Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Predimerization of recombinant platelet-derived growth factor receptor extracellular domains increases antagonistic potency

Platelet-derived growth factor (PDGF) is a dimeric growth factor acting through tyrosine kinase alpha- and beta-receptors. In both receptors, the extracellular parts are composed of five Ig-like domains. Functional mapping of the extracellular part of the receptors have shown that ligand-binding occurs to Ig-like domains 2 and 3 and that Ig-like domain 4 is involved in receptor-receptor interactions. Recombinant GST-fusion proteins of PDGF alpha-receptor Ig-like domains 1-4 and beta-receptor Ig-like domains 1-3 (alphaRD1-4-GST and betaRD1-3-GST) were generated and compared with their cleaved counterparts (alphaRD1-4 and betaRD1-3) with regard to their ability to block PDGF binding to cell surface receptors. In the case of both the alpha- and the beta-receptors, 100-1000-fold lower concentrations of the GST-fusion proteins were required, as compared to the cleaved forms, for inhibition of PDGF binding to cell surface receptors. alphaRD1-4-GST and betaRD1-3-GST, in contrast to alphaRD1-4 and betaRD1-3, were shown to occur as ligand independent dimers. Covalently cross-linked alphaRD1-4 dimers displayed a 50-fold increased potency as compared to alphaRD1-4. We thus conclude that the dimeric nature of alphaRD1-4-GST and betaRD1-3-GST is responsible for the high antagonistic potency of the fusion proteins.     

Cell adhesion regulates ubiquitin-mediated degradation of the platelet-derived growth factor receptor beta

Cross-talk between integrin-mediated adhesion and growth factors has been described in many recent studies; however, the underlying mechanisms remain incompletely understood. We report here that detachment of cells from the extracellular matrix induced a decrease in both the autophosphorylation and protein levels of the platelet-derived growth factor receptor beta (PDGF-R beta), which was completely reversed upon replating cells on fibronectin. The effect occurred in all cells examined but to a greater extent in primary fibroblasts compared with established cell lines. Decreased PDGF-R levels in suspended cells correlated with ubiquitination of the PDGF-R and was blocked by treatment with inhibitors of the proteasome pathway. Unlike PDGF-induced down-regulation, detachment-induced degradation did not require receptor autophosphorylation, internalization, or tyrosine kinase activity. We conclude that cell detachment results in cellular desensitization to PDGF that is mediated by degradation of the PDGF-R via a novel ubiquitin-dependent pathway.     

In vivo gene transfer of an extracellular domain of platelet-derived growth factor beta receptor by the HVJ-liposome method ameliorates bleomycin-induced pulmonary fibrosis

A number of investigators have reported augmented expression of PDGF in lungs with idiopathic pulmonary fibrosis (IPF) or with other types of pulmonary fibrosis. To accomplish such a regulation of PDGF activity, we constructed an expression plasmid of the extracellular domain of PDGF receptor beta chain (XR), which lacks intracellular tyrosine kinase domain and transmembrane portions, and estimated the therapeutic effects of XR gene transfer through the trachea on bleomycin-induced lung fibrosis of C57BL/6 mice using the hemagglutinating virus of Japan(HVJ)-liposome method. The XR gene transfer ameliorated the increases in the wet weight and hydroxyproline content and the histopathologic changes of the lung induced by bleomycin. These findings suggest that PDGF plays a crucial role in the pathogenesis of pulmonary fibrosis, and that XR gene transfer using the HVJ-liposome method may limit the progression of pulmonary fibrosis.     

Phosphospecific antibodies reveal temporal regulation of platelet-derived growth factor beta receptor signaling

The platelet-derived growth factor beta receptor (betaPDGFR) is a receptor tyrosine kinase involved in multiple aspects of cell growth and differentiation. Upon activation, betaPDGFR is phosphorylated at up to nine different tyrosine residues. Phosphorylation of the receptor results in at least two different outcomes: recruitment of signaling molecules and activation of intrinsic receptor kinase activity. In order to evaluate the phosphorylation state of the receptor, phosphospecific antibodies were generated against peptides encompassing betaPDGFR phospho-Y751, phospho-Y771, or phospho-Y857. When phosphorylated, these sites enable the receptor to recruit signaling molecules PI3K or RasGAP, or enhance the receptor's kinase activity, respectively. We found that receptors phosphorylated at Y751, Y771, and Y857 display distinct temporal and spatial distribution by immunofluorescence. Subsequent biochemical studies revealed that receptor function corresponding to each of the phosphorylated sites was regulated as a function of time. Within the first 10 min, PDGF enhanced the receptor's kinase activity and initiated recruitment of PI3K and RasGAP. After prolonged exposure to PDGF, PI3K binding persisted to approximately 85% of the amount bound at 10 min, whereas binding of RasGAP and the exogenous kinase activity of the receptor diminished to less than 15% of the levels displayed at 10 min. We conclude that the phosphorylation state of the receptor, as well as its signaling capacity, is dynamic and changes as cells are continuously exposed to PDGF.     

Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase

The platelet-derived growth factor (PDGF) beta receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF beta receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF beta receptor, we compared PDGF beta receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF beta receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cgamma1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cgamma1 activity and migratory hyperresponsiveness to PDGF. PDGF beta receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPepsilon ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF beta receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.     

Role of alpha beta receptor heterodimer formation in beta platelet-derived growth factor (PDGF) receptor activation by PDGF-AB.

We investigated the ability of highly purified recombinant platelet-derived growth factor (PDGF) AB to interact with the products of alpha and beta receptor genes expressed in cells independently or concurrently. Although PDGF-AB lacked any detectable ability to bind or activate beta receptors in cells expressing only this receptor, efficient beta receptor activation by this ligand was readily observed in cells coexpressing alpha platelet-derived growth factor receptors (alpha PDGFRs). beta receptor activation induced by PDGF-AB was shown to be dependent upon in vivo physical association of this receptor with alpha PDGFRs. Moreover, cross-linking analysis established the existence of PDGF-AB-induced beta PDGFR dimers in vivo. All of these findings argue that initial PDGF-AB interaction with the alpha PDGFR induces conformational changes in the ligand or receptor that facilitates efficient recruitment of beta PDGFR by this PDGF isoform.     

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line

The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-beta), but no TGF-alpha production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-beta mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well as in (human) teratocarcinomas.     

Myc is an essential negative regulator of platelet-derived growth factor beta receptor expression

Platelet-derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as well as many other cell types. Interaction of PDGF BB with the PDGF beta receptor (PDGF-betaR) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-betaR downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-betar mRNA expression. Our studies show that pdgf-betar mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-betar mRNA and protein. Suppression of pdgf-betar mRNA in response to Myc is specific, since expression of the related receptor pdgf-alphar is not affected. We further show that Myc suppresses pdgf-betar mRNA expression by a mechanism which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts demonstrates that Myc is required for the repression of pdgf-betar mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-betar mRNA levels plays an important role in the regulation of basal pdgf-betar expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-betaR.     

Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.     

Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.     

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.     

Phylogenetic analysis of platelet-derived growth factor by radio- receptor assay

Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo.     

Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor beta receptor

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) beta receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3'-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short alpha-helix followed by a three-stranded beta-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second beta strands and along one face of the beta-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WW-like domain.     

Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells

Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels     

Regulation of platelet-derived growth factor receptor activation by afadin through SHP-2: implications for cellular morphology

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology.