A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation.

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.     

Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous beta receptor for platelet-derived growth factor in mouse C127 cells.

The bovine papillomavirus E5 protein is a 44-amino-acid membrane-associated protein that forms a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor in rodent and bovine fibroblasts, resulting in sustained receptor activation and cell transformation. We report here that high-level expression of the E5 protein caused a reduction in the level of the mature form of the PDGF beta receptor in acutely and stably transformed mouse C127 cells. To explore in more detail the interaction of the E5 protein and the PDGF beta receptor, we tested the abilities of various E5 point mutants to bind the PDGF receptor, to induce PDGF receptor down-regulation and tyrosine phosphorylation, and to transform cells. A transformation-competent mutant, like the wild-type E5 protein, bound the receptor and induced receptor tyrosine phosphorylation and down-regulation. Transformation-defective E5 proteins either failed to interact with the endogenous PDGF beta receptor in mouse fibroblasts or underwent an aberrant interaction with the receptor. Mutation of glutamine at position 17, aspartic acid at position 33, or both carboxyl-terminal cysteine residues required for E5 homodimerization interfered with stable complex formation with the PDGF receptor, tyrosine phosphorylation and down-regulation of the receptor, and cell transformation. Point mutations at several other carboxyl-terminal positions generated transformation-defective E5 proteins that formed a complex with the PDGF receptor and induced receptor tyrosine phosphorylation but did not induce PDGF receptor down-regulation. Either PDGF receptor activation is not sufficient for transformation of C127 cells or the receptors that are tyrosine phosphorylated in response to these mutant E5 proteins are not fully activated and therefore are not able to deliver a mitogenic signal.     

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.     

Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.     

Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signaling.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.     

Physiological Requirement for Both SH2 Domains for Phospholipase C-γ1 Function and Interaction with Platelet-Derived Growth Factor Receptors

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain

The epidermal growth factor receptor plays crucial roles throughout the development of multicellular organisms, and inappropriate activation of the receptor is associated with neoplastic transformation of many cell types. The receptor is thought to be activated by ligand-induced homodimerisation. Here, however, we show by chemical cross-linking and sucrose density-gradient centrifugation that in the absence of bound ligand the receptor has an ability to form a dimer and exists as a preformed dimer on the cell surface. We also analysed the receptor dimerisation by inserting cysteine residues at strategic positions about the putative alpha-helix axis of the extracellular juxtamembrane region. The mutant receptors spontaneously formed disulphide bridges and transformed NIH3T3 cells in the absence of ligand, depending upon the positions of the cysteine residue inserted. Kinetic analyses of the disulphide bonding indicate that EGF binding induces flexible rotation or twist of the juxtamembrane region of the receptor in the plane parallel with the lipid bilayer. The binding of an ATP competitor to the intracellular domain also induced similar flexible rotation of the juxtamembrane region. All the disulphide-bonded dimers had flexible ligand-binding domains with the same biphasic affinities for EGF as the wild-type. These results demonstrate that ligand binding to the flexible extracellular domains of the receptor dimer induce rotation or twist of the juxtamembrane regions, hence the transmembrane domains, and dissociate the dimeric, inactive form of the intracellular domains. The flexible rotation of the intracellular domains may be necessary for the intrinsic catalytic kinase to become accessible to the multiple tyrosine residues present in the regulatory domain and various substrates, and may be a common property of many cell-surface receptors, such as the insulin receptor.     

Strong oligomerization behavior of PDGFβ ?receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.     

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity.

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.     

Autoinhibition of the kit receptor tyrosine kinase by the cytosolic juxtamembrane region

Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.     

E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation

The E5 oncoprotein of bovine papillomavirus type 1 is a Golgi-resident, 44-amino acid polypeptide that can transform fibroblast cell lines by activating endogenous platelet-derived growth factor receptor beta (PDGF-R). However, the recent discovery of E5 mutants that exhibit strong transforming activity but minimal PDGF-R tyrosine phosphorylation indicates that E5 can potentially use additional signal transduction pathway(s) to transform cells. We now show that two classes of E5 mutants, despite poorly activating the PDGF-R, induce tyrosine phosphorylation and activation of phosphoinositide 3-kinase (PI 3-K) and that this activation is resistant to a selective inhibitor of PDGF-R kinase activity, tyrphostin AG1296. Consistent with this independence from PDGF-R signaling, the E5 mutants fail to induce significant cell proliferation in the absence of PDGF, unlike wild-type E5 or the sis oncoprotein. Despite differences in growth factor requirements, however, both wild-type E5 and mutant E5 cell lines form colonies in agarose. Interestingly, activation of PI 3-K occurs without concomitant activation of the ras-dependent mitogen-activated protein kinase pathway. The known ability of constitutively activated PI 3-K to induce anchorage-independent cell proliferation suggests a mechanism by which the mutant E5 proteins transform cells.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.     

PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex

Platelet-derived growth factor (PDGF) stimulated the tyrosine phosphorylation of the GTPase activating protein (GAP) in 3T3 cells and in CHO cells expressing wild-type PDGF receptors, but not in several CHO cell lines expressing mutant receptors defective in transmitting mitogenic signals. Following PDGF treatment of cells, GAP physically associated with the PDGF receptor and with Raf-1, phospholipase c-gamma, and PI-3 kinase, suggesting that PDGF induced the formation of complexes of signaling molecules. The association of GAP with the PDGF receptor and the phosphorylation of GAP with the PDGF receptor and the phosphorylation of GAP were reconstituted in vitro using purified protein and in insect cells expressing murine PDGF receptor and human GAP. However, in cells transformed by activated c-Ha-ras, which are defective in certain responses to PDGF, GAP failed to associate with the PDGF receptor or increase its phosphotyrosine content in response to PDGF. The association of GAP with ligand-activated PDGF receptors may directly link PDGF and ras signaling pathways.     

Regulation of the wild-type and Y1235D mutant Met kinase activation

Met receptor tyrosine kinase plays a crucial role in the regulation of a large number of cellular processes and, when deregulated by overexpression or mutations, leads to tumor growth and invasion. The Y1235D mutation identified in metastases was shown to induce constitutive activation and a motile-invasive phenotype on transduced carcinoma cells. Wild-type Met activation requires phosphorylation of both Y1234 and Y1235 in the activation loop. We mapped the major phosphorylation sites in the kinase domain of a recombinant Met protein and identified the known residues Y1234 and Y1235 as well as a new phosphorylation site at Y1194 in the hinge region. Combining activating and silencing mutations at these sites, we characterized in depth the mechanism of activation of wild-type and mutant Met proteins. We found that the phosphotyrosine mimetic mutation Y1235D is sufficient to confer constitutive kinase activity, which is not influenced by phosphorylation at Y1234. However, the specific activity of this mutant was lower than that observed for fully activated wild-type Met and induced less phosphorylation of Y1349 in the signaling site, indicating that this mutation cannot entirely compensate for a phosphorylated tyrosine at this position. The Y1194F silencing mutation yielded an enzyme that could be activated to a similar extent as the wild type but with significantly slower activation kinetics, underlying the importance of this residue, which is conserved among different tyrosine kinase receptors. Finally, we observed different interactions of wild-type and mutant Met with the inhibitor K252a that may have therapeutic implications for the selective inhibition of this kinase.     

Sphingosine 1-phosphate and platelet-derived growth factor (PDGF) act via PDGF beta receptor-sphingosine 1-phosphate receptor complexes in airway smooth muscle cells

Platelet-derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) act via PDGF beta receptor-S1P(1) receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S1P(1) antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF beta receptor-S1P(1) receptor complex. Third, treatment of cells with antisense S1P(1) receptor plasmid construct reduced the PDGF- and S1P-dependent activation of p42/p44 MAPK. Fourth, S1P and/or PDGF induced the formation of endocytic vesicles containing both PDGF beta receptors and S1P(1) receptors, which was required for activation of the p42/p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the presentation of sphingosine from other cell types and its conversion to S1P by the kinase exported from airway smooth muscle cells might enable S1P to act with PDGF on the PDGF beta receptor-S1P(1) receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-protein-coupled receptor agonists and/or sequential release and action of S1P in response to PDGF.     

The juxtamembrane region of MuSK has a critical role in agrin-mediated signaling

MuSK is a receptor tyrosine kinase expressed selectively in skeletal muscle and localized to neuromuscular synapses. Agrin activates MuSK and stimulates phosphorylation and clustering of acetylcholine receptors (AChRs) at synaptic sites. We expressed wild-type or mutant MuSK in MuSK(-/-) myotubes and identified tyrosine residues in the MuSK cytoplasmic domain that are necessary for agrin-stimulated phosphorylation and clustering of AChRs. The activation loop tyrosines and the single juxtamembrane tyrosine were found to be essential for agrin-stimulated phosphorylation and clustering of AChRs. Further, we show that the juxtamembrane tyrosine, contained within an NPXY motif, is phosphorylated in vivo by agrin stimulation. We constructed chimeras containing extracellular and transmembrane domains from MuSK and cytoplasmic sequences from TrkA and found that inclusion of 13 amino acids from the MuSK juxtamembrane region, including the NPXY motif, is sufficient to convert a phosphorylated but inactive MuSK-TrkA chimera into a phosphorylated active chimera. These data suggest that phosphorylation of the MuSK NPXY site leads to recruitment of a phosphotyrosine-binding domain-containing protein that functions to stimulate phosphorylation and clustering of AChRs.     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.