.C., unpublished). As with lasp-1, in vitro
phosphorylation of cortactin suppresses F-actin binding
(Huang et al., 1997
) and
cortactin undergoes phosphorylation-dependent translocation in intact cells
(Ozawa et al., 1995
).
Cortactin also has no direct homologue in yeast and is primarily localized
within in peripheral cell regions associated with dynamic actin assembly in
cultured cells (Wu and Parsons,
1993
). Most earlier studies of cortactin focused on pp60 src
kinase-dependent tyrosine phosphorylation. However, epidermal growth factor
induces the phosphorylation of cortactin on serine and threonine residues, and
it is the phosphorylation of these residues which induces a mobility shift on
western blots (Campbell et al.,
1999
). In addition, it has recently been proposed that cortactin
translocation is mediated by serine/threonine phosphorylation rather than by
tyrosine phosphorylation (Lopez et al.,
2001
). Thus, cortactin, like ezrin and possibly lasp-1 may be
differentially regulated by serine/threonine kinases and tyrosine kinase
dependent on the developmental stage and/or cell type involved.
Because lasp-1 lacks the conserved Arp 2/3 interaction site (the acidic `A'
domain (DD/EW), which is present in cortactin as well as WASP-family members
and several other Arp 2/3 binding proteins
(Olazabal and Machesky, 2001),
it appears unlikely that there is a direct interaction between lasp-1 and the
Arp 2/3 complex. However, lasp-1 could modulate this complex indirectly.
Several SH3 domain-containing proteins have been found to bind to proline-rich
sequences in WASP and N-WASP (Higgs and
Pollard, 2001
). In addition, the large GTPase, dynamin, which has
been implicated in endocytotic trafficking
(Kessels et al., 2000
), has
been shown to complex with WASP indirectly through an interaction with the
WASP-binding protein, syndapin I (Qualmann
et al., 1999
). Recent work has shown that lasp-1 associates with
dynamin II in vitro (Okamoto, 2001). Thus, it will be of interest to determine
whether or not lasp-1 has the ability to modulate the Arp 2/3 complex via
direct connections with WASP family members and/or indirect associations via
dynamin, for example. The answer to this question is particularly critical
given the current lack of knowledge of the molecular mechanisms involved in
directing membrane trafficking to sites of actin polymerization at the leading
edge of migrating cells and in the formation of microvillar extensions in
ion-transporting cells such as the gastric parietal cell. Another important
goal for future research will be to identify additional protein binding
partners for lasp-1 and to determine whether it is an indirect modulator of
actin assembly and/or membrane trafficking and cell adhesion and motility.
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Acknowledgments |
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