SWAP-70 is required for oncogenic transformation by v-Src in mouse embryo fibroblasts

SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src.     

A mutant of SWAP-70, a phosphatidylinositoltrisphosphate binding protein, transforms mouse embryo fibroblasts, which is inhibited by sanguinarine

SWAP-70, a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, has been suggested to be involved in transformation of mouse embryo fibroblasts (MEFs) as well as membrane ruffling after growth factor stimulation of the cells. A mutant, SWAP-70-374, was found to be able to bind to F-actin in vitro, whereas wild-type SWAP-70 failed to do so. This mutant was present at the plasma membrane without any stimulation while the wild-type protein was present only in the cytosol unless cells were stimulated with EGF. Expression of this mutant in MEFs resulted in morphologic transformation, fast growth, and loss of contact inhibition, suggesting that SWAP-70 with this mutation can transform the cells. ERK1/2 was activated in SWAP-70-374-transformed cells. Use of MEK inhibitors revealed that the ERK1/2 pathway does not affect the cell growth of MEFs but is responsible for loss of contact inhibition. To investigate the function of SWAP-70 further, drugs that can inhibit SWAP-70-dependent cell responses were screened. Among various drugs, sanguinarine was found to inhibit transformation of MEFs by SWAP-70-374. This drug was able to inhibit SWAP-70-mediated membrane ruffling as well, suggesting that its effect was closely related to the SWAP-70 signaling pathway. These results suggest that SWAP-70-374 can activate some signaling pathways, including the ERK1/2 pathway, to transform MEFs.     

Expression and localization of SWAP-70 in human fetomaternal interface and placenta during tubal pregnancy and normal placentation.

SWAP-70 has been demonstrated as a multiple functional signaling protein involved in formation of membrane ruffling induced by signal cascade of tyrosine kinase growth factor receptors. In the present study, the spatial and temporal expression pattern of SWAP-70 on human fetomaternal interface was investigated using specimens collected from tubal and normal pregnancies by in situ hybridization, immunohistochemistry, and Western blotting. Data showed an intense expression of SWAP-70 in trophoblasts at weeks 3-6 of fallopian implantation and at weeks 6-7 of normal pregnancy. The most intense expression was exhibited by those highly motile and invasive extravillous trophoblasts. From gestational week 8 on, the level of SWAP-70 in trophoblasts decreased significantly, and the signal was restricted in villous cytotrophoblast cells. In the in vitro cultured human trophoblast cell line, B6Tert-1, colocalization of SWAP-70 with F-actin was verified. Data in human placenta were similar to what we recently reported on rhesus monkey fetomaternal interface. Our results suggest that SWAP-70 may be involved in regulating migration and invasion of trophoblast cells during the processes of embryonic implantation and placentation in primates.     

Activity of beta3-beta4 loop of the PH domain is required for the membrane targeting of SWAP-70

SWAP-70 translocates to the plasma membrane in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner and contributes to membrane ruffling. It binds to phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) through its PH domain, which is essential for the membrane translocation after EGF stimulation. We examined the behavior of the SWAP-70s which have mutations in the beta3/beta4 loop of the PH domain. The two mutants fused to green fluorescent protein (GFP) carrying the mutations failed to translocate to the plasma membrane. The sole PH domains carrying the same mutations behaved similarly. The PtdIns(3,4,5)P(3) binding activity of two mutants was comparable to that of the wild-type protein. These results suggest that translocation of SWAP-70 largely depends on the activity of the PH domain, and that not only PtdIns(3,4,5)P(3) binding activity, but also some additional activity of the PH domain is required for the translocation.     

The Guanine Nucleotide Exchange Factor SWAP-70 Modulates the Migration and Invasiveness of Human Malignant Glioma Cells

The malignant glioma is the most common primary human brain tumor. Its tendency to invade away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Accordingly, the molecular pathogenesis of glioma invasion is currently under investigation. Previously, we examined a gene expression array database comparing human gliomas to nonneoplastic controls and identified several Rac guanine nucleotide exchange factors with differential expression. Here, we report that the guanine nucleotide exchange factor SWAP-70 has increased expression in malignant gliomas and strongly correlates with lowered patient survival. SWAP-70 is a multifunctional signaling protein involved in membrane ruffling that works cooperatively with activated Rac. Using a glioma tissue microarray, we validated that SWAP-70 demonstrates higher expression in malignant gliomas compared with low-grade gliomas or nonneoplastic brain tissue. Through immunofluorescence, SWAP-70 localizes to membrane ruffles in response to the growth factor, epidermal growth factor. To assess the role of SWAP-70 in glioma migration and invasion, we inhibited its expression withsmall interfering RNAs and observed decreased glioma cell migration and invasion. SWAP-70 overexpression led to increased levels of active Rac even in low-serum conditions. In addition, when SWAP-70 was overexpressed in glioma cells, we observed enhanced membrane ruffle formation followed by increased cellmigration and invasiveness. Taken together, our findings suggest that the guanine nucleotide exchange factor SWAP-70 plays an important role in the migration and invasion of human gliomas into the surrounding tissue.     

SWAP-70: A New Type of Oncogene

SWAP-70 is a protein that has been suggested to be involved in regulation of actin rearrangement. Having discovered that an artificially-derived mutant of SWAP-70 can transform mouse embryo fibroblasts, we searched for naturally-occurring mutations in the SWAP-70 gene, finding listings for several on the Web at www.sanger.ac.uk/genetics/CGP/cosmic/, including three mutations found in ovarian cancers. (The number of such mutations has now reached 13 out of 228 tumors). We created expression vectors for the mutant SWAP-70 proteins and introduced these into NIH3T3 cells. The cells expressing the mutant SWAP-70 constructs exhibited faster growth than the parental or wild-type SWAP-70-expressing cells. In most instances, cells that are able to grow in soft agar will form tumors in nude mice. While SWAP-70-transformed cells grew in soft agar, they failed to form tumors in nude mice. This result implies that transformation by the SWAP-70 mutants is unique. The cells bearing the mutant SWAP-70 genes were sensitive to nutrient starvation, supporting the idea that they are transformed cells. However, they failed to pile up and demonstrated contact inhibition, unlike most normal transformed cells. Upon expression of human SWAP-70 genes, MEK1 was activated. This activation appeared to contribute to the saturation density of the cells. As SWAP-70 has been shown to be the last protein to receive signals from cytokines, it is likely that there is a putative feedback signaling pathway, and that disorder of this signaling pathway can transform cells. Accordingly, this may explain why SWAP-70-transformed cells have different characteristics than most transformed cells.     

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.     

v-Crk induces Rac-dependent membrane ruffling and cell migration in CAS-deficient embryonic fibroblasts

Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.     

Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.     

Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src.

Two eps8 isoforms, p97eps8 and p68eps8, were previously identified as substrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine content in v-Src transformed cells (IV5) revealed that both isoforms were highly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97eps8 detected in cells coexpressing both p97eps8 and active Src relative to that in cells expressing p97eps8 alone supported our hypothesis. The existence of common phosphotryptic peptides between in vitro 32P-labeled p97eps8 and p68eps8 indicated that these two proteins shared the same Src-mediated sites. Further in vitro binding assays demonstrated that p68eps8 was the major eps8 isoforms that could be precipitated by bacterial fusion protein containing Src SH3. Interestingly, both p68eps8 and p97eps8 were preferentially expressed in v-Src transformed cells and the presence of p68eps8 appeared to depend on Src. Since p97eps8 has been implicated in mitogenesis and tumorigenesis, its readiness to be phosphorylated and induced by v-Src might attribute to v-Src-mediated transformation.     

Regulation of macropinocytosis by p21-activated kinase-1

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.     

Regulation of both apoptosis and cell survival by the v-Src oncoprotein

A number of oncogenes alter the regulation of the cell cycle and cell death, contributing to the altered growth of tumours. Expression of the v-Src oncoprotein in Rat-1 fibroblasts prevented cell cycle exit in response to growth factor withdrawal. Here we investigated whether survival of v-Src transformed cells in low serum is dependent on v-Src activity. We used a temperature sensitive v-Src to study the effect inactivating v-Src on transformed cells growing under low serum conditions. We found when we switched off v-Src the cells died by apoptosis characterised by activation of caspases and the stress-activated kinases, JNK (Jun N-terminal kinase) and p38 MAP (mitogen activated protein) kinase. We were able to prevent cell death by addition of serum or overexpression of Bcl-2. Thus v-Src transformed Rat-1 cells can be protected from apoptosis by serum, v-Src, or Bcl-2. We investigated how v-Src protects from apoptosis under these conditions. Amongst other effects, v-Src activates two kinases which have been shown to protect cells from apoptosis, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK1/2). We found that switching off v-Src led to a decrease in the activity of both PI3-K and ERK1/2, however, we found that adding a specific inhibitor of PI3-K (LY294002) to v-Src transformed Rat-1 cells grown in low serum induced apoptosis while a specific ERK kinase (MEK1) inhibitor (PD98059) had no effect. This suggests that v-Src protects from apoptosis under low serum conditions by activating PI3-K.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

Membrane-induced alteration of the secondary structure in the SWAP-70 pleckstrin homology domain

Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal, which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to D-myo-phosphatidylinositol 3,4,5-trisphosphate or D-myo-phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal α-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal α-helix of the PH domain. The unwinding of the C-terminal α-helix could regulate the functions of SWAP-70 at the plasma membrane surface.     

Membrane ruffling requires coordination between type Ialpha phosphatidylinositol phosphate kinase and Rac signaling

Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKIalpha isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKIalpha, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKIalpha and PIP2 participate in ruffling. To explore this, PIPKIalpha was overexpressed in serum-starved cells and stimulated with PDGF. In serum-starved cells, PIPKIalpha expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKIalpha-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKIalpha in PDGF-stimulated cells resulted in membrane ruffling. The PDGF- and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKIalpha. Combined, these data support a model where the localized production of PIP2 by PIPKIalpha is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.     

Dendritic Cell Podosome Dynamics Does Not Depend on the F-actin Regulator SWAP-70

In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.     

Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins

We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen- activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v- Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule- disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.     

Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.     

Syndecan-1 transmembrane and extracellular domains have unique and distinct roles in cell spreading

Raji cells expressing syndecan-1 (Raji-S1) adhere and spread when plated on heparan sulfate-binding extracellular matrix ligands or monoclonal antibody 281.2, an antibody directed against the syndecan-1 extracellular domain. Cells plated on monoclonal antibody 281.2 initially extend a broad lamellipodium, a response accompanied by membrane ruffling at the cell margin. Membrane ruffling then becomes polarized, leading to an elongated cell morphology. Previous work demonstrated that the syndecan-1 cytoplasmic domain is not required for these activities, suggesting important roles for the syndecan-1 transmembrane and/or extracellular domains in the assembly of a signaling complex necessary for spreading. Work described here demonstrates that truncation of the syndecan-1 extracellular domain does not affect the initial lamellipodial extension in the Raji-S1 cells but does inhibit the active membrane ruffling that is necessary for cell polarization. Replacement of the entire syndecan-1 transmembrane domain with leucine residues completely blocks the cell spreading. These data demonstrate that the syndecan-1 transmembrane and extracellular domains have important but distinct roles in Raji-S1 cell spreading; the extracellular domain mediates an interaction that is necessary for dynamic cytoskeletal rearrangements whereas an interaction of the transmembrane domain is required for the initial spreading response.