Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Adhesion to fibronectin stimulates proliferation of wild-type and bcr/abl-transfected murine hematopoietic cells

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased β1 integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of α5β1 integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G1 or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G1/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27Kip1. Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27Kip1, resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G1/S adhesion checkpoint.

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter resulted in a loss of β-galactosidase expression in the telencephalon and mesencephalon. Collectively, our experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development.

Measuring the forces involved in polyvalent adhesion of uropathogenic Escherichia coli to mannose-presenting surfaces

Mechanisms of bacterial pathogenesis have become an increasingly important subject as pathogens have become increasingly resistant to current antibiotics. The adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is a plausible target for new antiinfective agents. Examination of bacterial adhesion has been difficult both because it is polyvalent and because bacterial adhesins often recognize more than one type of cell-surface molecule. This paper describes an experimental procedure that measures the forces of adhesion resulting from the interaction of uropathogenic Escherichia coli to molecularly well defined models of cellular surfaces. This procedure uses self-assembled monolayers (SAMs) to model the surface of epithelial cells and optical tweezers to manipulate the bacteria. Optical tweezers orient the bacteria relative to the surface and, thus, limit the number of points of attachment (that is, the valency of attachment). Using this combination, it was possible to quantify the force required to break a single interaction between pilus and mannose groups linked to the SAM. These results demonstrate the deconvolution and characterization of complicated events in microbial adhesion in terms of specific molecular interactions. They also suggest that the combination of optical tweezers and appropriately functionalized SAMs is a uniquely synergistic system with which to study polyvalent adhesion of bacteria to biologically relevant surfaces and with which to screen for inhibitors of this adhesion.

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the α subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gzα also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Giα family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor β receptor

The bovine papillomavirus E5 protein is a 44-aa transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor and induces constitutive tyrosine phosphorylation and activation of the receptor, resulting in cell transformation. The E5 protein does not resemble PDGF, but rather activates the receptor in a ligand-independent fashion, thus providing a unique system to examine activation of receptor tyrosine kinases. Here, we used a variety of approaches to explore the mechanism of receptor activation by the E5 protein. Chemical cross-linking experiments revealed that the E5 protein activated only a small fraction of the endogenous PDGF β receptor in transformed fibroblasts and suggested that this fraction was constitutively dimerized. Coimmunoprecipitation experiments using extracts of cells engineered to coexpress full-length and truncated PDGF β receptors confirmed that the E5 protein induced oligomerization of the receptor. Furthermore, in cells expressing the E5 protein, a kinase-active receptor was able to trans-phosphorylate a kinase-negative mutant receptor but was unable to catalyze intramolecular autophosphorylation. These results indicated that the E5 protein induced PDGF β receptor activation by forming a stable complex with the receptor, resulting in receptor dimerization and trans-phosphorylation.

Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.

Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.

Forced unfolding modulated by disulfide bonds in the Ig domains of a cell adhesion molecule

Cell adhesion molecules (CAMs) mediate cell attachment and stress transfer through extracellular domains. Here we forcibly unfold the Ig domains of a prototypical Ig superfamily CAM that contains intradomain disulfide bonds. The Ig domains of all such CAMs have conformations homologous to cadherin extracellular domains, titin Ig-type domains, and fibronectin type-III (FNIII) domains. Atomic force microscopy has been used to extend the five Ig domains of Mel-CAM (melanoma CAM)—a protein that is overexpressed in metastatic melanomas—under conditions where the disulfide bonds were either left intact or disrupted through reduction. Under physiological conditions where intradomain disulfide bonds are intact, partial unfolding was observed at forces far smaller than those reported previously for either titin's Ig-type domains or tenascin's FNIII domains. This partial unfolding under low force may be an important mechanism for imparting elasticity to cell–cell contacts, as well as a regulatory mechanism for adhesive interactions. Under reducing conditions, Mel-CAM's Ig domains were found to fully unfold through a partially folded state and at slightly higher forces. The results suggest that, in divergent evolution of all such domains, stabilization imparted by disulfide bonds relaxes requirements for strong, noncovalent, folded-state interactions.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria

Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.

A thrombin receptor function for platelet glycoprotein Ib–IX unmasked by cleavage of glycoprotein V

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V −/− platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib–IX–V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib–IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (Gq-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib–IX–V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib–IX via thrombin acting as a ligand, resulting in platelet activation.

Integrin activation controls metastasis in human breast cancer

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin αvβ3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin αvβ3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Sp1 phosphorylation regulates inducible expression of platelet-derived growth factor B-chain gene via atypical protein kinase C-ζ

Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-ζ blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-ζ (on residue Thr410). These findings demonstrate for the first time that PKC-ζ and Sp1 phosphorylation mediate the inducible expression of this growth factor.