Getting RIP'd Stunts Your Growth

The p75 neurotrophin receptor (p75NTR) collaborates with the Nogo receptor (NgR) and LINGO-1 to activate RhoA in response to myelin-based growth inhibitors such as myelin-associated glycoprotein (MAG). In this issue of Neuron, Domeniconi et al., in a surprising turn, show that MAG induces intramembrane proteolysis (RIP) of p75NTR and find that p75NTR cleavage is required for MAG-induced RhoA activation and growth inhibition.     

Effects of S1 Cleavage on the Structure,Surface Export,and Signaling Activity of Human Notch1 and Notch2

Background

Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.

Principal Findings

The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.

Conclusions/Significance

S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.     

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival

Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.     

Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.     

FGFR3 intracellular mutations induce tyrosine phosphorylation in the Golgi and defective glycosylation

Mutations of the Fibroblast Growth Factor Receptor 3 (FGFR3) gene have been implicated in a series of skeletal dysplasias including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The severity of these diseases ranges from mild dwarfism to severe dwarfism and to perinatal lethality, respectively. Although it is considered that the mutations give rise to constitutively active receptors, it remains unclear how the different mutations are functionally linked to the severity of the different pathologies. By examining various FGFR3 mutations in a HEK cell culture model, including the uncharacterized X807R mutation, it was found that only the mutations affecting the intracellular domain, induced premature receptor phosphorylation and inhibited receptor glycosylation, suggesting that premature receptor tyrosine phosphorylation of the native receptor inhibits its glycosylation. Moreover, these mutations appeared to be associated with elevated receptor signaling in the Golgi apparatus. In conclusion, although pathological severity could not be correlated with a single factor arising from FGFR3 mutations, these results suggest that intracellular domain mutations define a distinct means by which mutated FGFR3 could disrupt bone development.     

Phorbol 12-myristate 13-acetate-induced release of the colony-stimulating factor 1 receptor cytoplasmic domain into the cytosol involves two separate cleavage events

The colony-stimulating factor 1 (CSF-1) receptor is a protein-tyrosine kinase that regulates cell division, differentiation, and development. In response to phorbol 12-myristate 13-acetate (PMA), the CSF-1 receptor is subject to proteolytic processing. Use of chimeric receptors indicates that the CSF-1 receptor is cleaved at least two times, once in the extracellular domain and once in the transmembrane domain. Cleavage in the extracellular domain results in ectodomain shedding while the cytoplasmic domain remains associated with the membrane. Intramembrane cleavage depends on the sequence of the transmembrane domain and results in the release of the cytoplasmic domain. This process can be blocked by gamma-secretase inhibitors. The cytoplasmic domain localizes partially to the nucleus, displays limited stability, and is degraded by the proteosome. CSF-1 receptors are continuously subject to down-modulation and regulated intramembrane proteolysis (RIP). RIP is stimulated by granulocyte-macrophage-CSF, CSF-1, interleukin-2 (IL-2), IL-4, lipopolysaccharide, and PMA and may provide the CSF-1 receptor with an additional mechanism for signal transduction.     

Bent bone dysplasia-FGFR2 type, a distinct skeletal disorder, has deficient canonical FGF signaling

Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia.     

Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.

Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor tyrosine kinase with the transmembrane domains of wild-type and mutant FGFR3, the Arg380 mutation in FGFR3 is shown to activate both the kinase and transforming activities of this chimeric receptor. Residues with side chains capable of participating in hydrogen bond formation, including Glu, Asp, and to a lesser extent, Gln, His and Lys, were able to substitute for the activating Arg380 mutation. The Arg380 point mutation also causes ligand-independent stimulation of the tyrosine kinase activity of FGFR3 itself, and greatly increased constitutive levels of phosphotyrosine on the receptor. These results suggest that the molecular basis of achondroplasia is unregulated signal transduction through FGFR3, which may result in inappropriate cartilage growth plate differentiation and thus abnormal long bone development. Achondroplasia may be one of the number of cogenital disorders where constitutive activation of a member of the FGFR family leads to development abnormalities.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains

Fibroblast growth factor receptors (FGFR) 1 and 3 have distinct mitogenic activities in vitro. In several cultured cell lines, FGFR1 transmits a potent mitogenic signal, whereas FGFR3 has little or no mitogenic activity. However, in other in vitro assays the FGFR3 intracellular domain is comparable with that of FGFR1. In vivo, FGFR3 negatively regulates chondrocyte proliferation and differentiation, and activating mutations are the molecular etiology of achondroplasia. By contrast, FGFR1 transmits a proliferative signal in various cell types in vivo. These observations suggest that inhibition of the proliferating chondrocyte could be a unique property of FGFR3 or, alternatively, a unique property of the proliferating chondrocyte. To test this hypothesis, FGFR1 signaling was activated in the growth plate in cells that normally express FGFR3. Comparison of transgenic mice with an activated FGFR1 signaling pathway with an achondroplasia-like mouse that expresses a similarly activated FGFR3 signaling pathway demonstrated that both transgenes result in a similar achondroplasia-like dwarfism. These data demonstrate that suppression of mitogenic activity by FGFR signaling is a property that is unique to growth plate chondrocytes. Surprisingly, we observed that in transgenic mice expressing an activated FGFR, some synovial joints failed to develop and were replaced by cartilage. The defects in the digit joints phenocopied the symphalangism that occurs in Apert syndrome and the number of affected joints was dependent on transgene dose. In contrast to the phenotype in the growth plate, the joint phenotype was more severe in transgenic mice with an activated FGFR1 signaling pathway. The failure of joint development resulted from expanded chondrification in the presumptive joint space, suggesting a crucial role for FGF signaling in regulating the transition of condensed mesenchyme to cartilage and in defining the boundary of skeletal elements.     

Activating FGFR3 mutations cause mild hyperplasia in human skin,but are insufficient to drive benign or malignant skin tumors

Fibroblast growth factor receptor 3 (FGFR3) activating mutations are drivers of malignancy in several human tissues, including bladder, lung, cervix, and blood. However, in skin, these mutations are associated predominantly with benign, common epidermal growths called seborrheic keratoses (SKs). How epidermis resists FGFR3 mediated transformation is unclear, but previous studies have suggested that FGFR3 activation in skin keratinocytes may serve a tumor-suppressive role by driving differentiation and antagonizing Ras signaling. To define the role of FGFR3 in human normal and neoplastic epidermis, and to directly test the hypothesis that FGFR3 antagonizes Ras, we engineered human skin grafts in vivo with mutant active FGFR3 or shRNA FGFR3 knockdown. We show that FGFR3 active mutants drive mild hyperproliferation, but are insufficient to support benign or malignant tumorigenesis, either alone, or in combination with G₁–S checkpoint release. This suggests that additional cell-intrinsic or stromal cues are required for formation of benign SKs with FGFR3 mutations. Further, FGFR3 activation does not alter the growth kinetics or differentiation status of engineered human epidermal SCCs driven by Ras, and FGFR3 protein itself is dispensable for Ras-driven SCC. To extend these findings to patients, we examined a uniquely informative human tumor in which SCC developed in continuity with a SK, raising the hypothesis that one of the tumors evolved from the other. However, mutational analysis from each tumor indicates that the overlapping SK and SCC evolved independently and supports our conclusion that FGFR3 activation is insufficient to drive SCC.     

Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14. Characterization of a new receptor binding partner

Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 binding partner. A kinase-inactive mutant of FGFR1 failed to interact with Grb14, indicating that activation of FGFR1 is necessary for binding. Deletion of the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to phenylalanine abolished binding, and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites. Co-immunoprecipitation and in vitro binding assays demonstrated that binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor activation by fibroblast growth factor-2 (FGF-2). Deletion of the Src homology 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and eliminated dependence on receptor activation. The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb14 bound only FGFR1, suggesting that regions upstream of the SH2 domain confer specificity for FGFR1. Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this phosphorylation. Expression of exogenous Grb14 inhibited FGF-2-induced cell proliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis. These data demonstrate an interaction between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.     

The Pathogenic A391E Mutation in FGFR3 Induces a Structural Change in the Transmembrane Domain Dimer

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.     

Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.     

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.     

Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases.

The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFalpha-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but our analysis suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to our knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and our results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.     

Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.     

The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr(402), the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr(402), indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr(402) of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.