FGFR2IIIb is the resident FGFR isotype in differentiated epithelial cells in most multi-compartment adult squamous and glandular epithelia. Exclusive expression of exon IIIb instead of IIIc gene in epithelial cells confers specificity of the ectodomain of the FGFR2 transmembrane receptor tyrosine kinase for FGF7 that fails to activate other FGFR isoforms [29
]. Unlike numerous other FGFs, the expression of FGF7 is limited to the stromal cell compartment in adult tissues where FGFR2IIIb is expressed in the epithelium [28
]. Among the cellular FGFs, access of stromal-derived FGF7 to epithelial FGFR2IIIb is stringently regulated by abundant heparan sulfate oligosaccharides with a spectrum of charge density in the extracellular matrix between stroma and epithelium. The structural requirements for a heparan sulfate oligosaccharide to form an active oligomeric complex of FGFR2IIIb and FGF7 is the most stringent of those studied to date [5
]. Consistent with stringent co-factor control and its resident status in differentiated epithelial cells, FGFR2IIIb plays a role in maintenance of cellular and compartmental homeostasis in epithelial tissues [30
]. FGFR2IIIb is capable of supporting epithelial cell proliferation, but the proliferation is self-limited and often concurrent with differentiation [28
]. This property has been exploited for application of FGF7 (Palifermin, Kepivance™), the first FDA approved FGF for clinical use, as an adjuvant in cancer chemotherapy to alleviate severe mucosititis and restoration of damaged epithelia [58
]. The net homeostasis-promoting activity of FGFR2IIIb in epithelial cells is in marked contrast to the normally mesenchymal or stromal cell-associated homologue FGFR1 that largely has been implicated in cell proliferation and ancillary cellular activities most commonly associated with mesenchymal-derived cells as motility and migration. Ectopic expression or appearance of FGFR1 is observed in numerous epithelial-derived tumor cells and in a variety of tumor progression models, which imparts properties associated with normal mesenchymal cells constitutively to epithelial cells [34
]. These properties are predominantly proliferation and multiple phenotypes that drive malignancy. In contrast, loss of stromal-epithelial compartmental homeostasis concurrent with loss of FGFR2IIIb or FGFR2 expression altogether is often associated with progression to malignancy [29
]. Restoration of FGFR2 to tumor epithelial cells restores responsiveness to stroma, differentiation in the epithelial compartment, and balance between stroma and epithelium that results in a net limitation in tumor growth and malignancy [32
]. Consequently, FGFR2IIIb may serve as a “tumor suppressor” and a potential avenue for anti-tumor therapy by restoration of normal compartmental homeostasis or even cell death [31
In this study, we have produced new clues and new potential downstream targets that shed light on molecular aspects of the multiple functions of growth stimulation, growth inhibition and induction of differentiation supported by FGFR2IIIb signaling relative to FGFR1 through tyrosine phosphoproteomic analysis. In addition to the “priming” strategy, we employed the same cell model for a better comparison to oncogenic FGFR1, the HEK293 cells that were used in an FGFR1 phosphoproteomic study. These cells appear to have undetectable or very low tyrosine phosphorylation background after short-term serum restriction. FGFR2IIIb was introduced into the naïve HEK293 cells with no previous history of expression of FGFR2IIIb by controlled expression of an inducible stably transfected FGFR2IIIb gene. This preserved an absolute dependence on and quick response to exogenous FGF7, a ligand that only activates FGFR2IIIb. This prevented adaptation and selection for only the growth stimulatory elements of FGFR2IIIb signaling. An FGF7-dependent response of the pTyr proteome to FGFR2IIIb required “priming” of the naïve HEK293 cells to the transient presence of activated FGFR2IIIb followed by reduction of pTyr proteins to negligible levels by medium restrictions. The mechanism underlying this short-term priming phenomenon in cells originally unresponsive to the FGF7/FGFR2IIIb pair is of considerable interest. The priming occurs in an insufficient time frame for cell division and evolution via selection of a responsive population. Although all isotypes of FGFRs including ectodomain splice variants IIIb and IIIc are expressed at the mRNA level, our preliminary results further indicate that FGFR1 is the predominant endogenous FGFR isotype expressed, albeit at very low but a still functional level in HEK293 cells. Thus it is likely that the cell population has undergone long-term adaptation and selection for the proliferative response driven by FGFR1 stimulated pathways even though it appears to be no longer dependent on it (unpublished results).
FGFR2 exhibits all seven homologous tyrosines that have been observed to be phosphorylated in the intracellular domain of FGFR1 [19
] (). Five FGFR1 pTyr counterparts [21
] were observed in FGFR2 in our analysis compared to three in the FGFR1 analysis [42
]. The role of FGFR1 tyrosines other than the counterparts of FGFR2 Tyr 656, 657 and 769 in FGF signaling is unclear. FGFR1 counterparts of FGFR2 Tyr 656 and 657 are involved in repression/derepression via transactivating FGFR kinase phosphorylation rather than recruitment of diverse signaling mediators [19
]. The FGFR1 counterpart of FGFR2 Tyr 769 is the only direct binding site for a signal mediator (PLCγ) validated to date. Although FGFR2 pTyr 769 was observed neither in our analysis nor FGFR1 Tyr766 in the FGFR1 analysis [42
], FGF-stimulated pTyr-PLCγ was observed in both. Although low yield of the pTyr 769-containing peptide cannot be eliminated in both the FGFR1 and FGFR2 analyses, the results raise the possibility that PLCγ may access FGFR by mechanisms other than recruitment to pTyr 769. There are no clear differences between tyrosine phosphorylations on FGFR2 and FGFR1 that would suggest differences in signaling between the two FGFR isotypes.
Unlike other receptor tyrosine kinases, FGFR signaling is largely mediated through a single membrane-bound tyrosine phosphorylated substrate, FRS2, that organizes diverse signal pathway mediators that are recruited to its pTyr sites directly or bind to it independently of these sites [25
]. Although we did not detect FRS2 pTyr peptides in the proteomic screen, separate immunoprecipitation indicated that FRS2α is phosphorylated in response to FGF7. Detection of pTyr-SHP2 that is a major substrate of specific pTyr residues in pTyr-FRS2α [48
] further suggested that FRS2α is a major mediator of FGF7/FGFR2IIIb signaling in cells including the HEK293 cells utilized as host in this study. In contrast, the FGFR1 proteomic study in the same cells also failed to detect pTyr-FRS2α causing the authors to suggest that FRS2α may be deficient in the HEK293 cells [42
]. Separate results not shown here also indicate that FRS2α levels are significant in this cell line.
Consistent with its growth promotion properties and overlapping with FGF2/FGFR1 [42
], the FGF7/FGFR2IIIb pair induced tyrosine phosphorylation of the canonical ERK2, a central mediator of the RAF-RAS pathway that is a hallmark correlate of both compensatory growth and constitutive oncogenic growth [60
]. In addition the FGF7/FGFR2IIIb pair elicits tyrosine phosphorylation of IRS4. IRS4 has been implicated in mediating the growth-associated elements of growth hormone, insulin and IGF-1 in diverse tissues and models [55
]. Notably the FGF7/FGFR2IIIb pair elicited phosphorylation of cell cycle promoter CDK2 ser/thr kinase on Y15 which was not apparent in the reported FGFR1 analyses [42
]. pTyr15-CDK2 is an inhibitory event in respect to CDK2 ser/thr kinase activity and thus an inhibitory event on cell cycle in respect to the role of CDK2 [56
]. Therefore phosphorylation of Tyr15 on CDK2 may contribute to the growth controlling aspects of the FGF7/FGFR2IIIb pair observed in epithelial cells [33
The FGF7/FGFR2IIIb pair elicited tyrosine phosphorylation of tyrosine phosphatases SHP2, SHIP2 and PTPN18. SHP2 and SHIP2, but not PTPN18 were also detected in the FGF2/FGFR1 analyses [42
]. pTyr phosphatases intuitively are predicted to attenuate positive aspects of phosphotyrosine signaling and in particular tyrosine kinase driven cell growth cascades. However, through activation of its phosphatase activity via pTyr 62, SHP2 is a strong enhancer of the canonical RAS-MAP kinase pathway and hyperactivation has been shown to be oncogenic [49
]. Although pTyr-SHP2 is predicted to be a part of the growth promoting function of FGFR2IIIb along with the RAS-MAP kinase pathway, participation of SHP2 and the associated pathway in the growth attenuation and differentiation functions of FGFR2IIIb cannot be eliminated. The selective phosphorylation of PTPN18 may further add to the selective growth attenuation function of FGFR2 through downregulation of growth-stimulatory signaling including that of the EGFR family [53
A number of pTyr proteins observed in FGF2/FGFR1 signaling were notably absent from the FGF7/FGFR2IIIb pTyr proteome. These substrates included a number of kinases or kinase-modifying factors (RSK2, PIX, GIT, FAK, PI3K, ShcA) as well as cytoskeletal components or modifiers (ODIN, paxillin, p130Cas, Nck-associated protein, vimentin, annexin) that largely are related to cell adhesion and motility. Alterations in most are generally associated with tumorigenic phenotypes. Instead the FGF7/FGFR2 pair caused tyrosine phosphorylation of several factors that maintain nuclear structure and nuclear interactions with cytosolic elements (emerin and LAP2), transcription regulation, protein folding and other cellular fine structures as well as novel proteins of unknown function. Among them are several pTyr-proteins associated with growth limitation and tumor suppression and their disruption is associated with tumor promotion [62
In summary, our results indicate that FGF7-activated FGFR2IIIb shares a limited subset of phosphotyrosine targets generally associated with FGFR1 and other growth promoting kinases, but also exhibit novel FGFR2IIIb-specific targets associated with growth limitation in the same context. This is consistent with observed differences among the two isotypes in growth, migration, growth limitation and differentiation. Our results provide specific targets for further study of differences between the two FGFRs in diverse biological contexts and associated diseases. The “induction and prime” approach used here should be useful for dissection of differences in the phosphoproteome elicited by combinations of FGFs, FGFR and co-factors heparan sulfate and klothos associated with the family’s increasingly diverse roles in both cellular and metabolic homeostasis [6
]. A major challenge is to dissect how the overlapping and unique subsets of pTyr substrates observed among the FGFR isotypes are generated from co-factor and cell context-dependent conformational changes within the ecto- and intracellular domains of the transmembrane FGFR signaling complex.