Direct Binding of Syndecan-4 Cytoplasmic Domain to the Catalytic Domain of Protein Kinase Calpha (PKCalpha ) Increases Focal Adhesion Localization of PKCalpha *

Ssang-Taek LimDagger , Robert L. Longley§, John R. Couchman, and Anne WoodsDagger ||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Calpha (PKCalpha ) activity. Syndecan 4V peptide directly potentiates PKCalpha 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 PKCalpha . Full-length PKCalpha weakly interacted with 4V by yeast two-hybrid assays, but PKCalpha 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 PKCalpha , 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 PKCalpha (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 PKCalpha appears unique since PKCdelta and epsilon  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 PKCalpha localization to focal adhesions. The data support a mechanism where syndecan-4 binds PKCalpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 5beta 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).

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 PKCalpha in vitro and partially activates it (11). The 4V peptide also binds phosphatidylinositol 4,5-bisphosphate (PIP2), which also partially activates PKCalpha . The combination of 4V and PIP2 results in a superactivation of PKCalpha 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 PKCalpha (12, 13).

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 PKCalpha (11). It can superactivate both PKCalpha and PKM (the free catalytic domain derived after calpain cleavage), and both PKCalpha 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. PKCalpha , 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/PKCalpha complex may form during adhesion. To understand the molecular basis for the pathway of PKCalpha 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 PKCalpha isoform and to identify 1) the site in the PKCalpha 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 PKCalpha with syndecan-4 and increased PKCalpha localization in focal adhesions. This is dependent on the V region of syndecan-4 and specifically requires tyrosine 192.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid DNAs-- Standard PCR techniques (23) were used to generate syndecan-4 cytoplasmic domain constructs (see Fig. 1A) and deletion mutants of PKCalpha (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 PKCalpha , -delta , and -epsilon cDNA were obtained from ATCC. For yeast two-hybrid constructs, all PKCalpha 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.

The cDNA sequences encoding various fragments of syndecan-4 cytoplasmic domain and human PKCalpha for yeast two-hybrid assays were generated by PCR. Tables I and II show the syndecan-4 and PKCalpha 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 PKCepsilon ended with XhoI sites.


                              
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Table I
Primers used for syndecan-4 yeast two-hybrid constructs shown in Fig. 1
Forward (F) and reverse (R) primers are shown containing EcoRI (underlined) and XhoI (italic) sites (bold indicates stop codon).


                              
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Table II
PKC primers used to generate constructs for yeast two-hybrid assays
Italics, underlined and bold represent XhoI, EcoRI, and stop codon as in Table I. PKCdelta and varepsilon  constructs only contained their catalytic domains, and the amino acid sequences are numbered in the legend of Fig. 6.

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.


                              
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Table III
Primers used to generate PKCalpha and its deletion constructs for S2 cell expression
KpnI sites are underlined. Primers not described here are listed in Table II.

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 (MATalpha , 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 beta -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).

In Vitro Binding Assays-- His6- and Xpress epitope-tagged full-length PKCalpha , its regulatory domain, and PKM were expressed in S2 Drosophila cells (Invitrogen). For small PKCalpha 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.

Immunoprecipitation-- Rat embryo fibroblasts (REFs), REFs overexpressing wild type syndecan-4 (S4), or truncated at arginine 175 (S4Delta R), isoleucine 191 (S4Delta I), or glutamic acid 198 (S4Delta E) were subjected to immunoprecipitation as previously (11). These constructs were cloned into LK444 vector under the control of a beta -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-PKCalpha (Upstate Biotechnology), rabbit anti-PKCdelta , or goat anti-PKCepsilon (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.

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-PKCalpha , rabbit polyclonal anti-PKCdelta , goat polyclonal anti-PKCepsilon , 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

PKCalpha Directly Interacts with Syndecan-4-- Yeast two-hybrid analysis confirmed a direct interaction between the V region of syndecan-4 cytoplasmic domain and PKCalpha . The syndecan-4 constructs used for these and self-association assays are shown in Fig. 1A, and the structure of PKCalpha 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 PKCalpha construct (Fig. 1C). This is consistent with previous biochemical data indicating a lack of PKCalpha 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 PKCalpha but interacted less strongly with full-length PKCalpha . These results are consistent with a need for prior activation of PKCalpha 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 PKCalpha regulatory domain (Fig. 1C). However, a clear interaction was detected between syndecan-4V and PKCalpha catalytic domain (PKM). Colony growth assays were complemented by quantitative beta -galactosidase assays (Fig. 1D), which confirmed these interactions.


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Fig. 1.   Interaction between syndecan-4 cytoplasmic domain and PKCalpha constructs. A, syndecan bait constructs used in yeast two-hybrid analyses of PKCalpha 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 PKCalpha . 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. PKCalpha indicates full-length PKCalpha . -PS, construct where the inhibitory N-terminal (pseudosubstrate) sequence has been deleted; Reg, regulatory domain of PKCalpha ; RegC1, the first constant region of PKCalpha ; RegC2, the second constant region of PKCalpha ; PKM, catalytic domain of PKCalpha which is a freely active enzyme in the absence of phospholipid or Ca2+. Each construct is numbered according to the amino acid sequence of PKCalpha . D, quantitative interaction assay monitoring relative beta -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 PKCalpha . B, quantitative interaction assay monitoring relative beta -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).

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 beta -galactosidase assays (Fig. 2B). The PKM5-binding site for syndecan-4 (amino acids 513-672) contains an important module for activation of PKCalpha , two autophosphorylation sites at threonine 638 and serine 657 (25, 26).

To confirm the interactions between syndecan-4 cytoplasmic domain and PKCalpha detected in yeast two-hybrid assays, recombinant PKCalpha from Drosophila S2 cells and GST·PKM subdomains from bacteria were subjected to pull-down assays with syndecan-4V-conjugated beads. Full-length PKCalpha 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 PKCalpha , Reg, or PKM to beads blocked with cysteine as a negative control (not shown). Previous studies showed that PKCalpha does not bind to beads conjugated with the syndecan-2V region (11). A higher proportion of input PKM than PKCalpha 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 PKCalpha 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.

Syndecan-4 Oligomerization Is Needed for PKCalpha 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 PKCalpha 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 PKCalpha 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 PKCalpha , 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 PKCalpha 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 beta -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).

To confirm the results from yeast two-hybrid assays and affinity chromatography, subdomains of PKM were tested for the ability to displace intact PKCalpha from syndecan-4 immunoprecipitated from REF cells (Fig. 5). Since fully spread REF cells show little coprecipitation of PKCalpha unless treated with phorbol ester (Fig. 6D) (11), REF cells overexpressing syndecan-4 were used. PKCalpha (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 PKCalpha 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-PKCalpha (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 PKCalpha was monitored with monoclonal anti-PKCalpha . Arrowheads indicate PKCalpha , 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-PKCalpha (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, PKCdelta immunoblots of cell lysates or material coprecipitating with syndecan-4 from REF cells and those expressing S4 or truncated constructs S4Delta R, S4Delta E, or S4Delta I. Bands marked with * represent IgGs. B, PKCepsilon immunoblots of cell lysates or material coprecipitating with syndecan-4 from REF and transfected REF. IgG is not visible since anti-PKCepsilon is a goat antibody and secondary donkey anti-goat (Santa Cruz Biotechnology) was used. C, quantitative yeast two-hybrid assays measuring relative beta -galactosidase activity. 4V constructs were used as baits, and constructs containing the catalytic domains of PKCdelta (318-676) or PKCepsilon (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 PKCalpha requires the V region. Immunoblotting for PKCalpha in syndecan-4 immunoprecipitates from normal REF cells and those transfected with syndecan-4 constructs. PKCalpha that is associated with syndecan-4 migrates more slowly, indicative of phosphorylation. * indicates IgGs.

Syndecan-4 V Region Specifically Binds PKCalpha and Localizes This Isoform to Focal Adhesions-- Three PKC isotypes have been implicated in focal adhesion formation (28), PKCalpha , PKCdelta , and PKCepsilon . To determine whether isoforms other than PKCalpha could interact with syndecan-4, further coimmunoprecipitation and yeast two-hybrid assays were performed. Unlike PKCalpha (Fig. 6D) (11), PKCdelta and PKCepsilon 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 PKCdelta and -epsilon with the syndecan-4 cytoplasmic domain (Fig. 6C). In addition, neither PKCdelta or PKCepsilon 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 PKCalpha isoform in focal adhesions. Normal REFs show little labeling for PKCalpha 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 beta -actin promoter (B). Vinculin labeling confirms the presence of focal adhesions in both REF (C) and S4 (D) cells. PKCdelta 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.

Increased syndecan-4 expression in S4 REF cells correlates with increased association with PKCalpha (Fig. 6D, compare REFs and S4). Interestingly, migration of the PKCalpha coprecipitating with syndecan-4 was decreased, indicative of phosphorylation.3 The PKM region that interacts with syndecan-4 contains two autophosphorylation sites implicated in PKCalpha activation and stabilization (25, 26). The association of PKCalpha with syndecan-4 required the presence of the V region since PKCalpha coimmunoprecipitation did not occur in lysates from cells expressing syndecan-4 with a partial (S4Delta I) or entire (S4Delta R) truncation of the cytoplasmic domain. PKCalpha /syndecan-4 association did not, however, require the C-terminal FYA sequence since PKCalpha was immunoprecipitated from cells expressing the S4Delta E constructs. These data suggest that interactions of PDZ domain-containing proteins with syndecan-4 are not required for PKCalpha /syndecan-4 association within cells.

Since coimmunoprecipitation experiments indicated specific association with PKCalpha through the syndecan-4 V region, we also determined whether overexpression of syndecan-4 specifically altered PKCalpha distribution. REFs grown for 24 h showed limited focal adhesion staining for PKCalpha (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 PKCalpha 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 PKCdelta 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 PKCepsilon was dim and diffuse (not shown), and no PKCepsilon was detected in focal adhesions.

A mutational approach was used to address whether the tyrosine 192 mutation to phenylalanine in syndecan-4, which still allowed self-association but reduced PKCalpha association, abrogated the increased PKCalpha 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 PKCalpha into these adhesions (compare Fig. 8, A and B and C and D), as seen with constructs under a beta -actin promoter. However, cells expressing S4(YF) showed unusual vinculin labeling of wider areas at cell edges (Fig. 8E) with no increase in PKCalpha labeling (Fig. 8F) in focal adhesions.


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Fig. 8.   Overexpression of syndecan-4 mutant (YF) fails to target PKCalpha into focal adhesions. REF transfected with pcDNA3 vector alone shows some labeling for vinculin (A) and PKCalpha (B) in focal adhesions of cells grown for 24 h. Vinculin (C) and PKCalpha (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

Previous studies indicated direct binding of PKCalpha to the V region of syndecan-4 and superactivation of PKCalpha 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 PKCalpha ; 2) determines the requirements for self-association and tyrosine 192 of syndecan-4; 3) determines the specificity for association with PKCalpha but not other isoforms; and 4) demonstrates increased localization of PKCalpha to focal adhesions in cells overexpressing syndecan-4, but not S4(YF).

Syndecan-4 at the cell surface may activate PKCalpha in a novel way. The classical scheme is that diacylglycerol, derived through activation of phospholipase C, binds the regulatory domain of PKCalpha , 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 PKCalpha 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" PKCalpha that now lacks the requirement for calcium ions (12). Here we confirm a direct interaction between the catalytic domain of PKCalpha (PKM) and syndecan-4 V region. PKCalpha 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 PKCalpha are rare, although PICK1 has been found by yeast two-hybrid analysis to bind the C-terminal 4 amino acids of PKCalpha (669QASV672) through the PDZ domain of PICK1 (30, 31).

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 PKCalpha , could compete for interactions between the full-length endogenous PKCalpha and syndecan-4 and displace PKCalpha from syndecan-4 immunoprecipitates. This is similar to the reduction of coimmunoprecipitated PKCalpha when syndecan-4 V region peptide was present (11). Thus, the direct interaction of PKCalpha 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 PKCalpha contains autophosphorylation sites, and coimmunoprecipitation results indicate that syndecan-4-associated PKCalpha may be phosphorylated.

Previous studies have shown that self-association of syndecan-4 cytoplasmic domain is needed for PKCalpha 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 PKCalpha in cells. Interactions of syndecan-4 with PDZ domain-containing partners in cells may, therefore, regulate syndecan-4 self-association and, in turn, PKCalpha 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 PKCalpha . 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 PKCalpha . 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 PKCalpha localization to focal adhesions, unlike wild-type syndecan-4.

PKCalpha 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 PKCalpha , and yeast two-hybrid analysis and coimmunoprecipitation experiments strongly suggest that neither PKCdelta nor PKCepsilon can interact with this proteoglycan. Interestingly, whereas PKCdelta appears to phosphorylate serine 183 in the syndecan-4 cytoplasmic domain (13), thereby reducing multimerization and superactivation of PKCalpha (12, 14), these interactions are not sufficiently stable to be detectable by yeast two-hybrid analysis. The lack of interaction seen here between PKCdelta and PKCepsilon with syndecan-4 is consistent with recent biochemical data showing that only PKCalpha activity, not that of PKCbeta I, -delta , -gamma , or -epsilon , can be strongly up-regulated by the combination of syndecan-4 and PIP2 (27). Reciprocal coimmunoprecipitations (not shown) confirmed association of syndecan-4 with PKCalpha but not PKCdelta , and immunofluorescence confirmed specific increases in PKCalpha , not in PKCdelta or -epsilon , in focal adhesions. This is consistent with a previous report that PKCdelta localizes to the Golgi complex and PKCepsilon is perinuclear (36).

There has been controversy concerning whether syndecan-4 binds directly to PKCalpha (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 PKCalpha , 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 PKCalpha . Our current hypothesis remains that PIP2 binds the regulatory region of PKCalpha , 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.

    FOOTNOTES

* 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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RESULTS
DISCUSSION
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