Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

Roles of fibroblast growth factor receptors in carcinogenesis

The fibroblast growth factor receptors (FGFR) play essential roles both during development and in the adult. Upon ligand binding, FGFRs induce intracellular signaling networks that tightly regulate key biological processes, such as cell proliferation, survival, migration, and differentiation. Deregulation of FGFR signaling can thus alter tissue homeostasis and has been associated with several developmental syndromes as well as with many types of cancer. In human cancer, FGFRs have been found to be deregulated by multiple mechanisms, including aberrant expression, mutations, chromosomal rearrangements, and amplifications. In this review, we will give an overview of the main FGFR alterations described in human cancer to date and discuss their contribution to cancer progression.     

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.     

Quantitative assessment of FGF regulation by cell surface heparan sulfates

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.     

Structural basis for fibroblast growth factor receptor activation

FGF signaling plays a ubiquitous role in human biology as a regulator of embryonic development, homeostasis and regenerative processes. In addition, aberrant FGF signaling leads to diverse human pathologies including skeletal, olfactory, and metabolic disorders as well as cancer. FGFs execute their pleiotropic biological actions by binding, dimerizing and activating cell surface FGF receptors (FGFRs). Proper regulation of FGF-FGFR binding specificity is essential for the regulation of FGF signaling and is achieved through primary sequence variations among the 18 FGFs and seven FGFRs. The severity of human skeletal syndromes arising from mutations that violate FGF-FGFR specificity is a testament to the importance of maintaining precision in FGF-FGFR specificity. The discovery that heparin/heparan sulfate (HS) proteoglycans are required for FGF signaling led to numerous models for FGFR dimerization and heralded one of the most controversial issues in FGF signaling. Recent crystallographic analyses have led to two fundamentally different models for FGFR dimerization. These models differ in both the stoichiometry and minimal length of heparin required for dimerization, the quaternary arrangement of FGF, FGFR and heparin in the dimer, and in the mechanism of 1:1 FGF-FGFR recognition and specificity. In this review, we provide an overview of recent structural and biochemical studies used to differentiate between the two crystallographic models. Interestingly, the structural and biophysical analyses of naturally occurring pathogenic FGFR mutations have provided the most compelling and unbiased evidences for the correct mechanisms for FGF-FGFR dimerization and binding specificity. The structural analyses of different FGF-FGFR complexes have also shed light on the intricate mechanisms determining FGF-FGFR binding specificity and promiscuity and also provide a plausible explanation for the molecular basis of a large number craniosynostosis mutations.     

Intracellular retention, degradation, and signaling of glycosylation-deficient FGFR2 and craniosynostosis syndrome-associated FGFR2C278F

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are known to play a critical role in a variety of fundamental processes, including wound healing, angiogenesis, and development of multiple organ systems. Mutations in the FGFR gene family have been linked to a series of syndromes (the craniosynostosis syndromes) whose primary phenotype involves aberrant development of the craniofacial skeleton. Craniosynostosis syndrome-linked FGFR mutations have been shown to be gain of function in terms of receptor activation and have been presumed to result in increased levels of FGF/FGFR signaling. Unfortunately, studies attempting to link expression of mutant FGFRs with changes in cellular phenotype have yielded conflicting results. In an effort to better understand the biochemical consequences of these mutations on receptor function, here we have investigated the effect of the FGFR2C278F mutation of Crouzon craniosynostosis syndrome on receptor trafficking, ubiquitination, degradation, and signaling. We find that FGFR2C278F exhibits diminished glycosylation, increased degradation, and limited cellular sublocalization in the osteoblastic cell line, MC3T3E1(C4). Additionally, we show that trafficking and autoactivation of wild type FGFR2 is glycosylation-dependent. Both FGFR2C278F and unglycosylated wild type FGFR2 signal through phospholipase Cgamma in a ligand-independent manner as well as exhibit dramatically increased binding to the adaptor protein, Frs2. These findings suggest that autoactive FGFR2 can signal from intracellular compartments. Based upon our results, we propose that the functional signaling of craniosynostosis mutant, autoactive receptors is limited in some cell types by protective cellular responses, such as increased trafficking to lysosomes and proteasomes for degradation.     

Role of fibroblast growth factors in elicitation of cell responses

Fibroblast growth factors (FGFs) are signalling peptides that control important cell processes such as proliferation, differentiation, migration, adhesion and survival. Through binding to different types of receptor on the cell surface, these peptides can have different effects on a target cell, the effect achieved depending on many features. Thus, each of the known FGFs elicits specific biological responses. FGF receptors (FGFR 1–5) initiate diverse intracellular pathways, which in turn lead to a variety of results. FGFs also bind the range of FGFRs with a series of affinities and each type of cells expresses FGFRs in different qualitative and quantitative patterns, which also affect responses. To summarize, cell response to binding of an FGF ligand depends on type of FGF, FGF receptor and target cell, all interacting in concert. This review aims to examine properties of the FGF family and its members receptors. It also aims to summarize features of intracellular signalling and highlight differential effects of the various FGFs in different circumstances.     

The physical basis of FGFR3 response to fgf1 and fgf2

Fibroblast growth factors (fgfs) play important roles in embryonic development and in adult life by controlling cell proliferation, differentiation, and migration. There are 18 known fgfs which activate four fibroblast growth factor receptors (FGFRs), with different isoforms due to alternative splicing. The physical basis behind the specificity of the biological responses mediated by different fgf-FGFR pairs is currently unknown. To gain insight into the specificity of FGFR3c, a membrane receptor which is critical for bone development, we studied, analyzed, and compared the activation of FGFR3c over a wide range of fgf1 and fgf2 concentrations. We found that while the strength of fgf2 binding to FGFR3c is lower than the strength of fgf1 binding, the fgf2-bound dimers exhibit higher phosphorylation of the critical tyrosines in the activation loop. As a result, fgf1 and fgf2 elicit a similar FGFR3c response at low, but not at high, concentrations. The results demonstrate the versatility of FGFR3c response to fgf1 and fgf2 and highlight the complexity in fgf signaling.     

14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival

Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3ζ is phosphorylated on Tyr¹⁷⁹ in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3ζ can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr¹⁷⁹. The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3ζ^-/- mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3ζ^-/- mice when reconstituted with wild type 14-3-3ζ, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.The ability of a cell to respond to extrinsic stimuli critically hinges on its ability to regulate specific intracellular protein-protein interactions in a reversible manner. Such signals are relayed within the cell through the assembly of signaling complexes that are built using protein scaffolds. One important mechanism by which this occurs is via the binding of Src homology 2 (SH2)⁵ or phosphotyrosine-binding (PTB) domains to phosphotyrosine residues (1, 2). Importantly, the ability of individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in different proteins enables the assembly of purpose-built signaling complexes that promote signaling via specific pathways (3). In some cases, signaling proteins not only contain more than one SH2 and/or PTB domain but are also themselves tyrosine-phosphorylated, leading to a network of phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are also important for the assembly of signaling complexes. For example, the 14-3-3 family of proteins is able to bind phosphoserine/threonine residues in a sequence-specific context (RSX(S/T)XP and RXXX(S/T)XP, where (S/T) is phosphoserine/threonine) (4, 5). The 14-3-3 proteins have been proposed to function as “modifiers” or “sequestrators”; however, because of their dimeric structure, they have also been proposed to function as “adaptor” or “scaffold” proteins through their ability to bring together two serine/threonine phosphorylated proteins (4–7). Additionally, a number of phosphoserine/threonine-binding modules such as tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and BRCA1 C-terminal (BRCT) domains have been shown to interact with phosphoserine/threonine residues within a sequence-specific context and have also been proposed to be important for the assembly of multi-protein signaling complexes (8).The genes/cassettes encoding each phosphotyrosine- and phosphoserine/threonine-binding protein/module arose as a separate evolutionary event, and the DNA encoding these modules has been subject to frequent duplication and shuffling. For example, the 14-3-3 family of proteins is ubiquitously expressed in mammalian tissues and is composed of seven different isoforms, each encoded by a separate gene (6). In addition, duplication and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their wide distribution among signaling proteins. Yet, despite the frequent duplication and shuffling of the DNA encoding these domains throughout evolution, proteins that contain both a phosphotyrosine-binding cassette (e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette (e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This is perhaps surprising given the highly integrated nature of phosphotyrosine and phosphoserine/threonine signaling and would suggest that alternative strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to interact with Shc and provide a scaffold for the assembly of signaling complexes via both phosphoserine/threonine and phosphotyrosine residues. Our results show that Tyr¹⁷⁹ of 14-3-3ζ directly binds to the SH2 domain of Shc and that this interaction is critical for the assembly of a phosphatidylinositol (PI) 3-kinase signaling complex in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Moreover, we show that Tyr¹⁷⁹ of 14-3-3ζ is necessary and sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived from 14-3-3ζ^-/- mice with wild type (wt) or mutant 14-3-3ζ demonstrated an important role for Tyr¹⁷⁹ in cytokine-mediated Akt phosphorylation and cell survival. These multivalent 14-3-3:Shc scaffolds provide a novel mechanism by which phosphoserine/threonine and phosphotyrosine pathways can be integrated for the regulation of specific cellular responses.     

Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate. Implications for dynamic assembly of a ternary signaling complex

Heparan sulfate (HS) regulates the kinetics of fibroblast growth factor 2 (FGF2)-stimulated intracellular signaling and differentially activates cell proliferation of cells expressing different FGF receptors (FGFRs). Evidence suggests that HS interacts with both FGFs and FGFRs to form active ternary signaling complexes. Here we compare the interactions of two FGFRs with HS. We show that the ectodomains of FGFR1 IIIc and FGFR2 IIIc exhibit specific interactions with different characteristics for both heparin and porcine mucosal HS. These glycans are both known to activate FGF signaling via these receptors. FGFR2 interacts with a higher apparent affinity than FGFR1 despite both involving 6-O-, 2-O-, and N-sulfates. FGFR1 and FGFR2 bind heparin with mean association rate constants of 1.9 x 10(5) and 2.1 x 10(6) m(-1)s(-1), respectively, and dissociation rate constants of 1.2 x 10(-2) and 2.7 x 10(-2) s(-1), respectively. These produced calculated affinities of 63 and 13 nm, respectively. Hence, FGFR1 and FGFR2 bind to heparin chains with markedly different kinetics and affinities. We propose a mechanistic model where the kinetic parameters of the HS/FGFR interaction are a key element regulating the formation of ternary complexes and the resulting FGF signaling outcomes.     

Functional roles of fibroblast growth factor receptors (FGFRs) signaling in human cancers

The fibroblast growth factor receptors (FGFRs) regulate important biological processes including cell proliferation and differentiation during development and tissue repair. Over the past decades, numerous pathological conditions and developmental syndromes have emerged as a consequence of deregulation in the FGFRs signaling network. This review aims to provide an overview of FGFR family, their complex signaling pathways in tumorigenesis, and the current development and application of therapeutics targeting the FGFRs signaling for treatment of refractory human cancers.     

Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis

Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.     

Small molecule inhibition of fibroblast growth factor receptors in cancer

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs), which are a sub-family of the superfamily of receptor tyrosine kinases, to regulate human development and metabolism. Uncontrolled FGF signaling is responsible for diverse array of developmental disorders, most notably skeletal syndromes due to FGFR gain-of-function mutations. Studies in the last few years have provided significant evidence for the importance of FGF signaling in the pathogenesis of diverse cancers, including endometrial and bladder cancers. FGFs are both potent mitogenic and angiogenic factors and can contribute to carcinogenesis by stimulating cell proliferation and tumor angiogenesis. Gene knockout and pharmacological inhibition of FGFRs in in vivo and in vitro models validate FGFRs as a target for cancer treatment. Considerable efforts are being expended to develop specific, small-molecule inhibitors for treating FGFR-driven cancers. Recent reviews on the FGF/FGFR system have focused primarily on signaling, pathophysiology, and functions in cancer. In this article, we review the key roles of FGFR in cancer, provide an update on the status of clinical trials with small-molecule FGFR inhibitors, and discuss how the current structural data on FGFR kinases guide the design and characterization of new FGFR inhibitors.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Roles of FGFR in oral carcinogenesis

Fibroblast growth factor receptors (FGFRs) play essential roles in organ development during the embryonic period, and regulate tissue repair in adults. Accumulating evidence suggests that alterations in FGFR signalling are involved in diverse types of cancer. In this review, we focus on aberrant regulation of FGFRs in pathogenesis of oral squamous cell carcinoma (OSCC), including altered expression and subcellular location, aberrant isoform splicing and mutations. We also provide an overview of oncogenic roles of each FGFR and its downstream signalling pathways in regulating OSCC cell proliferation and metastasis. Finally, we discuss potential application of FGFRs as anti‐cancer targets in the preclinical environment and in clinical practice.     

Variant heparan sulfates synthesized in developing mouse brain differentially regulate FGF signaling

Heparan sulfates (HSs) exert critical regulatory actions on many proteins, including growth factors, and are essential for normal development. Variations in their specific sulfation patterns are known to regulate binding and signaling of fibroblast growth factors (FGFs) via tyrosine kinase receptors (FGFRs). We previously reported differences in sulfation patterns between HS species expressed by embryonic day 10 (E10) and E12 mouse neural precursor cells. We have examined the abilities of the different HS species to support signaling of the relevant FGF-FGFR combinations expressed early during brain development. For FGF8, which only functions early (E8-E11), E10 HS showed preferential activation. The most potent signaling for FGF8 was via FGFR3c, for which E10 HS was strongly active and E12 HS had no activity. For FGF2, which functions from E10 to E13, HS from both stages showed similar activity and were more potent at activating FGFR1c than the other receptors. Thus, we find a stage-specific correlation with activation. To explore the potential mechanisms for the generation of these stage-specific HS species, we investigated the expression of the HS sulfotransferase (HSST) isozymes responsible for creating diverse sulfation motifs in HS chains. We find that there are stage-specific combinations of HSST isozymes that could underlie the synthesis of different HS species at E10 and E12. Collectively, these data lead us to propose a model in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.     

Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors

SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.