The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.     

Disruption of gap junctional communication by the platelet-derived growth factor is mediated via multiple signaling pathways

The platelet-derived growth factor (PDGF) mediates its cellular functions via activation of its receptor tyrosine kinase followed by the recruitment and activation of several signaling molecules. These signaling molecules then initiate specific signaling cascades, finally resulting in distinct physiological effects. To delineate the PDGF signaling pathway responsible for the disruption of gap junctional communication (GJC), wild-type PDGF receptor beta (PDGFRbeta) and a series of PDGFRbeta mutants were expressed in T51B rat liver epithelial cells. In cells expressing wild-type PDGFRbeta, PDGF induced disruption of GJC and phosphorylation of a gap junctional protein, connexin-43 (Cx43), which required activation of mitogen-activated protein kinase, although involvement of additional factors was also evident. In the F5 mutant lacking binding sites for phosphatidylinositol 3-kinase, GTPase-activating protein, SHP-2, and phospholipase Cgamma1 (PLCgamma1), PDGF induced mitogen-activated protein kinase, but failed to affect GJC or Cx43, indicating involvement of additional signals presumably initiated by one or more of the mutated binding sites. Examination of the single-site mutants revealed that PDGF effects were not mediated via a single signaling component. This was confirmed by the "add-back" mutants, which showed that restoration of either SHP-2 or PLCgamma1 binding was sufficient to propagate the GJC inhibitory actions of PDGF. Further analysis showed that activation of PLCgamma1 is involved in Cx43 phosphorylation, which surprisingly failed to correlate with GJC blockade. The results of our study demonstrate that PDGF-induced disruption of GJC can be mediated by multiple signaling pathways and requires participation of multiple components.     

Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.     

SHP-1 requires inhibitory co-receptors to down-modulate B cell antigen receptor-mediated phosphorylation of cellular substrates

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and SLP-65.     

A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways.

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation.     

Biological properties of the deer papillomavirus E5 gene in mouse C127 cells: growth transformation, induction of DNA synthesis, and activation of the platelet-derived growth factor receptor.

We determined the biological activities of the 44-amino-acid deer papillomavirus (DPV) E5 protein in mouse C127 cells. The DPV E5 gene can induce focus formation, stable and acute morphologic transformation, and DNA synthesis, and it activates the platelet-derived growth factor (PDGF) beta receptor as assessed by increased constitutive tyrosine phosphorylation of mature and precursor receptor forms. Thus, the DPV E5 protein has biological activities similar to those of the closely related E5 protein from bovine papillomavirus type 1. Moreover, like the bovine papillomavirus type 1 E5 protein, the DPV E5 protein shares a short region of sequence similarity with the B chain of PDGF. These results show that activation of the PDGF receptor is a general property of fibropapillomavirus E5 proteins, lending support to our previous proposal (L. Petti, L. Nilson, and D. DiMaio, EMBO J. 10:845-855, 1991) that activation of the PDGF receptor may play a central role in transformation of fibroblasts by E5 proteins.     

Novel binding site for Src homology 2-containing protein-tyrosine phosphatase-1 in CD22 activated by B lymphocyte stimulation with antigen

CD22, a B lymphocyte membrane glycoprotein, contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and recruits Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) to the phosphorylated ITIMs upon ligation of B lymphocyte antigen receptor (BCR), thereby negatively regulating BCR signaling. Among the three previously identified ITIMs, both ITIMs containing tyrosine residues at position 843 (Tyr(843)) and 863 (Tyr(863)), respectively, are shown to be required for CD22 to recruit SHP-1 and regulate BCR signaling upon BCR ligation by anti-Ig antibody (Ab), indicating that CD22 has the SHP-1-binding domain at the region containing Tyr(843) and Tyr(863). Here we address the requirement of CD22 for SHP-1 recruitment and BCR regulation upon BCR ligation by antigen, which induces much stronger CD22 phosphorylation than anti-Ig Ab does. We demonstrate that the CD22 mutant in which both Tyr(843) and Tyr(863) are replaced by phenylalanine (CD22F5/6) recruits SHP-1 and regulates BCR signaling upon stimulation with antigen but not anti-Ig Ab. This result strongly suggests that CD22 contains another SHP-1 binding domain that is specifically activated upon stimulation with antigen. Both of the flanking sequences of Tyr(783) and Tyr(817) fit the consensus sequence of ITIM, and the CD22F5/6 mutant requires these tyrosine residues for SHP-1 binding and BCR regulation. Thus, these ITIMs constitute a novel conditional SHP-1-binding site of CD22 that is activated upon BCR ligation by antigen but not by anti-Ig Ab.     

The Lipoxin A4 receptor is coupled to SHP-2 activation: implications for regulation of receptor tyrosine kinases

Mesangial cell proliferation is pivotal to the pathology of glomerular injury in inflammation. We have previously reported that lipoxins, endogenously produced eicosanoids with anti-inflammatory and pro-resolution bioactions, can inhibit mesangial cell proliferation in response to several agents. This process is associated with elaborate receptor cross-talk involving modification receptor tyrosine kinase phosphorylation (McMahon, B., Mitchell, D., Shattock, R., Martin, F., Brady, H. R., and Godson, C. (2002) FASEB J. 16, 1817-1819). Here we demonstrate that the lipoxin A(4) (LXA(4)) receptor is coupled to activation and recruitment of the SHP-2 (SH2 domain-containing tyrosine phosphatase-2) within a lipid raft microdomain. Using site-directed mutagenesis of the cytosolic domain of the platelet-derived growth factor receptor beta (PDGFRbeta), we report that mutation of the sites for phosphatidylinositol 3-kinase (Tyr(740) and Tyr(751)) and SHP-2 (Tyr(763) and Tyr(1009)) recruitment specifically inhibit the effect of LXA(4) on the PDGFRbeta signaling; furthermore inhibition of SHP-2 expression with short interfering RNA constructs blocked the effect of LXA(4) on PDGFRbeta phosphorylation. We demonstrate that association of the PDGFRbeta with lipid raft microdomains renders it susceptible to LXA(4)-mediated dephosphorylation by possible reactivation of oxidatively inactivated SHP-2. These data further elaborate on the potential mechanisms underlying the anti-inflammatory, proresolution, and anti-fibrotic bioactions of lipoxins.     

Intrinsic signaling functions of the beta4 integrin intracellular domain

A key issue regarding the role of alpha6beta4 in cancer biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling, including growth factor-mediated signaling. One approach is to evaluate the intrinsic signaling capacity of the unique beta4 intracellular domain in the absence of contributions from the alpha6 subunit and tetraspanins and to assess the ability of growth factor receptor signaling to cooperate with this domain. Here, we generated a chimeric receptor composed of the TrkB extracellular domain and the beta4 transmembrane and intracellular domains. Expression of this chimeric receptor in beta4-null cancer cells enabled us to assess the signaling potential of the beta4 intracellular domain alone or in response to dimerization using brain-derived neurotrophic factor, the ligand for TrkB. Dimerization of the beta4 intracellular domain results in the binding and activation of the tyrosine phosphatase SHP-2 and the activation of Src, events that also occur upon ligation of intact alpha6beta4. In contrast to alpha6beta4 signaling, however, dimerization of the chimeric receptor does not activate either Akt or Erk1/2. Growth factor stimulation induces tyrosine phosphorylation of the chimeric receptor but does not enhance its binding to SHP-2. The chimeric receptor is unable to amplify growth factor-mediated activation of Akt and Erk1/2, and growth factor-stimulated migration. Collectively, these data indicate that the beta4 intracellular domain has some intrinsic signaling potential, but it cannot mimic the full signaling capacity of alpha6beta4. These data also question the putative role of the beta4 intracellular domain as an "adaptor" for growth factor receptor signaling.     

The tyrosine phosphatase SHP-1 inhibits proliferation of activated hepatic stellate cells by impairing PDGF receptor signaling

The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-β and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-β activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.     

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.     

Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins

The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5-transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein.     

Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.

In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.     

Platelet-derived growth factor receptor can mediate tumorigenic transformation by the bovine papillomavirus E5 protein.

We showed previously that the beta receptor for platelet-derived growth factor (PDGF) is constitutively activated in fibroblasts transformed by the 44-amino-acid bovine papillomavirus type 1 (BPV) E5 protein and that the E5 protein and the PDGF receptor exist in a stable complex in E5-transformed fibroblasts. On the basis of these results, we proposed that activation of the PDGF receptor by the BPV E5 protein generates a sustained proliferative signal, resulting in fibroblast transformation. In this study, we used a gene transfer approach to provide functional evidence that the PDGF receptor can mediate transformation by the E5 protein. We show that normal mouse mammary gland (NMuMG) cells, a murine mammary epithelial cell line that does not express PDGF receptors, are not susceptible to transformation by the E5 protein. Coexpression of the PDGF beta receptor and E5 genes in these cells results in markedly increased tyrosine phosphorylation of an immature PDGF receptor species and the formation of a stable complex between the E5 protein and this immature PDGF receptor form. Importantly, introduction of the PDGF receptor gene into NMuMG cells renders them highly susceptible to E5-mediated tumorigenic transformation. In contrast, the E5 protein does not induce transformation via the endogenous epidermal growth factor receptor pathway in these cells. These results demonstrate that the PDGF receptor, a cellular protein with a well-characterized role in the positive control of cell proliferation, can mediate transformation by a DNA virus transforming protein.     

Differential induction of the c-fos promoter through distinct PDGF receptor-mediated signaling pathways.

The multiple isoforms of PDGF induce fibroblastic mitogenesis through two distinct PDGF receptors, alpha and beta. The molecular mechanisms by which these alpha and beta PDGF receptors regulate gene expression are poorly understood. We present data which indicates that differential induction of c-fos gene expression by PDGF isoforms occurs through distinct PDGF alpha and beta receptor-mediated signaling pathways. Comparison of PDGF-AA with PDGF-BB stimulation showed that PDGF-BB induced prolonged expression of the c-fos gene in BALB/c-3T3 cells, but that PDGF-AA induced more potent activation of the serum response element (SRE) in transient transfection assays. PDGF-AA, which binds alpha but not beta PDGF receptors, could only induce the SRE through a protein kinase C (PKC)-dependent pathway, whereas PDGF-BB, which binds both alpha and beta PDGF receptors, could also induce the SRE through a PKC-independent pathway. These results suggest that PDGF alpha receptors activate the PKC-dependent signaling pathway while PDGF beta receptors also activate a PKC-independent pathway. In addition, we found that PDGF-BB could induce another c-fos promoter element within the -90 to +10 region, suggesting that the more potent mitogenic effect and prolonged c-fos gene expression induced by PDGF-BB may result from cooperativity between more than one c-fos promoter elements.     

Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymes that are recruited to the PDGF beta receptor.

Since the extracellular matrix (ECM) can promote platelet-derived growth factor (PDGF)-dependent responses, we hypothesized that the ECM mediates this effect by preventing the PDGF beta receptor (betaPDGFR) from associating with the negative regulator, RasGAP (the GTPase-activating protein of Ras). We found that binding of RasGAP to the wild-type betaPDGFR was decreased; the activation of Ras and Erk was enhanced, and [3H]thymidine uptake was better in cells cultured on fibronectin than in cells cultured on polylysine. To investigate the mechanism by which culturing cells on fibronectin diminished the recruitment of RasGAP to the betaPDGFR, we focused on SHP-2 since it dephosphorylates the betaPDGFR at the phosphotyrosine required for binding of RasGAP. Culturing cells on fibronectin increased the amount of SHP-2 that associated with the betaPDGFR. Furthermore, cells expressing receptor mutants that failed to associate with SHP-2 were insensitive to fibronectin. The ECM enhances PDGF-dependent responses by increasing the association of SHP-2 with the betaPDGFR, which in turn decreases the time that RasGAP interacts with the receptor. Thus, fibronectin changes PDGF-dependent signaling and biological responses by altering the signal relay enzymes that are recruited to the receptor.     

Activation of downstream signals by the long form of the leptin receptor

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.     

Tyrosine phosphatase SHP-2 is involved in regulation of platelet-derived growth factor-induced migration.

SHP-2 is a ubiquitously expressed Src homology-2-containing cytosolic tyrosine phosphatase that binds to and becomes tyrosine-phosphorylated by the activated platelet-derived growth factor receptor-beta (PDGFR-beta). Removal of the binding site on the receptor, by mutation of Tyr1009 to Phe1009 (denoted Y1009F), led to loss of PDGF-stimulated phosphatase activity in cells expressing the mutated receptor, and these cells failed to form membrane edge ruffles and to migrate toward PDGF. Furthermore, treatment with phosphatase inhibitors phenylarsine oxide (PAO) and orthovanadate led to loss of PDGF-stimulated phosphatase activity and attenuated PDGF-stimulated migration of wild type PDGFR-beta cells. Treatment of wild type PDGFR-beta cells with combinations of PAO or orthovanadate and phosphatidylinositol 3-kinase inhibitors wortmannin or LY294002 resulted in a synergistic inhibition of PDGFR-beta-mediated cell migration. PDGF stimulation of wild type PDGFR-beta cells led to induction of p125 focal adhesion kinase (FAK) activity at low concentrations of the growth factor and a decrease at higher concentrations. In the mutant Y1009F cells and in wild type PDGFR-beta cells treated with PAO and orthovanadate, FAK activity was not increased in response to PDGF. These results suggest that SHP-2 activity is involved in regulation of FAK activity and thereby of cell migration through PDGFR-beta, independently of phosphatidylinositol 3-kinase.     

The protein tyrosine phosphatase SHP-2 is required for EGFRvIII oncogenic transformation in human glioblastoma cells

Oncogenic EGFRvIII is a naturally occurring oncoprotein and is expressed in about 40-50% of human glioblastomas, particularly those that arise de novo. To understand the molecular mechanisms by which this oncoprotein alters transforming phenotypes, and since our previous work indicated that SHP-2 protein tyrosine phosphatase activity modulated EGFRvIII activation and downstream signaling, we examined whether SHP-2 plays a role in EGFRvIII-induced oncogenesis by using both PTEN-deficient U87MG.EGFRvIII and PTEN-intact LN229.EGFRvIII cells. Inhibition of SHP-2 expression by Shp-2 siRNA inhibited cell growth, transformation and altered morphology of these EGFRvIII transformed GBM cells. Ectopic expression of a PTPase-inactive form of SHP-2, SHP-2 C459S, but not its wild-type SHP-2 or either of two SH2 domain mutants, abrogated transformation of EGFRvIII-expressing glioblastomas in soft agar and in nude mice. SHP-2 C459S cells grew slower and exhibited a more flattened morphology with more organized actin stress fibers under both full growth and low serum conditions. Furthermore, shp-2+/− and −/− mouse embryonic fibroblasts (MEFs) could not be transformed by EGFRvIII while shp-2+/+ MEFs displayed a fully transformed phenotype upon introduction of EGFRvIII, again indicating a requirement for functional SHP-2 in EGFRvIII transformation. Moreover, the SHP-2 PTPase activity inhibitor NSC-87877 inhibited endogenous SHP-2 activity, Erk phosphorylation and transformation in both GBM cell lines. EGFRvIII expression recruited SHP-2 to the receptor complex to transduce signals and also increased SHP-2 phosphorylation at Tyr542. Inhibition of EGFRvIII-induced cell growth and transformation by SHP-2 C459S or shp-2 siRNA was mediated by its ability to block cell cycle progression at different phases in these GBM cells. These data indicate that differential activation of SHP-2 phosphorylation at Tyr542 in these two GBM cell lines likely results in increased different PTPase activity and distinct mechanisms of cell cycle progression and SHP-2, in particular its PTPase activity, plays a critical role in EGFRvIII-mediated transformation.