1 Department of Cell Biology, Emory University School of Medicine, Room 100, 1648 Pierce Drive, Atlanta, GA 30322, USA
2 Department of Microbiology, Mount Sinai School of Medicine, Box 1124, One Gustave L. Levy Place, New York, NY 10029, USA
* These authors contributed equally to this work
Author for correspondence (e-mail: barry{at}cellbio.emory.edu)
Accepted March 29, 2001
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SUMMARY |
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The yeast two-hybrid screen identified two distinct clones (1.12 and 2.52) that showed identity to portions of SSeCKS (Src Suppressed C Kinase Substrate). SSeCKS is a previously defined kinase and cytoskeleton scaffolding protein whose subcellular distribution and functions are remarkably similar to those attributed to GalT. Both SSeCKS and GalT have been localized to the perinuclear/Golgi region as well as to filopodia/lamellipodia. SSeCKS and GalT have been implicated in regulating cell growth, actin filament dynamics, and cell spreading. Interestingly, 1.12 and 2.52-GFP constructs were localized to subcellular domains that correlated with the two purported subcellular distributions for GalT; 2.52 being confined to the Golgi, whereas 1.12 localized primarily to filopodia. Coimmunoprecipitation assays demonstrate stable binding between the GalT cytoplasmic domain and the 1.12 and 2.52 domains of SSeCKS in appropriately transfected cells. Similar assays demonstrate binding between the endogenous GalT and SSeCKS proteins also. Coimmunoprecipitation assays were performed in both directions and produced similar results (i.e. using either anti-GalT domain or anti-SSeCKS domain antibodies as the precipitating reagent). A functional interaction between the GalT cytoplasmic domain and SSeCKS was illustrated by the ability of either the 1.12 or 2.52 SSeCKS domain to restore a normal adhesive phenotype in cells overexpressing the TL-GFP dominant negative construct. TL-GFP is composed of the GalT cytoplasmic and transmembrane domains fused to GFP, and leads to a loss of cell adhesion on laminin by displacing the endogenous GalT from its cytoskeleton binding sites. This is the first reported interaction between a glycosyltransferase and a scaffolding protein, and suggests that SSeCKS serve to integrate the various functions ascribed to the GalT cytoplasmic domain.
Key words: Galactosyltransferase, SSeCKS, Cell surface, AKAP
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INTRODUCTION |
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GalT is one of at least seven polypeptides that synthesize ß1,4-galactosyl linkages (Amado et al., 1999). GalT is a type II membrane glycoprotein and, similar to other glycosyltransferases, resides in the Golgi complex where, in the presence of the sugar donor UDP-galactose, it is responsible for the galactosylation of complex glycoconjugates terminating in N-acetylglucosamine. However, GalT is unusual in that a portion of the cellular pool is expressed on the surface of specific cell types, possibly owing to the presence of specific cytoplasmic domain sequences, but this has not yet been resolved (Shur et al., 1998). In any event, GalT is not unique in having multiple subcellular distributions, since recent immunolocalization studies have documented the presence of an N-acetylgalactosaminyl transferase, a sialyltransferase and a fucosyltransferase on various cell surfaces (Mandel et al., 1999; Close and Colley, 1998; Borsig et al., 1996).
On the plasma membrane, GalT functions as a lectin-like matrix receptor owing, in part, to the absence of UDP-galactose, which enables low affinity, stable binding between GalT and its extracellular glycoside ligand (Begovac et al., 1991). Cell adhesion to basal lamina matrices results in an increase in GalT on the leading edge of the cell, where it facilitates cell spreading and migration through an association with the cytoskeleton (Eckstein and Shur, 1989; Eckstein and Shur, 1992). GalT is also expressed on the sperm surface (Lopez et al., 1985), where it is responsible for the recognition and binding to egg coat glycosides that stimulate induction of the acrosome reaction (Miller et al., 1992; Lu and Shur, 1997).
Ligand-induced aggregation of GalT results in a range of cell-type specific intracellular signal cascades. The binding of ZP3 glycosides to sperm GalT causes activation of a heterotrimeric G-protein cascade (Gong et al., 1995) whereas, on fibroblasts, clustering of surface GalT with multivalent ligands results in the subsequent tyrosine phosphorylation of focal adhesion kinase (FAK) and disorganization of actin stress fibers (Wassler and Shur, 2000). The ability of GalT to function as a signal transducing receptor has most recently been demonstrated by the ectopic expression of murine GalT on the surface of Xenopus oocytes. GalT-expressing oocytes selectively bind ZP3, but not an irrelevant egg coat glycoprotein. ZP3 binding to GalT-expressing eggs leads to G-protein activation, cortical granule exocytosis and fertilization envelope elevation, all of which can be inhibited by pertussis toxin (Shi et al., 2001). The cytosolic effectors of GalT-dependent signaling remain unknown in these systems, but require association with the GalT cytoplasmic domain (Gong et al., 1995; Shur et al., 1998; Wassler and Shur, 2000, Shi et al., 2001).
The yeast two-hybrid system was used in this study to identify proteins that interact with the cytoplasmic domain of GalT. Two clones, 1.12 and 2.52, were isolated from a testicular cDNA library. 1.12 and 2.52 encode two distinct regions of SSeCKS, an intracellular protein that modulates cell morphology and cytoskeletal organization, presumably through a kinase scaffolding function (Lin et al., 1995). The gene was originally isolated from a screen of transcripts that are downregulated in response to Src activation and the polypeptide product was subsequently found to be a PKC substrate (Lin et al., 1995; Lin et al, 1996). The major 280 and 290 kDa SSeCKS isoforms have predicted rod-like structures that localize to the cortical cytoskeleton and plasma membrane sites, most likely via myristylation (Lin et al., 1996). Upon PKC activation, SSeCKS relocates from the cell cortex to perinuclear sites. Inducible overexpression of SSeCKS results in cell flattening, elaboration of the cortical cytoskeleton and an increase in integrin-independent FAK phosphorylation and growth arrest (Gelman et al., 1998). These studies suggest that SSeCKS may exert its effects through modulation of the cytoskeleton. Recently, it has been demonstrated that SSeCKS exerts its tumor suppressor effects by interacting with cyclin D in the cytoplasm and thus regulating cyclin D activity in the cell and regulating the cell cycle progression from G1 to S (Lin et al., 2000).
GalT and SSeCKS show many similarities: (1) both are localized to the perinuclear/Golgi complex and to tips of lamellipodia; (2) both associate with the cytoskeleton; and (3) both inhibit cell growth when overexpressed (Hinton et al., 1995; Nelson et al., 1999; Gelman et al., 1998; Gelman et al., 2000). In this study, we show that the SSeCKS domains isolated by yeast two-hybrid screens colocalize with GalT, coimmunoprecipitate with GalT in a variety of cell types, and functionally interact with GalT to modulate GalT-dependent cellular adhesion. These studies suggest that the scaffolding function of SSeCKS serves to orchestrate the various signaling and cytoskeletal functions associated with GalT.
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MATERIALS AND METHODS |
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Rabbit (#9807) antibodies to GalT were made against affinity-purified murine GalT catalytic domain that was expressed in Escherichia coli as a His fusion protein, as previously described (Nguyen et al., 1994). Rabbit polyclonal antibodies to SSeCKS were prepared as described previously (Lin et al., 1996). Rabbit polyclonal and mouse monoclonal antibodies to GFP were purchased from Clontech. Chicken anti-GFP antibodies were a gift from Henry Tomasiewicz (Emory University, Atlanta, GA). Horseradish peroxidase secondary antibodies (Santa Cruz Biotechnology Inc.) were used unless otherwise stated. All vectors were purchased from Clontech (La Jolla, CA) and, unless stated otherwise, all chemicals were from Sigma (St Louis, MO).
Construction of the GAL4-GalT cytoplasmic domain two-hybrid vector
A yeast two-hybrid DNA binding (DB) domain vector was constructed such that the cytoplasmic domain of GalT was located upstream of the GAL4 DB domain, thus mimicking the physiological orientation of the GalT cytoplasmic domain. A 75 bp oligomer encoding the N-terminal portion (amino acids 1-24) of GalT (Lopez et al., 1991) was ligated into the BamHI and NcoI site of a modified GAL4 DB plasmid (D151; kindly supplied by Robert Brazas, University of California, San Francisco, CA). This vector (D151-GT) was screened against an oligo(dT) and random-primed mouse testis cDNA library in pGAD10 vector (Clontech) pooled from 200 BALB/c mice, age 9-11 weeks. As controls, GAL4 activation domain (AD) fusion proteins containing Raf, E12 or SNF1 (kindly supplied by Stevan Marcus, MD Anderson Cancer Center, Houston, TX) were used.
Two-hybrid screening
Transformation of bait and prey was done by a modification of published methods (Schiestl and Gietz, 1989). Yeast strain HF7C (MATa ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2: GAL1, GAL1-HIS3, URA3: (GAL4 17-mers)3-CYC1-LacZ) were grown in 100 ml of YPD to an OD600 of 0.5-0.7, harvested by centrifugation, resuspended in 50 ml of sterile water and centrifuged again. The washed cells were washed again with 20 ml LiTE (100 mM LiOAc, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspended in 8 ml of LiTE. After a brief incubation (10 minutes) at room temperature, the cell suspension was mixed with 10 mg of denatured salmon sperm carrier DNA, 150 µg GT-D151 and 200 µg of mouse testis library cDNA, described earlier, was added. After incubation at 30°C for 10 minutes, sterile LiPEG (40% PEG 3350, 1.0 M LiOAc, 1xTE, pH 8.0) was added and mixed. The cell suspension was incubated in a 500 ml flask at 30°C, 30 minutes, with shaking at 200 r.p.m. DMSO was added to a final concentration of 10% (v/v) and the cell suspension was incubated at 42°C for 15 minutes, chilled on ice and the cells were resuspended in 1x TE buffer. 200 µl of the suspension was plated on 15-cm drop-out agar plates (SC-Trp, Leu, His) containing 5 mM 3-AT.
Protein interactions were identified using a modified ß-galactosidase filter assay (Clontech). His+ colonies were transferred to nitrocellulose membrane, permeabilized in liquid nitrogen, and placed on Whatman 3 mm filter paper soaked in Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM MgCl2, 50 mM ß-mercaptoethanol) containing 1.0 mg/ml X-gal (Gibco-BRL). Colonies that turned blue after 1-5 hours were screened for interaction again, as described above. To identify positive clones that did not activate the lacZ gene by themselves, colonies were repetitively replica-plated on drop-out agar plates (-Leu) and screened for loss of bait. Specific clones were harvested in drop-out media (-Leu, -His) and the GAL4AD plasmid cDNA (prey) was isolated by electroporation and amplification in E. coli. Prey plasmids were re-transformed into yeast together with D151-GT or GAL4 DB fusion plasmids containing Raf, E12 or SNF 1, and tested for specificity.
To quantify interactions, various yeast cell colonies were grown in an appropriate minimal medium and analyzed for ß-galactosidase activity using a 4-methyl umbelliferyl ß-o-galactopyranoside (4-MUG) assay (Meng et al., 1996). Activity was normalized to cell number as determined by OD600. To obtain the DNA sequence of a specific clone, PCR primers directed against the N-terminus and C-terminus of pGAD10 were used (Clontech). The isolated PCR products were either directly sequenced or ligated into the EcoRV site in Bluescript KS (Stratagene) and transformed into E. coli DH5 (Promega). Sequencing was performed using an ABI Fluorescence Sequencer and the identity of the sequences determined using a NCBI BLAST search program.
Construction of GFP fusion proteins
To analyze the distribution of the two interacting clones, either GFP (green fluorescence protein) or BFP (blue fluorescence protein) were fused to 1.12 and 2.52 cDNA sequences. A Bluescript vector containing 1.12 and 2.52 was digested with XhoI and PstI and the fragments were gel purified (Qiagen) and ligated into pEGFP-C1 or pEBFP-C1 (Clontech) using the same restriction enzymes. GFP was also fused to the GalT cytoplasmic and transmembrane domains, using a vector described earlier (pGEM TL; Evans et al., 1993). After digestion with SpeI, the 170 bp fragment was gel-purified and blunt-ended with Klenow. The fragments were then cut with AccI, gel-purified and cloned into the SmaI/AccI site of pEGFP-N3. After propagation in E. coli, all constructs were isolated from colonies selectively grown on LB plates containing 30 µg/ml kanamycin.
Separation of cell lysate into soluble and insoluble fractions
NIH 3T3 cells were grown to 80% confluence and the cells were scraped from the dishes and fractionated into soluble and insoluble components (Kiley and Jaken, 1990). Equal concentrations (25 µg) of total lysate, soluble fraction and insoluble fraction were loaded onto a 6% SDS-PAGE gel and the resulting gel was blotted onto nitrocellulose membranes (Protran BA 85, Schleicher & Schuell). Blots were immunolabeled with either polyclonal SSeCKS antibody or monoclonal E-cadherin antibody (Transduction Labs).
Western blotting
Cells grown to 80% confluence were gently removed from the dishes with a calcium, magnesium-free balanced salt solution containing 2 mM EDTA and chicken serum. Approximately 4x105 cells were washed twice with PBS (Gibco-BRL) and lysed in 1 ml of modified RIPA lysis buffer [10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.2 mM CaCl2, 0.1 mM MgCl2, 10 mM NaF, 10 mM Na4PO3, 4 mM Na3VO4 (-ortho) and protease inhibitor cocktail (Boehringer Mannheim)]. When indicated, 0.5 mM of CaCl2, MgCl2 or 0.31 mg/ml of phosphoserine (PS) were added to the lysis buffer. After a brief sonication (2-3 seconds), lysates were centrifuged at 15,500 g for 15 minutes and the pellet discarded. Proteins were resolved on 6% or 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Protran BA 85, Schleicher & Schuell). The membranes were blocked with 5% dry milk or 5% BSA/5% normal goat serum (NGS) when indicated, in TBS (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Tween 20). After 1 hour at room temperature, the filters were washed and incubated with antibodies against SSeCKS, GalT or GFP for 1 hour, at room temperature. Finally, mouse or rabbit IgG conjugated to horseradish peroxidase was added for an additional 45 minutes and the blots were developed using ECL (Pharmacia Biotech).
Cell transfection and immunoprecipitation
As indicated, NIH 3T3 fibroblasts and NMuMG cells were transfected with GFP-1.12, BFP-2.52, and/or TL-GFP cDNAs using Transfast reagent (Promega). Cells were plated onto 100-mm culture dishes and transfections were performed using a 1:1 reagent:DNA ratio (15 µg DNA total). 48 hours after transfection, the cells were replated at various dilutions in medium containing 8 µl/ml Geneticin (Gibco-BRL). Individual clones were chosen using trypsin-soaked cloning disks, expanded, and maintained in the presence of Geneticin.
Samples (250 µl) of various cell lysates were diluted twofold with NET buffer (50 mM Tris-HCl, pH 7.5, 0.1% NP-40, 0.25% (w/v) gelatin, 150 mM NaCl, protease inhibitor cocktail) and incubated with 2.5 µl of antibodies against SSeCKS, GFP or GalT from 4 hours to overnight on a nutator at 4°C. A volume of 30 µl of protein A-agarose (Santa Cruz) was added to the lysates and mixed for 1 hour. The beads were pelleted briefly and washed three times with 500 µl RIPA. The proteins were released from the beads by boiling in reducing Laemmli buffer and subjected to SDS-PAGE and western blotting as described above.
Indirect immunofluorescence and confocal microscopy
Cells were grown to 80% confluence, dissociated and plated on cell culture-treated chamber glass slides (Nalge Nunc Intl., Naperville, IL) coated with 10 µg/ml of fibronectin. After 24 hours, cells were washed twice with PBS and immediately fixed with 4% paraformaldehyde/PBS for 30 minutes at room temperature. For colocalization experiments, cells were washed three times with PBS and permeabilized with 0.1% saponin/PBS for 15 minutes at room temperature and blocked with PBS/saponin/5% normal goat serum (NGS) for an additional 20 minutes.
Cells that express GFP fusion proteins were incubated with chicken anti-GFP (1:200) and rabbit anti-GalT (1:100) for 2 hours, 37°C. After washing, cells were stained with goat anti-mouse-FITC (1:200) and goat anti rabbit-TRITC (1:200) for an additional 2 hours. After extensive washing, cells were blocked an additional time with 5% NRS in saponin/PBS to bind goat antibodies. Cells were then incubated with rabbit anti-chicken IgY-FITC for 45 minutes at room temperature. After mounting, stained cells were viewed under a Zeiss LSM 510 confocal microscope.
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RESULTS |
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The ß-galactosidase filter lift assay showed a specific interaction between the cytoplasmic tail of GalT and clones 1.12 and 2.52, but the control clones Ras, E12 and SNF1 showed no interaction. Since the ß-galactosidase filter-lift assay does not normalize for cell number, a MUG (4-methyl umbelliferyl ß-o-galactopyranoside) assay was used to quantify the relative strengths of the interactions by normalizing ß-galactosidase activity to OD600 (Fig. 1). The interaction between 1.12 or 2.52 and GalT is not as strong as that produced by the interaction of the positive control, Ras and Raf. However, the interaction between GalT and 1.12 or 2.52 still produces 10-15-fold more ß-galactosidase activity than the negative controls that pair 1.12 or 2.52 with Ras. Sequence analysis revealed that clone 1.12 is 840 bp in length and corresponds to amino acids 51-330 of mouse SSeCKS. Clone 2.52 is 854 bp in length and corresponds to amino acids 1278-1562 of mouse SSeCKS (GenBank accession no. AB020886) (Fig. 2).
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Initially, we tested the ability of anti-GalT antibodies to coprecipitate the GFP-1.12 and/or BFP-2.52 fusion proteins. 3T3 cells were stably transfected with either GFP-1.12 or BFP-2.52, and their lysates were prepared and subjected to immunoprecipitation with antibody raised against recombinant murine GalT. The immunoprecipitates were resolved under reducing conditions and immunoblotted with anti-GFP antibody. The GFP-1.12 fusion protein (Fig. 4A), as well as the BFP-2.52 fusion protein (Fig. 4B) coprecipitate with GalT. The association of GFP-1.12 with GalT is somewhat dependent on the presence of calcium, magnesium and phosphatidyl serine in the lysis buffer.
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GalT association with intact SSeCKS
Since 1.12 and 2.52 are only fragments of the intact SSeCKS protein, we determined whether these interactions were representative of the ability of the endogenous, intact SSeCKS protein to bind GalT. As previously reported, SSeCKS occurs as a 290/280 kDa doublet when 3T3 cell lysates are subjected to western blot analysis (Fig. 5). Lysates of 3T3 cells were immunoprecipitated with anti-GalT antibodies and immunoblotted with antibodies against SSeCKS. Immunoprecipitation of GalT coprecipitates SSeCKS from 3T3 cell lysate (Fig. 5). Although SSeCKS occurs as a 290/280 kDa doublet, incubation with anti-GalT antibodies generally coprecipitated only one of the two isoforms, often in a cell-type specific manner. We have not yet determined whether this reflects cell type differences in phosphorylation, glycosylation or some other posttranslational modification that influences GalT-SSeCKS affinities.
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SSeCKS is enriched in the insoluble/membrane fractions of cell lysates
If SSeCKS and GalT do associate with one another, then SSeCKS should fractionate with cell membranes and cytoskeletal material, since GalT is a well-defined integral membrane protein. As shown in Fig. 7, a majority of the SSeCKS in cell lysates partitions with the insoluble fraction of membranes and cytoskeletal material, as opposed to the soluble cytosolic material. A parallel blot was probed with antibody to E-cadherin to demonstrate that the soluble components were effectively resolved from the insoluble/membrane components.
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A previous study has shown that when the TL construct (GalT cytoplasmic and transmembrane domains) is sufficiently overexpressed, it competes with the endogenous full-length GalT for binding sites to the actin cytoskeleton, leading to a loss of cell spreading on laminin and a rounded morphology (Evans et al., 1993). We thought this dominant-negative GalT phenotype would serve as a useful tool for testing the requirement of SSeCKS in GalT-dependent cell spreading. We hypothesized that overexpression of TL-GFP may effectively consume all of the available endogenous SSeCKS, preventing full-length GalT from associating with SSeCKS and the actin cytoskeleton. We reasoned that overexpressing fragments of SSeCKS that bind the GalT cytoplasmic domain (1.12 or 2.52) should compete for binding to the TL-GFP fusion protein, making the endogenous SSeCKS available for binding GalT and rescuing the dominant-negative phenotype.
To test this possibility, NMuMG TL-GFP cells were stably transfected with either the 1.12 or 2.52 cDNAs. Fig. 8A shows that wild-type, nontransfected NMuMG cells grow well on tissue culture dishes and have a stellate morphology. However, NMuMG cells expressing TL-GFP show the expected dominant negative phenotype (Evans et al., 1993), that is, they are rounded and less stellate with a subsequent loss of cell adhesion and spreading on laminin surfaces (Fig. 8B). If this TL-GFP cell line is subsequently co-transfected with either the 1.12 or 2.52 (not shown) cDNAs, the dominant negative phenotype is abolished in that the cell morphology returns to a wild-type stellate appearance (Fig. 8C). Transfection of the TL-GFP cell line with the BFP vector alone has no effect on the dominant negative phenotype (Fig. 8D). Immunoblotting with anti-GFP antibodies confirm that the rescued cells (clones 3 and 8) express the GFP-1.12 fusion proteins (Fig. 8E). These results are consistent with the hypothesis that SSeCKS facilitates the interaction of the GalT cytoplasmic domain with the cellular cytoskeleton.
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DISCUSSION |
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The yeast two-hybrid screen has been used successfully to identify cytosolic partners for the cytoplasmic domains of integrin ß subunits (Shattil et al., 1995; Kolanus et al., 1996) and syndecan-4 (Baciu et al., 2000). In this study, the yeast two-hybrid system has allowed us to identify SSeCKS as a cytoplasmic partner for the GalT cytoplasmic domain. Two independent clones were isolated, 1.12 and 2.52, which show identity to the mouse SSeCKS cDNA sequence. SSeCKS has interesting characteristics in common with GalT. Both GalT and SSeCKS are found associated with the plasma membrane, both localize to sites of focal adhesion, and both associate with the actin cytoskeleton. In addition, overexpressing GalT decreases the growth rate of cells (Hinton et al., 1995), while inducible overexpression of SSeCKS causes growth arrest in fibroblasts, possibly through an alteration of the cytoplasmic distribution of ERK2 (Gelman et al., 1998).
SSeCKS is closely homologous to Gravin, both of which are members of the AKAP (A-kinase anchoring proteins) family of proteins. AKAPs are believed to function as scaffolding proteins for specific kinases, phosphatases and other signaling effectors, routing them to discrete subcellular sites where they have access to appropriate substrates (Nauert et al., 1997; Faux and Scott, 1996). SSeCKS has binding sites for both protein kinase C and A, and serves as a substrate for PKC phosphorylation (Lin et al., 1996).
It is somewhat surprising that two independent domains of the SSeCKS protein were found to interact with the GalT cytoplasmic domain. However, given the large size of SSeCKS and its putative role as a scaffolding protein, this may not be completely surprising. The two SSeCKS domains are separated by over 900 amino acids, which could easily reflect functionally separable domains. Although the current data show that both 1.12 and 2.52 interact with GalT, we do not know whether they interact with GalT independently of one another, are capable of interacting at the same time, or whether each domain interacts with distinct GalT proteins to facilitate clustering required for GalT-induced signaling. Furthermore, pairwise alignment of the 1.12 and 2.52 sequences illustrates some potential regions of similarity (two 17 amino acid regions showed 35.3% identity and 76% similarity to each other) and secondary structure prediction algorithms suggest that both 1.12 and 2.52 consist of a number of -helical segments interspersed with ß-strands or random regions. However, it is unclear at this time whether these structural motifs are at all important for interaction with the GalT cytoplasmic domain.
Immunolocalization studies reveal that the 1.12 is localized predominantly to cell processes, whereas 2.52 is localized to the perinuclear region. This difference in subcellular distribution of 2.52 and 1.12 is consistent with SSeCKS localization to both subcellular regions, and may reflect a loss of complementary targeting sequences in each fragment that are present in the intact SSeCKS protein. In any event, the dual subcellular distribution of SSeCKS, whether or not it is due to differential targeting sequences in the 1.12 and 2.52 domains, is remarkably coincident with the dual subcellular distribution of GalT. This then raises the possibility that SSeCKS is the cytosolic effector that is responsible for GalT being routed from the Golgi complex to the cell surface. Similarly, since PKC activation is thought to release SSeCKS from the cell surface, allowing it to translocate back to the perinuclear region (Lin et al., 1996), it is possible that SSeCKS regulates the differential transport of GalT between the Golgi and the cell surface. This interesting possibility awaits further testing.
As a scaffolding protein, SSeCKS could serve to direct the necessary signaling molecules (e.g. PKC, Rho family members) to the GalT microenvironment in order to affect the ability of GalT to bind the actin cytoskeleton (Barry and Critchley, 1994; Chapline et al., 1998). It is also possible that the binding of GalT to SSeCKS alters the ability of SSeCKS to bind other cytoplasmic signaling molecules. Regulating the interaction between SSeCKS and GalT may be one method the cell uses to receive information on the status of its interaction with the extracellular matrix. The cell could then regulate the organization of the actin cytoskeleton in response to signals from GalT.
One mechanism by which SSeCKS has been suggested to regulate the organization of the actin cytoskeleton is by affecting the tyrosine phosphorylation/activation of FAK. The activation of FAK by Src-family kinases plays a central role in LPA-, neuropeptide- and integrin-mediated signal transduction and cytoskeletal reorganization (Rozengurt, 1995). Earlier reports have demonstrated that aggregation of cell surface GalT with synthetic multivalent substrates or with anti-GalT antibodies induces a transient tyrosine phosphorylation of FAK and a loss of stress fibers (Wassler and Shur, 2000). Similarly, increasing the level of SSeCKS, by a tetracycline-regulated promoter, elevates the level of integrin-independent FAK tyrosine phosphorylation causing cells to flatten with a subsequent reduction of stress fibers (Gelman et al., 1998). In this respect, it is interesting to note that fibroblasts overexpressing GFP-1.12 show a reduction in actin stress fiber staining compared with cells expressing GFP only (M.J.W. et al., unpublished). These observations suggest that SSeCKS binding to the cytoplasmic domain of GalT is coupled, directly or indirectly, with the transient phosphorylation of FAK and reorganization of the actin cytoskeleton that are induced by GalT aggregation (Wassler and Shur, 2000). Whether or not this is the case, the identification of the SSeCKS scaffolding protein as a cytoplasmic partner for the GalT cytoplasmic domain suggests that it is partly responsible for the ability of GalT to associate with the cytoskeleton and induce signal transduction cascades.
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ACKNOWLEDGMENTS |
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