From the Department of Cell Biology,
University of Alabama at Birmingham, Alabama 35294, § Schering-Plough Research Institute, Union, New Jersey
07083, and the ¶ Division of Biomedical Sciences, Imperial
College London, London SW7 2AZ, United Kingdom
Received for publication, August 13, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Syndecan-4 is a transmembrane heparan sulfate
proteoglycan that acts as a coreceptor with integrins in focal adhesion
formation. The central region of syndecan-4 cytoplasmic domain (4V;
LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together
they regulate protein kinase C Syndecans are transmembrane heparan sulfate proteoglycans that
play roles in cell adhesion, growth factor binding and presentation, regulation of lipases, and bacterial and viral entry among other functions (reviewed in Refs. 1-3). Of the four mammalian syndecans, syndecan-4 appears to have a unique role in cell adhesion, acting in
concert with the integrin family of matrix receptors (4). Syndecan-4 is
a focal adhesion component in a range of cell types adherent to several
different matrix molecules (4). When primary fibroblasts adhere
to the RGD-containing, cell-binding domain of fibronectin through
Syndecan-4 overexpression in Chinese hamster ovary cells leads to the
formation of larger focal adhesions and thicker stress fibers, whereas
transfection with cytoplasmic domain truncation mutants lacking the
central variable (V) region prevents their formation (9). Recently,
syndecan-4 gene disruption in mice was reported (10). Fibroblasts from
this mouse formed focal adhesions on intact fibronectin. However, these
cells could not respond to soluble HepII domain of fibronectin by
forming focal adhesions when seeded on substrates of the cell-binding
domain (10), emphasizing a biological role for syndecan-4 in this process.
All syndecans have short cytoplasmic tails with conserved amino acid
sequences proximal (C1) and distal (C2) to the membrane with an
intervening sequence unique to each family member (V region) (1-3).
The variable region of syndecan-4, whose amino acid sequence is
LGKKPIYKK, directly binds PKC Unlike other proteins that regulate PKC subtypes (e.g. RACK)
(16,17), syndecan-4 V region appears to bind the catalytic, not the
regulatory domain, of PKC Plasmid DNAs--
Standard PCR techniques (23) were used to
generate syndecan-4 cytoplasmic domain constructs (see Fig.
1A) and deletion mutants of PKC
The cDNA sequences encoding various fragments of syndecan-4
cytoplasmic domain and human PKC
For Drosophila S2 cell expression, PCR products were
subcloned into pcDNA3.1/HisC at the KpnI and
XhoI sites, and secondary PCR products including N
terminus His6 and Xpress tags were inserted into
SpeI and XhoI sites of pMT/V5-HisA vector
(Invitrogen). To construct the His6/Xpress-containing tag
from the pcDNA3.1 vector, HisCF primer
(5'-CAAGCTGACTAGTGTTTAAAC-3') was used (the SpeI site is underlined). The ligation products were sequenced and cotransfected into S2 cells with pCohygro selection vector at a plasmid
ratio of 19:1 (w/w) using calcium phosphate (as per the manufacturer's
protocol). Stable transfectants were selected with DES® medium
containing 300 µg/ml hygromycin (Invitrogen) for 3 weeks, and protein
expression was induced with 500 µM CuSO4 for
3 days. The primers used are listed in Table
III.
Yeast Two-hybrid Analysis--
pLexA-LaminC and cotransformants
of pLex A and pB42AD empty vectors were used as negative controls,
whereas those of pLex-p53 and pB42AD-T served as a positive control
(Clontech). To test interactions, both bait and
prey were transformed into yeast strain EGY48 (MAT In Vitro Binding Assays--
His6- and Xpress
epitope-tagged full-length PKC Immunoprecipitation--
Rat embryo fibroblasts (REFs), REFs
overexpressing wild type syndecan-4 (S4), or truncated at arginine 175 (S4 Site-directed Mutagenesis and Transfection--
Site-directed
mutagenesis was performed with template rat syndecan-4 in pcDNA3
vector using the QuikChange kit (Stratagene, La Jolla, CA) and PCR (30 s at 94 °C, 16 min at 68 °C, 30 s at 55 °C with 15 cycles) for amplification. Tyrosine 192 in the central V region
was changed to phenylalanine using complementary forward 5'-gcaagaaacccatcttcaaaaaagccc-3' and reverse
5'-tgggcttttttgaagatggctttcttgc-3' primers. The underlined
sequences are mutation targets. After PCR, parental (template) plasmid
DNAs were digested with the restriction enzyme DpnI for
1 h at 37 °C and transformed into XL-Blue competent cells.
Mutation was confirmed by DNA sequencing using the 5'-upstream sequencing primer, 5'-ctgaggtcttggcagctc-3'. Six µg of mutated and
wild type syndecan-4 constructs were transfected into REF cells using
LipofectAMINE (Invitrogen) for 18 h in serum-free media.
Transfected cells were selected with 600 µg/ml G418 (Invitrogen) for
3 weeks. Increased cell surface expression of syndecan-4 was confirmed
by fluorescence-activated cell sorter analysis (not shown) at
University of Alabama at Birmingham flow cytometry core facility and by
staining with 150.9 monoclonal antibody.
Immunostaining--
REFs and stable transfectants
were seeded onto coverslips for 24 h and fixed and permeabilized
with 3.5% paraformaldehyde containing 0.1% Tween 20 for 10 min (11).
Cells were stained with monoclonal anti-PKC PKC
To determine more precisely the interaction site(s),
syndecan 4V or C14V baits were used in further experiments with smaller PKM fragments. Yeast transformed with PKM1 or 4 showed no growth, but
colony growth assays indicated an interaction of PKM5 with syndecan-4
(Fig. 2A). PKM2 showed occasional slight growth activity. The growth assays were again confirmed by
To confirm the interactions between syndecan-4 cytoplasmic domain and
PKC Syndecan-4 Oligomerization Is Needed for PKC
To confirm the results from yeast two-hybrid assays and affinity
chromatography, subdomains of PKM were tested for the ability to
displace intact PKC Syndecan-4 V Region Specifically Binds PKC
Increased syndecan-4 expression in S4 REF cells correlates with
increased association with PKC
Since coimmunoprecipitation experiments indicated specific association
with PKC
A mutational approach was used to address whether the tyrosine 192 mutation to phenylalanine in syndecan-4, which still allowed self-association but reduced PKC Previous studies indicated direct binding of PKC Syndecan-4 at the cell surface may activate PKC Confirmation of the yeast two-hybrid data was obtained through
pull-down assays with recombinant kinase and synthetic syndecan-4 V
peptide and by coimmunoprecipitation and competition experiments. GST
fusion proteins containing PKM5, which represents the C-terminal 160 amino acids of PKC Previous studies have shown that self-association of syndecan-4
cytoplasmic domain is needed for PKC PKC There has been controversy concerning whether syndecan-4 binds directly
to PKC (PKC
) activity. Syndecan 4V
peptide directly potentiates PKC
activity, leading to
"superactivation" of the enzyme, apparently through an interaction
with its catalytic domain. We now have performed yeast two-hybrid and
in vitro binding assays to determine the interaction sites
between 4V and PKC
. Full-length PKC
weakly interacted with 4V by
yeast two-hybrid assays, but PKC
constructs that lack the
pseudosubstrate region or constructs of the whole catalytic domain
interacted more strongly. A mutated 4V sequence (4V(YF):
LGKKPIFKK) did not interact with PKC
, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this
interaction. Further assays identified a novel interaction site in the
C terminus of the catalytic domain of PKC
(amino acid sequence
513-672). This encompasses the autophosphorylation sites, which
are implicated in activation and stability. Yeast two-hybrid data were
confirmed by in vitro binding and coimmunoprecipitation
assays. The interaction of syndecan-4 with PKC
appears unique since
PKC
and
did not interact with 4V in yeast two-hybrid assays or
coimmunoprecipitate with syndecan-4. Finally, overexpression of
syndecan-4 in rat embryo fibroblast cells, but not expression of the YF
mutant, increased PKC
localization to focal adhesions. The data
support a mechanism where syndecan-4 binds PKC
and localizes it to
focal adhesions, whose assembly may be regulated by the kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5
1 integrin, they attach and spread well but do not form focal adhesions or stress fibers (5, 6). These do form
when the high affinity heparin-binding domain (HepII) of fibronectin is
added, syndecan-4 is clustered by antibody against the ectodomain of
the core protein, or protein kinase C
(PKC)1 is activated by
phorbol ester (6, 7). The HepII domain of fibronectin binds to
syndecan-4 and drives syndecan-4 into forming focal adhesions (8).
in vitro and partially
activates it (11). The 4V peptide also binds phosphatidylinositol
4,5-bisphosphate (PIP2), which also partially activates PKC
. The
combination of 4V and PIP2 results in a superactivation of PKC
and
removes the dependence on calcium for activation (12, 13). PIP2, whose levels increase during fibronectin-mediated cell adhesion, promotes syndecan-4 cytoplasmic domain oligomerization (12, 14, 15), which is
also needed to bind and superactivate PKC
(12, 13).
(11). It can superactivate both PKC
and
PKM (the free catalytic domain derived after calpain cleavage), and
both PKC
and PKM interact with syndecan-4 cytoplasmic domain as
shown by coimmunoprecipitation, affinity chromatography, and solid
phase assays (11). These interactions require prior PKC activation,
which can translocate this kinase to the plasma membrane. PKC
, in
addition to syndecan-4, is present in some focal adhesions (4, 18-20)
and is coclustered with syndecan-4 when spreading cells are treated
with antibodies against syndecan-4 ectodomain (11). PKC activation is
required for spreading and focal adhesion formation in both the
integrin- and syndecan-4-mediated signaling pathways (21, 22), leading
to the concept that a ternary PIP2/syndecan-4/PKC
complex may form
during adhesion. To understand the molecular basis for the pathway of
PKC
activation, identification of the interaction sites in the
kinase and in syndecan-4 cytoplasmic domain is essential. In this
study, we used yeast two-hybrid assays to confirm the specificity of
interaction of 4V with the PKC
isoform and to identify 1) the site
in the PKC
catalytic domain that interacts with the syndecan-4 V
region, 2) the requirements for syndecan-4 cytoplasmic domain
self-association, and 3) a specific residue within syndecan-4 that is
needed for the interaction. Furthermore, we show that this interaction
is biologically relevant since overexpression of syndecan-4 in REF cells results in increased association of PKC
with syndecan-4 and
increased PKC
localization in focal adhesions. This is dependent on
the V region of syndecan-4 and specifically requires tyrosine 192.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(see Figs. 1C
and 2A). For the yeast two-hybrid assays, syndecan-4
cytoplasmic tail constructs were cloned into pLexA vector for baits,
and PKC constructs were cloned into pB42AD vector as preys
(Clontech). Human PKC
, -
, and -
cDNA
were obtained from ATCC. For yeast two-hybrid constructs, all PKC
deletion and syndecan-4 cytoplasmic tails were generated with pairs of degenerate PCR primers having terminal EcoRI and
XhoI restriction enzyme sites. To generate GST·PKC
constructs, yeast two-hybrid inserts were excised and ligated into
pGEX-5X-1 vector (Amersham Biosciences), and ligation products were
sequenced and transformed into the bacterial strain JM109 for protein expression.
for yeast two-hybrid assays were
generated by PCR. Tables I and II show
the syndecan-4 and PKC
primers respectively, and Figs. 1,
A and C and 2A show the constructs.
For syndecan-4 cytoplasmic constructs,
sense and antisense primers were annealed, or PCR products were cut by
restriction enzymes and cloned into EcoRI and
XhoI sites, although primers for PKC
ended with
XhoI sites.
Primers used for syndecan-4 yeast two-hybrid constructs shown in Fig. 1
PKC primers used to generate constructs for yeast two-hybrid assays
and
constructs only contained their catalytic domains, and the amino acid
sequences are numbered in the legend of Fig. 6.
Primers used to generate PKC and its deletion constructs for S2
cell expression
, ura3,
his3, trp1, LexAop(x6)-leu2) harboring p8op-lacZ reporter plasmids by the LiCl2/ssDNA/PEG method
(24). Transformants were plated on selection media lacking uracil,
histidine, and tryptophan and incubated at 30 °C for 3 days. Colony
growth was monitored by the X-Gal whole plate method on selection
plates containing 80 µg/ml X-Gal lacking uracil, tryptophan,
histidine, and leucine and by liquid
-galactosidase assays with
O-nitrophenylglycoside (Sigma) for quantification
(as per Clontech manufacturer's protocol). Statistical significance was determined by Student's t
test. Expression of prey constructs in yeast was confirmed by
immunoblotting (not shown) using hemagglutinin polyclonal antibody (Invitrogen).
, its regulatory domain, and PKM were
expressed in S2 Drosophila cells (Invitrogen). For small
PKC
constructs, the GST fusion expression and purification system
was used (Amersham Biosciences). His6/Xpress constructs were purified by Ni2+ agarose affinity chromatography
(Qiagen, Valencia, CA), and GST constructs were purified by glutathione
bead chromatography (as per the manufacturer's instructions). Except
for PKM1, all small fragments showed a tendency for degradation.
Synthetic 4V peptide (CLGKKPIYKKA; Synpep, Dublin, CA) was immobilized
to Sulfo-Link agarose beads (Pierce). Purified proteins were subjected
to 4V-conjugated bead pull-down assays (11) followed by SDS-PAGE and
Western blotting using monoclonal Xpress antibody (Invitrogen) or
polyclonal GST antibody (Upstate Biotechnology, Lake Placid, NJ) to
detect bound protein.
R), isoleucine 191 (S4
I), or glutamic acid 198 (S4
E) were
subjected to immunoprecipitation as previously (11). These
constructs were cloned into LK444 vector under the control of a
-actin promoter.2
Subconfluent cells were washed with phosphate-buffered saline and
scraped into lysis buffer (1% Triton X-100, phosphate-buffered saline,
pH 7.3, containing protease inhibitor mixture (Sigma)), incubated on
ice for 30 min, and centrifuged at 1000 × g for 10 min. Supernatants were transferred into new tubes and precleared with
10% fetal bovine serum-blocked protein-A conjugated Sepharose beads
for 30 min at 4 °C. Preclearing beads were removed, and the
supernatants were incubated with monoclonal antibody 150.9 against
syndecan-4 core protein for 45 min at 4 °C. Rabbit anti-mouse antibodies (Dako, Carpinteria, CA) were added for 30 min prior to
further addition of protein A-coated beads for 30 min at 4 °C. For
displacement assays, immunoprecipitates were incubated with 0.2 µg/ml
GST·PKM subdomain proteins for 30 min at 4 °C. After extensive
washing, bound complexes were separated by 3-15% SDS-PAGE and
immunoblotted using monoclonal anti-PKC
(Upstate Biotechnology),
rabbit anti-PKC
, or goat anti-PKC
(Santa Cruz Biotechnology,
Santa Cruz, CA). The blot was then incubated with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM
Tris-HCl, pH 6.7) at 50 °C for 30 min, washed three times for 10 min
in TBST buffer (50 mM Tris-HCl, pH 7.4, 0.05% Tween, 100 mM NaCl), and reprobed with anti-GST.
, rabbit polyclonal
anti-PKC
, goat polyclonal anti-PKC
, monoclonal anti-vinculin
(Sigma). Secondary antibodies were F(ab')2 goat anti-mouse
IgG or goat-anti-rabbit IgG (Cappel, Durham, NC), rabbit anti-goat
(Santa Cruz Biotechnology), or goat anti-rabbit IgG, conjugated with
fluorescein isothiocyanate or Texas Red dependent on the combination of
primary antibodies. Cells were viewed on a Nikon Optiphot
microscope and photographed with Ilford HP5 film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Directly Interacts with Syndecan-4--
Yeast two-hybrid
analysis confirmed a direct interaction between the V region of
syndecan-4 cytoplasmic domain and PKC
. The syndecan-4 constructs
used for these and self-association assays are shown in Fig.
1A, and the structure of
PKC
and its constructs are shown in Fig. 1, B and
C. Monitoring colony growth on selective media, 4L, which
codes for the full-length cytoplasmic domain, did not interact with any
PKC
construct (Fig. 1C). This is consistent with previous
biochemical data indicating a lack of PKC
by 4L in the absence of
PIP2 (11). 4V (which codes for the peptide sequence LGKKPIYKK)
interacted well (Fig. 1C) with the
PS construct, which
lacks the pseudosubstrate site that masks the catalytic site and is an
active form of PKC
but interacted less strongly with full-length
PKC
. These results are consistent with a need for prior activation
of PKC
before binding to the 4V peptide in in vitro
assays (11) and PKC activation during cell spreading (21, 22). 4V(YF),
where tyrosine 192 of 4V was mutated to phenylalanine, did not show
interaction in colony growth assays (Fig. 1C). Deletion of
the C2 domain in 4L (C14V) did allow interaction (Fig.
2A). Despite the fact that
most activators of PKCs bind to the regulatory domain (Fig.
1B), no interaction was detected between syndecan-4 baits
and PKC
regulatory domain (Fig. 1C). However, a clear
interaction was detected between syndecan-4V and PKC
catalytic
domain (PKM). Colony growth assays were complemented by
quantitative
-galactosidase assays (Fig. 1D),
which confirmed these interactions.
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Fig. 1.
Interaction between
syndecan-4 cytoplasmic domain and PKC
constructs. A, syndecan bait constructs used in
yeast two-hybrid analyses of PKC
interaction and self-association.
4L, complete cytoplasmic tail of syndecan-4;
C14V, cytoplasmic tail lacking the C2 domain;
C1(SD)4V, serine 183 mutated to
aspartic acid mimicking phosphorylated serine, underlined;
4V, central V region of syndecan-4;
4V(YF), 4V with tyrosine 192 changed to
phenylalanine, underlined; 4VC2, cytoplasmic tail
lacking the C1 domain;
FYA, cytoplasmic tail lacking the
C-terminal FYA sequence. B, schematic structure of PKC
.
C2 contains phospholipid-binding sites (e.g. DAG, PIP2).
C, yeast two-hybrid assay results, monitoring colony growth
using syndecan 4V, 4L, and 4V(YF) in nutrient lacking selection media; + indicates growth,
indicates no growth. PKC
indicates
full-length PKC
.
PS, construct where the
inhibitory N-terminal (pseudosubstrate) sequence has been deleted;
Reg, regulatory domain of PKC
; RegC1, the
first constant region of PKC
; RegC2, the second constant
region of PKC
; PKM, catalytic domain of PKC
which is a
freely active enzyme in the absence of phospholipid or
Ca2+. Each construct is numbered according to
the amino acid sequence of PKC
. D, quantitative
interaction assay monitoring relative
-galactosidase enzyme activity
using 4V bait. (n = 3, S.E.; *, p < 0.02, **, p < 0.005, p value was calculated
using empty vectors as baseline).
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Fig. 2.
Interaction between syndecan-4 cytoplasmic
domain and PKM subdomains. A, PKM subdomain constructs
and results from colony growth assays using 4V and C14V baits. PKM
subdomain constructs are numbered from the amino acid sequence of
PKC . B, quantitative interaction assay monitoring
relative
-galactosidase enzyme activity using 4V bait.
(n = 3, S.E.; *, p < 0.03, **,
p < 0.005, p value was calculated using
empty vectors as baseline).
-galactosidase assays (Fig. 2B). The PKM5-binding site for syndecan-4 (amino acids
513-672) contains an important module for activation of PKC
, two
autophosphorylation sites at threonine 638 and serine 657 (25, 26).
detected in yeast two-hybrid assays, recombinant PKC
from
Drosophila S2 cells and GST·PKM subdomains from bacteria were subjected to pull-down assays with syndecan-4V-conjugated beads.
Full-length PKC
bound to immobilized 4V peptides (Fig. 3A), but consistent with the
yeast two-hybrid analysis, no binding of the regulatory domain (Reg)
was detected. There was no binding of PKC
, Reg, or PKM to beads
blocked with cysteine as a negative control (not shown). Previous
studies showed that PKC
does not bind to beads conjugated with the
syndecan-2V region (11). A higher proportion of input PKM than PKC
bound to 4V-conjugated beads. Neither PKM1 nor PKM4 bound to
4V-conjugated beads (Fig. 3B), whereas PKM5 did bind, albeit
to a lesser extent than intact PKM. PKM3, which contains PKM4 and PKM5,
bound to a higher extent than PKM5, possibly due to increased stability
(not shown). Further truncation of the PKM5 construct indicated that a
peptide containing the C-terminal 41 amino acid sequence still
interacted with the syndecan-4 V peptide (not shown), but results were
less reproducible, and the isolated peptide appeared to be
unstable.
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Fig. 3.
Pull-down assays with 4V peptide-coupled
beads. As shown in A, recombinant PKC and
recombinant peptides Reg and PKM were purified from
Drosophila S2 cells, and their ability to bind 4V-conjugated
beads was monitored by immunoblotting with anti-Xpress. The regulatory
domain did not show any binding to 4V-conjugated beads. As shown in
B, recombinant GST·PKM peptides 1, 4, and 5 were purified
from bacteria and tested for binding, monitoring bound material by
immunoblotting with anti-GST. PKM1 and -4 did not show any binding to
the beads. GST is GST alone as a negative control.
Lane I, 10% of input amount; lane B, amount
bound to the beads.
Interaction--
Previous in vitro studies using synthetic
peptides demonstrated that 4V peptides oligomerized to form dimers and
larger oligomers up to octamer size, based on gel filtration
chromatography (14) and NMR spectroscopy (15), but 4L peptides remained
mostly as monomers with some dimers. Syndecan 4L peptides did not bind
added PKC
in in vitro assays except when induced to
oligomerize in the presence of PIP2 (11, 12, 14), and 4L baits in this present study did not interact with PKC
or PKM in yeast two-hybrid assays (Fig. 1C). We, therefore, monitored the
self-association activity of syndecan-4 tail baits by yeast two-hybrid
assays. When 4L was used as both prey and bait, no growth was detected in colony growth assays (Fig.
4A). In contrast,
transformants containing syndecan 4V constructs exhibited strong
growth, indicative of self-association. Interestingly, mutated 4V(YF),
which forms dimers but does not activate PKC in vitro (12),
also self-associated when analyzed by yeast two-hybrid analysis (Fig.
4). Thus, the central tyrosine 192 residue appears to be important in
binding to PKC
, rather than in self-association. A critical role for tyrosine 192 is confirmed by a lack of interaction with PKM in yeast
two-hybrid assays (Figs. 1C and 6C). C1(SD)4V
constructs, where serine 183 is mutated to aspartic acid to mimic
phosphorylation, did not self-associate (Fig. 4). Other studies
indicated that a 4L peptide phosphorylated in the same position had low
affinity for PIP2 (27), did not form a dimer in vitro (13),
and did not activate PKC
in the presence of PIP2 (13). The lack of self-association of syndecan 4L peptides may be due to the presence of
the C2 region since C14V constructs (Fig. 4, A and
B) showed association equal to, or higher than, those of 4V,
but 4VC2 peptides had decreased interaction. Deletion of the C-terminal
three amino acids (FYA), which interact with PDZ domain-containing
proteins, allowed association of syndecan 4L constructs, indicating
that this region may be inhibitory for self-association when free. This
raises the possibility that binding of PDZ domain-containing proteins
may regulate syndecan-4 oligomerization.
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Fig. 4.
Self-association between syndecan-4
cytoplasmic baits. As shown in A, yeast two-hybrid
assays with the same bait and prey constructs of syndecan-4 cytoplasmic
tail showed no growth with 4L or C1(SD)4V, slight growth with 4VC2, and
robust growth with 4V, 4V(YF) and 4L-FYA. B,
quantitative assays measuring relative -galactosidase activity of
self-association. Syndecan constructs are as in Fig. 1A.
p53, the tumor suppressor protein plus large T-antigen of SV40 were
used as a positive control, and empty vectors as a negative control.
(n = 3, S.E.; *, p < 0.02, **,
p < 0.005, p value was calculated using
empty vectors as baseline).
from syndecan-4 immunoprecipitated from REF
cells (Fig. 5). Since fully spread REF
cells show little coprecipitation of PKC
unless treated with
phorbol ester (Fig. 6D)
(11), REF cells overexpressing syndecan-4 were used. PKC
(Fig.
5A, arrowheads) remained in the complex following
syndecan-4 immunoprecipitation when washed beads were treated
with GST, GST·PKM1, or GST·PKM4. However, the addition of
GST·PKM5 reduced the amount of PKC
in the complex, and GST·PKM5
itself bound to the complex (arrow). The binding of
GST·PKM5 to the complex was confirmed by immunoblotting the stripped
blot with anti-GST (Fig. 5B). The monoclonal anti-PKC
(Upstate Biotechnology) used in these studies was originally raised against PKM, but the exact epitope was not known. Epitope mapping with
the GST·PKM subdomains shows that the epitope lies within PKM5 (Fig.
5C).
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Fig. 5.
Competition assays with GST·PKM subdomain
fusion proteins. As shown in A, syndecan-4 was
immunoprecipitated with monoclonal antibody 150.9 from cell lysates,
and GST·PKM fusion peptides were incubated with the washed beads. The
amount of bound intact PKC was monitored with monoclonal
anti-PKC
. Arrowheads indicate PKC
, the
arrow indicates PKM5, and * indicates IgG heavy and light
chains. As shown in B, the blot in A was stripped and
reprobed with anti-GST. Only GST·PKM5 was detected, indicating that
none of other GST·PKM subdomains bound to the syndecan-4 complex. As
shown in C, the epitope of monoclonal anti-PKC
(Upstate
Biotechnology) was mapped by immunoblotting. Equal amounts of GST or
GST fusion proteins PKM1, -4, and -5 were loaded in each lane.
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Fig. 6.
Specificity of syndecan-4 interaction with
PKC isoforms. A, PKC immunoblots of cell lysates or
material coprecipitating with syndecan-4 from REF cells and those
expressing S4 or truncated constructs S4
R, S4
E, or S4
I. Bands
marked with * represent IgGs. B, PKC
immunoblots of cell
lysates or material coprecipitating with syndecan-4 from REF and
transfected REF. IgG is not visible since anti-PKC
is a goat
antibody and secondary donkey anti-goat (Santa Cruz Biotechnology) was
used. C, quantitative yeast two-hybrid assays measuring
relative
-galactosidase activity. 4V constructs were used as baits,
and constructs containing the catalytic domains of PKC
(318-676) or
PKC
(406-737) were used as prey. 4V/PKM cotransformant was used as
positive controls. (n = 3, S.E.). As shown in
D, syndecan-4 association with PKC
requires the V region.
Immunoblotting for PKC
in syndecan-4 immunoprecipitates from normal
REF cells and those transfected with syndecan-4 constructs. PKC
that
is associated with syndecan-4 migrates more slowly, indicative of
phosphorylation. * indicates IgGs.
and Localizes This
Isoform to Focal Adhesions--
Three PKC isotypes have been
implicated in focal adhesion formation (28), PKC
, PKC
, and
PKC
. To determine whether isoforms other than PKC
could interact
with syndecan-4, further coimmunoprecipitation and yeast two-hybrid
assays were performed. Unlike PKC
(Fig. 6D) (11), PKC
and PKC
did not coimmunoprecipitate with syndecan-4 (Fig. 6,
A and B), even when syndecan-4 was overexpressed.
Similarly, yeast two-hybrid assays did not indicate interactions of
PKC
and -
with the syndecan-4 cytoplasmic domain (Fig.
6C). In addition, neither PKC
or PKC
were found to be
focal adhesion components (Fig. 7).
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Fig. 7.
Overexpression of syndecan-4, but not S4YF,
specifically increases the PKC isoform in
focal adhesions. Normal REFs show little labeling for PKC
in
focal adhesions of cells grown for 24 h (A), whereas
this is pronounced in S4 cells transfected with wild-type syndecan-4
under a
-actin promoter (B). Vinculin labeling confirms
the presence of focal adhesions in both REF (C) and S4
(D) cells. PKC
labeling (rabbit antibody followed by goat
anti-rabbit fluorescein isothiocyanate) is punctate with no detectable
focal adhesion labeling in REF (E) or S4 cells
(G), as monitored by vinculin labeling (F) and
(H), respectively (mouse anti-vinculin followed by goat
anti-mouse Texas Red). Bar = 10 µm.
(Fig. 6D, compare
REFs and S4). Interestingly, migration of the
PKC
coprecipitating with syndecan-4 was decreased, indicative of
phosphorylation.3 The PKM
region that interacts with syndecan-4 contains two autophosphorylation sites implicated in PKC
activation and stabilization (25, 26). The
association of PKC
with syndecan-4 required the presence of the V
region since PKC
coimmunoprecipitation did not occur in lysates from
cells expressing syndecan-4 with a partial (S4
I) or entire (S4
R)
truncation of the cytoplasmic domain. PKC
/syndecan-4 association did
not, however, require the C-terminal FYA sequence since PKC
was
immunoprecipitated from cells expressing the S4
E constructs. These
data suggest that interactions of PDZ domain-containing proteins with
syndecan-4 are not required for PKC
/syndecan-4 association within cells.
through the syndecan-4 V region, we also determined whether
overexpression of syndecan-4 specifically altered PKC
distribution.
REFs grown for 24 h showed limited focal adhesion staining for
PKC
(Fig. 7A), although vinculin labeling confirmed the
presence of focal adhesions (Fig. 7C). Labeling of S4 cells, which overexpress syndecan-4, showed prominent staining for PKC
in
focal adhesions (Fig. 7B) and increased vinculin labeling
(Fig. 7D). This did not occur in cells expressing truncated
syndecan-4 constructs that lack the V region (data not shown), which
form a reduced number of smaller focal adhesions (9). Labeling for PKC
in REF cells indicated a punctate distribution (Fig.
7E), which was not noticeably different in S4 cells (Fig.
7G), and double labeling confirmed no colocalization of this
PKC isoform with vinculin (Fig. 7, F and H).
Labeling for PKC
was dim and diffuse (not shown), and no PKC
was
detected in focal adhesions.
association, abrogated the
increased PKC
localization to the larger focal adhesions formed in
syndecan-4 overexpressing cells. REF cells overexpressing syndecan-4 in
pcDNA3 vector showed increased focal adhesions and insertion of
PKC
into these adhesions (compare Fig.
8, A and B and
C and D), as seen with constructs under a
-actin promoter. However, cells expressing S4(YF) showed unusual
vinculin labeling of wider areas at cell edges (Fig. 8E)
with no increase in PKC
labeling (Fig. 8F) in focal
adhesions.
View larger version (89K):
[in a new window]
Fig. 8.
Overexpression of syndecan-4 mutant (YF)
fails to target PKC into focal adhesions.
REF transfected with pcDNA3 vector alone shows some labeling for
vinculin (A) and PKC
(B) in focal adhesions of
cells grown for 24 h. Vinculin (C) and PKC
(D) are increased in REFs transfected with wild type
syndecan-4 (pcDNA-S4) but not (E and F) in
REF transfected with pcDNA3-S4 (YF). Vinculin labeling in
transfectants with the YF mutant (E) is abnormal with large areas of
the cell edges being stained (e.g. arrow).
Bar = 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to the V
region of syndecan-4 and superactivation of PKC
by syndecan-4 cytoplasmic domain (11, 12). More recent data (9) demonstrated a role
for this interaction in the control of morphology. The present study 1)
determines the binding site within PKC
; 2) determines the
requirements for self-association and tyrosine 192 of syndecan-4; 3)
determines the specificity for association with PKC
but not other
isoforms; and 4) demonstrates increased localization of PKC
to focal
adhesions in cells overexpressing syndecan-4, but not S4(YF).
in a novel way. The
classical scheme is that diacylglycerol, derived through activation of
phospholipase C, binds the regulatory domain of PKC
, localizing it
to the membrane and inducing autophosphorylation. Although PIP2 appears
to stabilize oligomers of the cytoplasmic domain of syndecan-4
cytoplasmic domain, which in turn allows PKC
binding (15, 29),
syndecan-4 oligomers can directly activate its catalytic site, and the
combination of phospholipid and the 4V region results in a
"superactivated" PKC
that now lacks the requirement for calcium
ions (12). Here we confirm a direct interaction between the catalytic
domain of PKC
(PKM) and syndecan-4 V region. PKC
in its
full-length form, which is less active (25, 26), showed a weak
interaction with the syndecan-4 cytoplasmic domain, as judged by yeast
two-hybrid analysis. However, once the pseudosubstrate or the
regulatory domain was deleted, a stronger activity was detected. The
affinity of interaction may be low since, in the quantitative assays,
the level of interaction activity was only maximally 20% of that
of the positive control (T-antigen and p53). However, this
represented an 8-9-fold increase in activity with PKM or PKM3 as
compared with that of negative control. PKM4 showed minimal
interaction, with most of the activity of PKM3 (containing PKM4 and
PKM5) being retained with PKM5 alone. Further truncation of the PKM5
construct resulted in less reproducible results. Protein interactions
with the PKM region of PKC
are rare, although PICK1 has been found
by yeast two-hybrid analysis to bind the C-terminal 4 amino acids of
PKC
(669QASV672) through the PDZ domain of
PICK1 (30, 31).
, could compete for interactions between the
full-length endogenous PKC
and syndecan-4 and displace PKC
from
syndecan-4 immunoprecipitates. This is similar to the reduction of
coimmunoprecipitated PKC
when syndecan-4 V region peptide was
present (11). Thus, the direct interaction of PKC
with syndecan-4 is
mediated via the C terminus of the catalytic domain of the enzyme to
the unique V region of syndecan-4. Interestingly, this region of PKC
contains autophosphorylation sites, and coimmunoprecipitation results
indicate that syndecan-4-associated PKC
may be phosphorylated.
association (11, 12, 14). The
C2 region of syndecan-4, known to interact with PDZ domain proteins
(32-35), may inhibit syndecan-4 oligomerization in the absence of
interacting partners. The full-length cytoplasmic domain could not
self-associate, but deletion of the FYA sequence or of the entire C2
region resulted in self-association. However, coimmunoprecipitation
experiments indicate that both full-length and
FYA constructs
associate with PKC
in cells. Interactions of syndecan-4 with PDZ
domain-containing partners in cells may, therefore, regulate syndecan-4
self-association and, in turn, PKC
interactions. In addition to the
need for self-association, we have now identified a critical amino acid
in syndecan-4 that is required for interaction with PKC
. Mutation of
tyrosine 192 to phenylalanine in the V region of the cytoplasmic domain
of syndecan-4, 4V(YF), diminished its interaction with PKM but still allowed self-association, suggesting that the central tyrosine residue
is critical for binding to PKC
. Expression of this construct appeared to result in abnormal vinculin distribution, and further analysis of other focal adhesion components is underway. Mutated S4(YF)
did not result in increased PKC
localization to focal adhesions,
unlike wild-type syndecan-4.
is a focal adhesion component along with syndecan-4, and
syndecan-4 may localize the kinase to these structures as they form.
Syndecan-4 appears to bind only PKC
, and yeast two-hybrid analysis
and coimmunoprecipitation experiments strongly suggest that neither
PKC
nor PKC
can interact with this proteoglycan. Interestingly,
whereas PKC
appears to phosphorylate serine 183 in the syndecan-4
cytoplasmic domain (13), thereby reducing multimerization and
superactivation of PKC
(12, 14), these interactions are not
sufficiently stable to be detectable by yeast two-hybrid analysis. The
lack of interaction seen here between PKC
and PKC
with syndecan-4
is consistent with recent biochemical data showing that only PKC
activity, not that of PKC
I, -
, -
, or -
, can be strongly
up-regulated by the combination of syndecan-4 and PIP2 (27). Reciprocal
coimmunoprecipitations (not shown) confirmed association of syndecan-4
with PKC
but not PKC
, and immunofluorescence confirmed specific
increases in PKC
, not in PKC
or -
, in focal adhesions. This is
consistent with a previous report that PKC
localizes to the Golgi
complex and PKC
is perinuclear (36).
(11, 12) or whether this is indirect through PIP2 (13). Our
previous data demonstrated direct binding and activation of PKM (11),
which lacks the regulatory domain that binds PIP2. Our current yeast
two-hybrid assays indicate direct interaction of syndecan-4V constructs
with PKM5. In these assays, the syndecan-4L constructs did not
self-associate or interact with PKC
, indicating that insufficient
PIP2 is supplied by the yeast for oligomerization (11). In contrast
4V-4V interactions can occur without PIP2 (15), and the syndecan-4 V
and C14V constructs showed both self-association and interaction with
PKM5 in yeast two-hybrid assays. Additional in vitro assays
confirmed 4V/PKM interactions in the absence of PIP2. Therefore, we
presume that PIP2 plays a role in oligomerization of syndecan-4
cytoplasmic tail rather than mediating interaction with PKC
. Our
current hypothesis remains that PIP2 binds the regulatory region of
PKC
, as demonstrated previously (37), whereas the catalytic domain interacts with syndecan-4. This results in a discrete ternary complex
at forming focal adhesions, where specific phosphorylation of
cytoskeletal or signaling proteins may occur.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM50194 (to A. W.) and Sankyo Co., Ltd. Additional support was provided by Wellcome Trust Program Grant 065940 (to J. R. C.).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: Dept. of Cell
Biology, University of Alabama at Birmingham, THT 946, 1530 3rd Ave.
S., Birmingham, AL 35294-0006; Tel.: 205-934-1548; Fax: 205-934-7029; E-mail: anwoods@uab.edu.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M208300200
1 The abbreviations used are: PKC, protein kinase C; PKM, protein kinase M; PIP2, phosphatidylinositol 4,5-bisphosphate; 4V, variable sequence of syndecan-4 cytoplasmic domain; REF, rat embryo fibroblast; GST, glutathione S-transferase; S4, syndecan-4.
2 R. L. Longley, S.-T. Lim, J. R. Couchman, and A. Woods, manuscript in preparation.
3 S.-T. Lim, J. R. Couchman, and A. Woods, unpublished observations.
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