(Received for publication, February 14, 1997)
From the Department of Cell Biology and the Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294
The transmembrane proteoglycan syndecan-4, which
is a coreceptor with integrins in cytoskeleton-matrix interactions,
appears to be multimerized in vivo. Both purified and
recombinant core proteins form sodium dodecyl sulfate-resistant
oligomers, and we now report that a synthetic peptide corresponding to
the central region of syndecan-4 cytoplasmic domain (4V) also
oligomerizes. The degree of oligomerization correlates with the
previously reported ability to bind protein kinase C (PKC) and regulate
its activity. Only multimeric recombinant syndecan-4 core protein, but
not the monomeric protein, potentiated the activity of PKC, and only oligomeric syndecan-4 cytoplasmic peptides were active. Changes in
peptide sequence caused parallel loss of stable oligomeric status and
ability to regulate a mixture of PKC
activity. A synthetic
peptide encompassing the whole cytoplasmic domain of syndecan-4 (4L)
containing a membrane-proximal basic sequence did not form higher order
oligomers and could not regulate the activity of PKC
unless
induced to aggregate by phosphatidylinositol 4,5-bisphosphate.
Oligomerization and PKC regulatory activity of the 4V peptide were both
increased by addition of N-terminal cysteine and reduced by
phosphorylation of the cysteine thiol group. Concentration of
syndecan-4 at sites of focal adhesion formation may enhance
multimerization and both localize PKC and potentiate its activity to
induce stable complex formation.
Extracellular matrix molecules such as fibronectin regulate many cellular processes through information encoded in the ligand-receptor interaction. Fibronectin has at least two distinct classes of cell-surface receptors: integrins and heparan sulfate proteoglycans. Integrins bind at several sites, but adhesion for many cell types is primarily through the classical RGD sequence in the tenth type III repeat of the molecule (1, 2). Clustering of specific integrins by either immobilized extracellular molecules or anti-integrin antibodies has many biological effects (reviewed in Refs. 3-6). It stimulates tyrosine phosphorylation (7, 8), elevates intracellular calcium (9), activates the Na+/H+ antiporter (10), and activates phosphatidylinositol 4-phosphate 5-kinase (11) with cytoskeletal rearrangement (3, 12). Ligand-induced dimerization or oligomerization is a key event in transmembrane signaling by hormone or growth factor receptors with tyrosine kinase activity. This leads to an increase in tyrosine kinase activity, autophosphorylation of receptors, and the induction of diverse biological responses (13, 14). Although integrins have no intrinsic tyrosine kinase activity, it is clear that their clustering is needed prior to subsequent tyrosine phosphorylation events (reviewed in Refs. 3-6). In addition, integrin-associated kinase(s) has been identified (15, 16).
Interactions between integrins and the cell-binding domain of
fibronectin are only sufficient for attachment and spreading in normal
primary fibroblasts (17), and the integrin 5
1, which is the
primary ligand for adhesion to fibronectin in this system (1, 3, 5),
remains in a diffusely punctuate distribution in the cell membrane (18,
19). An additional stimulus (17-19) is needed for cytoskeletal and
membrane reorganization to form stress fibers and focal adhesions
(reviewed in Ref. 3), which appear to be not only structural complexes
but also to have signaling functions. This can be provided by further
stimulation with the near C-terminal heparin-binding domain of
fibronectin (Hep II) (17), a synthetic peptide from this domain (18),
or by pharmacological activation of protein kinase C
(PKC)1 with phorbol esters (19). This
causes a redistribution of
5
1 into large clusters in focal
adhesions and a concomitant redistribution of the cytoplasmic
components vinculin and talin (18, 19). Circumstantial evidence has
indicated that a cell-surface heparan sulfate proteoglycan may
transduce the signal from the Hep II domain of fibronectin (reviewed in
Ref. 20). Syndecan-4 is one member of the syndecan family of
transmembrane heparan sulfate proteoglycans (20, 21) that is
selectively concentrated in the focal adhesions of a range of cells
(22) in a PKC-dependent manner (23) and may, therefore,
function as a coreceptor with integrins.
The four mammalian syndecans all have highly homologous transmembrane
and cytoplasmic domains, except for a short variable (V) region in the
center of the cytoplasmic domain (20, 21). The extracellular domains,
however, bear little homology. All syndecans form homologous dimers or
multimers that resist treatment with SDS (20-22, 24, 25). Asundi and
Carey (25) have shown that syndecan-3 core protein assembles into
stable, noncovalent multimers mediated by the transmembrane domain and
ectodomain flanking region and that this is independent of cysteine
residues. Multimerization of syndecan-4 has been seen in biochemical
studies (22, 24-27), and the high concentration of this molecule in
focal adhesions (22) should favor this. Recently, we showed that
syndecan-4 could directly activate PKC in the absence of
phospholipid (PL) and potentiate PL-induced activity (26). This ability
appears to reside in the central portion of the cytoplasmic domain,
which is unique to syndecan-4, based on studies with fusion proteins containing or lacking this region, and with synthetic peptides encompassing this amino acid sequence (26). The recombinant proteins
capable of activating PKC
were oligomeric, consistent with in
vivo data. Preliminary data also suggested that the variable region of syndecan-4 (4V) could also oligomerize. We now report that
the degree of oligomerization correlates with the ability to regulate
PKC
activity, indicating that clustering of syndecan-4 with ligand
may control signaling events.
Synthetic peptides with sequences corresponding
to regions of the cytoplasmic domain of syndecan-4 or syndecan-2
(see Table I) were synthesized and sequenced by the University of
Alabama at Birmingham (UAB) Comprehensive Cancer Center Peptide
Synthesis and Analysis Shared Facility and analyzed by the UAB
Comprehensive Cancer Center Mass Spectrometry Shared Facility. Some
peptides (e.g. Cys4V) had an additional cysteine at the N
terminus. PKC purified from rabbit brain was purchased from
Upstate Biotechnologies (Lake Placid, NY), recombinant PKC
from
Molecular Probes (Eugene, OR), [
-32P]ATP from DuPont
NEN, Sepharose CL-4B and glutathione-agarose beads from Pharmacia
Biotech Inc., and protein standards for size exclusion chromatography
from Bio-Rad. Phosphatidylserine and diolein were purchased from Avanti
Polar Lipids (Alabaster, AL). A peptide encompassing the PKC
phosphorylation site in the epidermal growth factor receptor and P-81
phosphocellulose paper were obtained from Biomol Research (Plymouth
Meeting, PA) and Whatman, respectively. Antibodies against glutathione
S-transferase (GST) were purchased from Molecular Probes,
and reduced glutathione was from Janssen Chimica (New Brunswick, NJ).
Sephadex G-50 (Fine), Sephadex G-150 (Fine),
N-lauroylsarcosine, isopropyl
-D-thiogalactopyranose, PIP2, inositol hexaphosphate,
and other chemicals were all purchased from Sigma.
|
A cDNA encoding the entire syndecan-4 core protein or
lacking the entire cytoplasmic domain was subcloned into GST expression vector pGEX-5X-1 (Pharmacia) and fusion protein in Escherichia coli induced with 0.1 mM isopropyl
-D-thiogalactopyranose (26). Bacteria were pelleted by
centrifugation for 2 min at 10,000 × g. The pellets
were resuspended with TBSE (Tris-buffered saline with EDTA; 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM
EDTA) containing 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine HCl,
sonicated on ice for 1 min, and N-lauroylsarcosine was added
to a final concentration of 1% in TBSE to solubilize recombinant
proteins. After 30 min of incubation on ice, insoluble materials were
removed by centrifugation at 10,000 × g for 10 min.
Supernatants were diluted 10 times with TBSE containing 4% Triton
X-100 and applied to 0.5-ml pre-equilibrated glutathione-agarose columns. After washing with TBSE containing 4% Triton X-100 to remove
unbound materials, the column was washed once with TBSE containing 1%
Triton X-100 and once with TBSE containing 0.1% Triton X-100 to
decrease the detergent concentration. Fusion proteins were eluted with
50 mM Tris-HCl, pH 8.0, containing 5 mM reduced glutathione.
Purified GST-syndecan-4
fusion proteins were loaded onto Sepharose CL-4B or Sephadex G-150
(0.7 × 100 cm) gel filtration columns pre-equilibrated with 50 mM HEPES (pH 7.3), 0.1% CHAPS, and 150 mM
NaCl. Proteins were eluted with the same buffer at a flow rate of 6 ml/h at room temperature and detected by measuring the absorbance at
280 nm. The column was calibrated using the protein standards thyroglobulin (670 kDa), bovine -globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin
B12 (1.3 kDa). Syndecan-4 peptides were loaded onto
Sephadex G-50 gel filtration columns (1.5 × 100 cm, or 0.7 × 100 cm for Fig. 7), and the columns were calibrated with standards
comprising carbonic anhydrase (29.0 kDa), equine myoglobin (17.0 kDa),
lysozyme (14.3 kDa), aprotinine (6.5 kDa), and vitamin B12
(1.3 kDa).
PKC Assays
Experiments were conducted using either
recombinant PKC or PKC
purified from rabbit brain, with
similar results in each case. PKC
or PKC
was incubated in
50 mM HEPES (pH 7.3) at room temperature for 10 min with or
without PL, phosphatidylserine, and diolein and with the different
syndecan peptides. These assays monitored the effect of peptides to
potentiate PL-induced activation of PKC
or to directly activate the
enzyme in the absence of PL. The reaction mixture (20 µl) contained 3 mM magnesium acetate, 750 µM
CaCl2, 200 µM ATP (0.5 µCi of
[32P]ATP), 4 µg of histone III-S, 8 ng of PKC
, 1 µg of peptide, with or without 4 µg of phosphatidylserine and 0.4 µg of diolein. SDS mixture (5×, direct activation) or
trichloroacetic acid (final concentration 20%) with 0.1% SDS
(potentiation) was added to stop the reaction. Phosphorylation was
quantitated on a PhosphorImager (Molecular Dynamics) after
electrophoresis on 20% SDS-PAGE. In assays using 0.1 mg/ml of the
epidermal growth factor receptor peptide of the sequence RKRTLRRL
(Biomol Research) as an alternative substrate (see Fig. 6), the
reaction mixture contained PIP2 (50 µM) and/or syndecan-4
peptide (4L; 0.25 mg/ml) in the absence of PL and calcium, and
PKC
mixture (3 ng) was used. The reaction was stopped by
spotting the whole reaction mixture onto phosphocellulose filters
(Whatman, P-81, 2.1 cm) and dropping these into 75 mM phosphoric acid. The phosphocellulose filters were washed three times
for 10 min each, immersed in 95% ethanol for 5 min, dried, and counted
with 4 ml of scintillation mixture in a scintillation counter (Wallac
model 1409).
Our previous studies showed that recombinant
syndecan-4, in which the core protein formed SDS-resistant oligomers,
could increase the ability of PKC to phosphorylate histone III-S
(26). Therefore, we investigated whether this multimeric form is
critical for its potentiation activity. Gel chromatography of pure
preparations of recombinant syndecan-4 on Sepharose CL-4B under
nondenaturing conditions showed that the major species had a
Mr ~500,000 (Fig. 1A), indicating that the major form of intact
syndecan-4 is oligomeric. We could sometimes also detect minor pools of
Mr ~350,000 and ~230,000 (data not shown).
These oligomeric core proteins migrated on SDS-PAGE gels as a single
population with apparent Mr ~150,000 (Fig.
1B). Since the deduced molecular mass of the fusion protein is 49 kDa, fusion protein resolves on denaturing gels as oligomers, possibly dimers. Reduction had no effect; the core protein of syndecan-4 in any case contained no cysteine. However, a single freeze-thaw cycle dissociated this oligomeric form into monomeric proteins migrating with Mr ~53,000 on Sephadex
G-150 gel chromatography (Fig. 1A) and ~50,000 on SDS-PAGE
gels (Fig. 1B). Although multimeric forms of intact
syndecan-4 core protein could potentiate PKC
activity to
phosphorylate histone III-S (Fig. 1C; compare lanes 1 and 2), monomeric forms of syndecan-4 core protein
did not have this activity (Fig. 1C, compare lanes
1 and 3). Similar potentiation was seen with both
purified PKC
mixture and recombinant PKC
(data not
shown).
PKC
As reported previously, mutants of syndecan-4
with partially or totally truncated cytoplasmic domains could not
increase PKC activity, although they did form oligomers (26). A
peptide from this domain was also active (26). Since oligomerization of
the core protein was needed for PKC regulatory activity, we monitored whether synthetic peptides with sequences from the cytoplasmic domain
could oligomerize and if this correlated with the ability to regulate
PKC activity. The apparent molecular masses of syndecan-4 peptides
encompassing the whole cytoplasmic domain (4L) or just the variable
region unique to syndecan-4 (4V) as estimated by Sephadex G-50 gel
chromatography are larger than predicted (Table I).
Peptide 4L eluted close to putative dimeric size, whereas 4V eluted as
a tetramer. Therefore, with respect to syndecan-4, two sites in the
core protein are involved in oligomerization: the transmembrane and/or
ectodomains as shown for syndecan-3 (25), and the central region of its
cytoplasmic domain. However, the highly basic region of syndecan-4
proximal to the membrane from the V region appears to limit
self-association of 4L compared with 4V in the absence of phospholipid
(see below). In agreement with a need for oligomerization for
PKC
regulatory activity, 4V but not 4L could potentiate
PL-induced activation of PKC
to phosphorylate histones (Fig.
2A) and directly activate PKC
in the
absence of PL (Fig. 2B). Equivalent syndecan-2 peptides, which lack PKC regulatory activity (26), were also examined, since
syndecan-2 is the closest homologue to syndecan-4 but lacks the
required cytoplasmic sequence for PKC regulation (20, 21, 26). Both gel
chromatography and SDS-PAGE indicated a lack of apparent
oligomerization of syndecan-2 peptide (Table I), confirming the unique
ability of syndecan-4 cytoplasmic domain to self-associate. GST fusion
proteins containing the whole syndecan-2 core protein, however, do
migrate with similar properties to syndecan-4 on SDS-PAGE gels,
confirming the general ability of syndecan core proteins to
self-associate (data not shown). Taken together, the results show that
although transmembrane regions of syndecans promote SDS-resistant
oligomer formation (putative dimers), further oligomerization to form
tetramers or higher oligomers, as seen with 4V peptides and whole
syndecan-4 fusion proteins, is required for PKC
(or PKC
)
activation.
We tested whether the addition of cysteine to the N terminus of 4V and
4L peptides could induce higher order clustering and concomitantly
increase PKC or PKC
regulatory activity. Cys4V eluted at
8.7 kDa (Table I), indicative of an octamer, even though electrospray
ionization mass spectrometry showed the molecular mass as 1.1 kDa (data
not shown).2 Thus, the N-terminal cysteine
on Cys4V may aid V region multimerization. This had no statistically
significant effect on the ability of 4V to potentiate PL-induced
PKC
activity (Fig. 2A), but direct activation of
PKC
by Cys4V (i.e. in the absence of PL) was greater than that seen with 4V (Fig. 2B). In the presence of
PL, both 4V and Cys4V could potentiate the normally maximal activity by
4-fold. In the absence of PL, when PKC
is normally inactive, 4V and Cys4V caused 1.7- and 3.0-fold direct activation. In contrast, Cys4L, having cysteine at the N terminus of 4L, still eluted at 7.4 kDa
as a dimer (Table I) and was no more active in regulating PKC
activity than 4L (Fig. 2). Calcium concentrations can affect protein-protein interactions, and phosphorylation experiments were
performed in 750 µM calcium to maximize PKC
activity (28). However, gel filtration at this calcium concentration
did not affect the multimeric status of the peptides (Table I). These multimeric peptides were resistant to SDS, although the apparent Mr on SDS-PAGE was lower than that seen by gel
filtration, being only 1.5- to 3-fold that deduced from the amino acid
sequence (Table I). For example, both 4V and Cys4V migrated with an
apparent Mr ~3000. Both methods of estimation
depend on the secondary structure of the molecules tested, with gel
filtration being less denaturing. Further control experiments with 2V
and 2L peptides showed that N-terminal cysteine residues did not induce
their oligomerization (Table I).
The results so far indicate that 1) syndecan-4 core protein
multimerization correlates with PKC potentiation; 2) the V region of
the cytoplasmic domain of syndecan-4 can itself form homopolymers as
does recombinant core protein; 3) multimerization is independent of
calcium concentration; 4) dimerization is not sufficient for PKC regulatory activity since 4L is inactive; and 5)
tetrameric 4V is sufficient for potentiation of PL-induced PKC
activity, but direct activation of PKC
is more effective with
octameric forms seen with Cys4V.
When incubated with PKC and
[32P]ATP in the absence of PL, Cys4V, but not 4V, was
itself phosphorylated even though it lacks serine or threonine amino
acids (Table I, Fig. 3A). Although the
phosphorylation of peptide Cys4V was 10-fold less than that of histone
III-S, it was time-dependent (Fig. 3A). Since 4V
was not phosphorylated, Cys4V phosphorylation may be on cysteine thiol groups (29). To investigate whether this phosphorylation affected the
direct activation of PKC
by Cys4V, peptides Cys4V, 4V, or 4L
were "cold-phosphorylated" by preincubation with PKC
and
ATP in the absence of PL (Fig. 3B, lanes 4-6).
Their capacities to subsequently activate PKC
were compared
with those of peptides similarly incubated with PKC
but
without ATP (Fig. 3B, lanes 1-3). Minimal
histone phosphorylation by PKC
was seen in the presence of 4L
(lane 1), and this was slightly reduced following incubation
with cold ATP and PKC
(lane 4). Peptide 4V promoted histone phosphorylation to a similar extent with (lane 5) or
without (lane 2) pretreatment. In contrast, the ability of
Cys4V to promote histone phosphorylation (lane 3) was
markedly reduced (lane 6) by preincubation with ATP and
PKC
. Thus, preincubation of Cys4V with cold ATP and
PKC
caused a loss of ability to activate PKC
. To
determine whether phosphorylation of Cys4V altered its oligomeric
structure, we incubated Cys4V with PKC
± 200 µM
cold ATP for 10 min at 37 °C and analyzed the peptide by Sephadex G-50 gel filtration chromatography (Fig. 4). Cys4V and
4V maintained their original elution profiles (8.7 and 4.6 kDa,
respectively) in the absence of enzyme, but elution of phosphorylated
Cys4V was markedly altered, with a minor population eluting at 8.7 kDa as before, but most at 3.9 kDa. Thus, phosphorylation appeared to cause
a reduction in the multimeric nature of Cys4V, paralleling a loss in
its ability to directly activate PKC
. A further control experiment was performed to analyze whether cysteine thiol group phosphorylation itself was inhibitory independent of peptide
multimerization. To do this, peptides based on syndecan 4V sequence
(CLGKPIYK or CLGKKPIKK, where
two lysine residues were substituted with arginine or the tyrosine
residue was substituted with phenylalanine) were used. These were
inactive in regulating PKC activity (26) but could be readily
phosphorylated. These peptides, when phosphorylated as described above,
had no effect on the subsequent ability of "wild type" Cys4V to
activate PKC
(data not shown). Therefore, reducing the
oligomerization status of Cys4V peptide was directly related to
decreased PKC
regulation.
Aggregation of Syndecan-4 Cytoplasmic Domain by PIP2 Results in PKC
As shown before, the sequence
KPIYK is critical in the up-regulation of PKC activity (26). This
sequence is present in both syndecan 4V and 4L, but 4L activated
PKC much less than 4V. Although this appears to parallel
multimerization status, it was also possible that the highly basic
membrane-proximal region of syndecan-4 cytoplasmic domain (Table I)
could directly inhibit the effects of 4V. This was ruled out by
addition of five times excess of 4L or of 2L, the latter having no
ability to regulate PKC
but possessing a highly homologous
membrane-proximal sequence. These had no effect on potentiation of
PL-induced PKC
activity by 4V (Fig.
5A) or on direct activation of PKC
by Cys4V (Fig. 5B). It was, therefore, most likely that it
was the lack of multimerization of 4L, rather than the presence of
membrane-proximal highly basic amino acids, that resulted in its
reduced activity. This was confirmed by increasing the oligomerization
of 4L by the addition of PIP2. After preincubating 4L peptide with PIP2
(1:1 molar ratio), 4L molecular weight was analyzed by Sephadex G-50
gel filtration. Approximately 30% of the 4L peptide eluted with higher
apparent molecular weight (Mr ~35,000), which
is approximately eight times greater than the deduced molecular weight
of 4L (3300) and PIP2 (1100) combined (Fig.
6A). No phosphorylation was seen in the absence of PIP2 and 4L (Fig. 6B, lane 1). Peptide
4L alone had no effect (lane 2), whereas PIP2 partially
activated PKC
(lane 3) as previously shown (30,
31). The PIP2-syndecan 4L complex resulted in a marked potentiation of
PKC
phosphorylation of epidermal growth factor receptor
peptide (Fig. 6B, lane 4) and histone III-S (data
not shown). The specificity of this interaction between 4L and PIP2
with respect to oligomerization and PKC
regulation was
demonstrated in several assays. First, the addition of PIP2 to
syndecan-2 cytoplasmic peptides was without effect on PKC
activity. Second, inositol hexaphosphate was unable to affect 4L
oligomerization (Fig. 6A) or PKC
activity (Fig. 6B), even though, like PIP2, inositol hexaphosphate can
interact with the 4L peptide.3 Third, both
phosphatidylinositol 3,4,5-triphosphate and inositol 1,3,4,5-tetraphosphate were also unable to replicate the
oligomerization or PKC
regulatory activity (data not
shown).
The Amino Acid Sequence KPIYK Is Needed for Multimerization
We have shown that the amino acid sequence KPIYK
is critical for both direct activation of PKC and for
potentiation of PL-induced activation (26), since peptides of altered
sequence showed reduced activity. Therefore, we investigated whether
this was due solely to the specific amino acid sequence or whether activity paralleled the amount of multimerization of the altered peptides. As shown in Fig. 7, although Cys4V eluted as a
putative octamer on Sephadex G-50 gel filtration, all the other
peptides (sequences shown in the legend to Fig. 7) eluted with a more
broad profile, indicative of a range of oligomerization states.
Although some substituted peptides had a little potentially octameric
content, most eluted with smaller mass. CysIS, in which the isoleucine was replaced by serine, eluted mainly as a tetramer, with some monomer.
CysPA, with proline replaced by alanine, eluted as a mixture of tetra-,
di-, and monomer, with 30% being monomeric. CysKR, in which two lysine
residues were replaced by arginine, was insoluble at the high
concentration needed for these experiments. CysYF, with tyrosine
replaced by phenylalanine, eluted as three peaks, the largest material
being of similar size to that of Cys4V (octameric). In parallel with
this, CysIS and CysPA showed very limited ability in our previous
studies (data not shown, but see Ref. 26) to directly activate
PKC
to phosphorylate histones or to potentiate PL-induced
activation. In contrast, CysYF showed more, although somewhat variable,
activity, but it was never as active as Cys4V or 4V. Since 4V is more
active than those peptides that also form tetramers (CysIS and CysPA)
and even CysYF, which forms some octamers, both multimerization state
and specific amino acid sequence seem to regulate PKC
activity.
It is a common characteristic of syndecans that their core proteins form homologous dimers or multimers. Isolated core proteins migrate on SDS-PAGE with apparent molecular masses significantly higher than those deduced from cDNA sequence analysis (21, 27). This occurs even with fusion proteins isolated from bacteria in which glycosylation can have no effect (24-26) and with fusion proteins lacking the cytoplasmic domain (25, 26). We confirm here that GST fusion proteins containing the core protein of syndecan-4 formed stable oligomers when analyzed by both gel chromatography and SDS-PAGE. Gel chromatography indicated that syndecan-4 core protein can form higher order oligomers (tetramers and perhaps octamers) under nondenaturing conditions. These forms of syndecan-4 had potent PKC up-regulatory activity (Ref. 26 and this study). Oligomerization did not involve cystine formation, since syndecan-4 lacks cysteine residues, and oligomers could be dissociated by a single freeze-thaw cycle, demonstrating that the interactions were noncovalent. Dissociation into monomeric forms correlated with the loss of ability of syndecan-4 fusion proteins to regulate PL-induced PKC activity. This supports previous data (25) that the cytoplasmic domain of syndecans is not necessary for self-association. These fusion proteins still contained the full ectodomain of syndecan-4, but the lysine critical for self-association of syndecan-3 is not present in syndecan-4 (21, 25, 31-33), suggesting that other interaction sites must exist in the ectodomain of syndecan-4. It is improbable that the GST portion of the syndecan-4 fusion protein was responsible for SDS-resistant self-association, since this is not a common occurrence in this type of fusion protein and other studies indicate that oligomerization is a common property of syndecans (21-27).
A major finding here was that, in addition to sites in the
transmembrane and/or ectodomain promoting oligomerization, the V region
of syndecan-4 cytoplasmic domain, but not of syndecan-2, could itself
oligomerize. Peptides corresponding to this region migrated on SDS-PAGE
gels with increased apparent Mr, but the full
extent of oligomerization was revealed by gel filtration, where
oligomeric status up to putative octamers was seen, particularly when
oligomerization was enhanced by addition of N-terminal cysteine. Direct
correlations between oligomerization and PKC regulatory ability were noted. Peptide Cys4V, which formed putative octamers by
gel filtration, was more active than 4V, which formed tetramers. Peptide 4V in turn was more active than 4L, which, in the absence of
PIP2, formed only dimers. In addition, when multimerization of Cys4V
was reduced by phosphorylation, its ability to regulate PKC
activity decreased. Peptides encompassing the whole cytoplasmic domain
showed less tendency to oligomerize than those containing just the V
region, probably due to the high charge of the C1 region normally
proximal to the membrane. However, the reduced tendency of the 4L
peptide to oligomerize was partly overcome by PIP2 in vitro,
since PIP2 induced higher oligomeric structures. This also increased
the ability of the 4L peptide to potentiate PKC activity. We believe
that the interaction of PIP2 with syndecan-4 may be of biological
significance, and this is under investigation in our laboratory. Of
particular relevance may be that PIP2 could partially activate PKC, and
this was further enhanced by 4V or 4L peptides. Thus, as seen
previously in vivo during cell adhesion, accumulated PIP2
resulting from PIP kinase activity induced on integrin-mediated
adhesion (reviewed in Refs. 3 and 6) may increase or stabilize the
oligomerization status of syndecan-4 cytoplasmic domain and induce the
potentiation of PKC activity. Regulation of other focal adhesion
components, such as vinculin and
-actinin, by PIP2 has also been
shown (3, 34-36), and we hypothesize that PIP2 may regulate syndecan-4
and PKC
, two additional focal adhesion components.
Biological activity of the syndecan-4 cytoplasmic domain is not due solely to oligomeric status, since peptides of altered sequence, even when tetrameric or partially octameric (e.g. CLGKKPIKK, where a single tyrosine was replaced by a phenylalanine residue), were much less active than the corresponding oligomeric forms of the native peptides. Preliminary data4 using circular dichroism spectroscopy also indicate that although the 4V peptide has secondary structure, this is partially or totally lost when residue substitutions are made or the peptide sequence is scrambled. Further long term studies with nuclear magnetic resonance spectroscopy are underway to examine peptide-peptide and peptide-PIP2 interactions.
Dimerization or oligomerization of membrane receptors often activate
signaling cascades with phosphorylated tyrosines binding SH2
domain-containing adapter proteins (3-6). Receptors with intrinsic
tyrosine kinase activity undergo transphosphorylation, whereas cytokine
receptors, which lack intrinsic kinase activity, can recruit and/or
activate kinases following oligomerization in response to ligand
binding (37). Indeed, integrin 1 can have associated kinases (15,
16). PKC activation is required for cell adhesion (38-40), migration
(41), and particularly for focal adhesion formation (19), and PKC
is
concentrated at the focal adhesions of normal but not transformed cells
(42), where syndecan-4 is also highly concentrated (22). Syndecan-4,
but not syndecan-2, V region can bind PKC and either directly activate it or potentiate conventional PL-induced activation (26), but, as shown
here, it can do so only when in oligomeric form. Our working model is
that syndecan-4, like other syndecans, is expressed on the cell surface
in dimeric form. Upon ligand binding, e.g. fibronectin's
heparin-binding (Hep II) domain (17, 18), further oligomerization takes
place. Indeed, focal adhesions have the appearance of cell surface
"patches" with clustering of transmembrane and actin-associated
cytoskeletal components (12). This oligomerization, perhaps facilitated
by PIP2 binding, the presence of which is increased on adhesion (11),
then localizes PKC
and activates the kinase in nascent adhesions.
What remains to be established is whether PKC interactions in the
presence of PIP2 require it to be preactivated or whether the
involvement of PIP2 is sufficient to form a ternary complex. The
requirement of PIP2 for focal adhesion formation, however, is clear
(35).
Syndecan-4 shares some characteristics with other enzyme-binding proteins but also has some differences. It is similar to proteins that interact with C kinase and receptors for activated C kinase in that it may localize specific enzymes to cellular target areas (43, 44). However, it appears to bind the catalytic domain of PKC and can enhance the activity of the isolated catalytic domain, PKM (26). In addition, PKC-syndecan-4 interactions are dependent on enzyme activation (26). One major difference from previously reported enzyme-binding proteins is that syndecan-4 is a transmembrane proteoglycan involved as a coreceptor with integrins in cell adhesion, the multimerization status of which, and therefore the biological activity of which, may be directly determined by interactions with extracellular ligands.
We thank Dr. J. T. Gallagher and his colleagues (University of Manchester, United Kingdom) for the provision of cDNAs encoding syndecan-4 core proteins. We also thank Marion Kirk at the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility for the mass spectrometry analysis of peptides.