Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins

Domain structure of FRS2 proteins

FRS2α and FRS2β are similar in structure. Both proteins contain amino acid residues MGSCCS, a consensus myristylation sequence (MGXXXS/T), at the N‐terminus for binding to lipids in the plasma membrane constitutively^{( 1} , ¹² , ^{19 )} (Fig. 2). Each has a PTB domain and multiple tyrosine phosphorylation sites at the C‐terminus. FRS2α has six tyrosine phosphorylation sites and FRS2β has five. The PTB domains are highly homologous and 72% of amino acids are identical. The residues surrounding each tyrosine phosphorylation site are similar but more than 10 amino acids from the sites there is no similarity. There is also a certain amount of similarity in the C‐terminus.

Cellular signaling upstream of FRS2 proteins

FRS2 proteins act as docking proteins downstream of limited species of RTKs, including FGF receptors, neurotrophin receptors,^{( 12} , ^{20 )} RET,^{( 21} , ²² , ^{23 )} and ALK.^{( 24 )} In particular, emerging evidence indicates that FRS2α is a ‘conning center’ for intracellular signaling in response to FGF as described in detail below. FRS2 proteins bind to these RTKs via the PTB domain and become phosphorylated on tyrosine residues upon activation of these RTKs. In contrast, FRS2 proteins are not good substrates for other RTKs, insulin receptors, platelet‐derived growth factor (PDGF) receptors and so on.^{( 25 )} FRS2α is phosphorylated on tyrosine residues by epidermal growth factor (EGF) receptor kinases in A431 cells where EGF receptors are overexpressed^{( 26 )} while EGF‐induced tyrosine phosphorylation of FRS2α seems to be not detectable in other cells that have modest amounts of EGF receptors.^{( 9} , ^{25 )} The specificity, in that FRS2 proteins choose only several tyrosine kinases to be phosphorylated, is characteristic of this protein family. The PTB domain is able to bind to specific peptides with or without tyrosine‐phosphorylated residues. The PTB domains of FRS2 proteins bind to unphosphorylated peptides at the juxtamembrane domain of the FGF receptor and the binding is stable with or without activation of the receptors^{( 13} , ^{20 )} (Fig. 3). In contrast, binding between the PTB domain of FRS2 and neurotrophin receptor TrkA and TrkB, or RET is mediated by tyrosine phosphorylated peptides in the receptors and is dependent on activation of the RTKs.^{( 20} , ²¹ , ²² , ²⁷ , ²⁸ , ²⁹ , ^{30 )} The t(2;5) chromosomal translocation found in anaplastic large‐cell lymphoma generates the nucleophosmin (NPM)‐ALK chimeric oncoprotein that carries portion of NPM and the tyrosine kinase domain of ALK.^{( 31 )} NPM‐ALK proteins have two FRS2 binding sites containing phosphorylated tyrosine residues and one FRS2 binding site without tyrosine phosphorylation.^{( 32 )} The crystallographic structure of the complex between the PTB domain of FRS2α and the peptides containing the binding sites of the FGF receptor or TrkA has revealed that there are two different binding pockets in the PTB domain for the phosphorylated and unphosphorylated peptides on tyrosine residues.^{( 19} , ²⁷ , ^{33 )} Due to constitutive binding between the FGF receptor and FRS2 proteins, it is possible that FRS2 is phosphorylated by the FGF receptor tyrosine kinase as efficiently as the receptor itself.

Laloo is one of the Src like kinases in Xenopus and functions in FGF signaling. It promotes tyrosine phosphorylation of xFRS2, whose PTB domain seems to bind to Laloo.^{( 16} , ^{17 )}

We recently found that the PTB domain of FRS2β binds to the EGF receptors. Moreover, FRS2β does not become phosphorylated by EGF receptor tyrosine kinases. Instead, it inhibits the tyrosine kinase activity of the receptors.^{( 34} , ^{35 )} Our finding clearly demonstrated that FRS2β is a unique scaffolding adaptor that serves as a negative as well as a positive regulator for RTK signaling dependent on the receptor species.

Cellular signaling downstream of FRS2 proteins

The four tyrosine phosphorylation sites on FRS2α and the three tyrosine phosphorylation sites on FRS2β bind to the Grb2 adaptor^{( 9} , ^{12 )} (Fig. 2). All these peptides contain the consensus sequence for the SH2 domain of Grb2, YXNX. The other two tyrosine phosphorylation sites on FRS2α and FRS2β bind to Shp2, the SH2‐containing tyrosine phosphatase.^{( 12} , ³⁶ , ^{37 )} Several proteins are constitutively bound via two SH3 domains of Grb2: SOS, Cbl, and Gab1.^{( 19} , ³⁸ , ^{39 )} SOS is a guanine nucleotide exchange factor (GEF) for Ras, and FRS2α‐mediated recruitment of Grb2‐SOS induces activation of the Ras/ERK pathway. Cbl functions as an E3 ubiquitin ligase and the ternary complex FRS2α‐Grb2‐Cbl results in ubiquitination and degradation of FRS2α and receptors. The ternary complex FRS2α‐Grb2‐Gab1 enables tyrosine phosphorylation of Gab1 followed by recruitment of PI‐3 kinase and activation of a cell survival pathway. SOS binds to the N‐terminal SH3 domain of Grb2, Gab1 binds to the C‐terminal SH3 domain of Grb2, and Cbl binds to both N‐ and C‐terminal SH3 domains of Grb2. Thus FRS2α assembles both positive and negative signaling proteins to mediate a balanced signal transduction. The FRS2α‐Shp2 complex induces tyrosine phosphorylation of Shp2 followed by strong activation of ERK in response to FGF.^{( 36 )} Tyrosine phosphorylated Shp2 activates its own tyrosine phosphatase, resulting in strong activation of the Ras/ERK pathway in numerous cell contexts, though the precise mechanisms by which Shp2 activates Ras are still controversial.^{( 40 )}

Growth factor‐induced activation of ERK is subdivided into two phases, a transient phase and a sustained phase.^{( 41 )} After stimulation with growth factors, the activation peaks within 5 min and returns to basal levels within 1 h in a transient phase, while it lasts for several hours with a gradual reduction in a sustained phase. Several growth factors such as EGF induce prominent levels of ERK activation in a transient manner but not in a sustained manner, while patterns of ERK activation induced by other growth factors such as FGF and NGF fit with a pattern of summation of both transient and sustained phases. In FGF signaling, FRS2α appears to be a critical mediator that is able to induce the sustained phase of ERK activation in cells lacking endogenous FRS2β.^{( 42 )} When FRS2β is exogenously expressed in FRS2α–null mouse embryonic fibroblasts, it compensates for the loss of FRS2α in FGF‐stimulated activation of ERK.^{( 12 )} Thus FRS2β is also able to activate ERK in a sustained manner in response to FGF. Compared with the strong activation levels of ERK via the Shp2‐binding sites of FRS2α, the Grb2‐binding sites of FRS2α activate ERK at moderate levels.^{( 42 )} Nevertheless, the Shp2‐binding sites and Grb2‐binding sites contribute to both phases.

FRS2α is phosphorylated by ERK at multiple threonine residues in response to a variety of ligands, FGF, insulin, EGF, and PDGF^{( 25 )} (Fig. 4a,b). These include extracellular stimuli that do not induce tyrosine phosphorylation of FRS2α. There are eight canonical ERK phosphorylation sites (PXTP motif) in FRS2α. Expression of a mutant FRS2α deficient in ERK phosphorylation sites resulted in enhanced tyrosine phosphorylation of FRS2α, ERK stimulation, cell migration, and cell proliferation. In addition, activated ERK binds to threonine phosphorylated FRS2α. Thus there is an ERK‐mediated negative feedback mechanism for the control of signaling pathways that are dependent on FRS2α.

In contrast, FRS2β may not have many sites of phosphorylation by ERK; however, it still binds to phosphorylated and activated ERK (M Watanabe and N Gotoh, unpublished data). We identified an ERK‐binding site on FRS2β and found that binding between FRS2β and phosphorylated ERK is important for inhibition of EGF receptor tyrosine kinase.^{( 35 )} The ternary complex, EGF receptor‐FRS2β‐phosphoryalted ERK, may be required for inhibition of EGF signaling. This functions to form a negative feedback loop after activation of ERK downstream of the activated EGF RTK or to maintain the EGF receptor in an inactive state after the activation of ERK by other stimuli.

Several other proteins are reported to bind to FRS2 proteins at sites different from phosphorylated tyrosine residues: SOS, Rnd, Cks1/Cks2, and Sprouty. Cks1 and Cks2 are mammalian homologs of p13^suc1. They are cell‐cycle inducers and trigger ubiquitination and degradation of p27^kip1. It is reported that Cks1 associates with the unphosphorylated FRS2α via the PTB domain of FRS2α.^{( 43 )} FGF‐dependent activation of FRS2α causes the release of Cks1 from FRS2α, and promotes degradation of p27^kip1. Sprouty is a negative regulator for the Ras/ERK pathway; however, the point at which Sprouty blocks ERK activation depends on the cellular context and/or the identity of the RTK.^{( 44} , ⁴⁵ , ^{46 )} It is reported that tyrosine phosphorylated Sprouty binds to and sequesters Grb2 from the Grb2‐SOS complex on FRS2 in response to FGF.^{( 47 )} However, another group has reported that there is constitutive interaction between Sprouty and FRS2, and the interaction is increased by FGF resulting in inhibition of ERK signaling.^{( 48 )} Rnd1 has low intrinsic GTPase activity and is antagonistic of RhoA signaling.^{( 37 )} It directly interacts with FRS2α and FRS2β. The interaction of FRS2β suppresses the inhibitory effect of Rnd1 on RhoA. Rnd1 binds to the C‐terminus of FRS2β including Shp2‐binding sites. When the FGF receptor is activated, it phosphorylates FRS2β, recruits Shp2, and releases Rnd1 from FRS2β. The liberated Rnd1 then inhibits RhoA activity.

Expression patterns in tissues and physiological functions of FRS2 proteins

In situ hybridization during mouse embryogenesis showed that Frs2α is strongly expressed in the extraembryonic ectoderm where trophoblast stem (TS) cells exist, stem cells for tissues of the placenta, at embryonic day 6 and is ubiquitously expressed during development, though Frs2β is strictly localized in several tissues including the neuroepithelium.^{( 12} , ^{49 )} In Xenopus, the transcripts of xFRS2 have an expression pattern that is quite similar to that of FGFR1 during development.^{( 16} , ^{18 )}

FGF stimulates cell growth and cell migration in fibroblasts and neurite outgrowth in PC12 cells. It seems that FRS2α plays critical roles in all these activities.^{( 36} , ^{42 )} FRS2 proteins bind to TrkA and B upon stimulation with NGF or brain derived growth factor (BDNF) and stimulate neurite outgrowth in neuronal cells such as PC12 cells.^{( 20} , ²⁹ , ^{30 )} The binding site on TrkA or TrkB is also known as a Shc‐binding site (Fig. 3). There may be competition for which protein binds to this site.^{( 50 )} Prolonged activation of ERK is associated with differentiation of PC12 cells, while transient activation of ERK is associated with the proliferation of these cells.^{( 41 )} Since FRS2 but not Shc induces prolonged activation of ERK, the competition may underlie molecular mechanisms to determine whether cells proliferate or differentiate. FRS2α is also involved in the neuronal differentiation of PC12 cells stimulated by activated ALK.^{( 24 )} The binding site of FRS2α on RET is a common binding site for several proteins carrying SH2 or/and PTB domains, including Shc, Dok1, 4, 5 and 6, and IRS1 and 2^{( 21} , ^{22 )} (Fig. 3). Experiments using a genetically engineered RET mutant that is able to bind FRS2α but not Shc and vice versa showed that Shc activates the cell survival pathway of neuroblastoma cells more strongly than FRS2α does.^{( 51 )}

Phenotypes of FRS2α mutant mice generated by gene targeting support the notion that FRS2α is a ‘conning center’ in FGF signaling in vivo (Fig. 5). It is well established that FGF signaling plays a fundamental role in many aspects of animal development. Frs2α‐null mouse embryos showed multiple developmental defects, resulting in early embryonic lethality by E8⁴⁹. This is due to a failure of maintenance of self‐renewing TS cells that are dependent on FGF4. Further, FGF4‐stimulated FRS2α‐mediated ERK activation in TS cells induces expression of Bmp4 that is important for maintenance of primordial germ cells. Using Frs2α‐null embryos we also showed that Bmp4 is important for cell proliferation in the inner cell mass and epiblast (M Murohashi and N Gotoh, unpublished data). In addition, FRS2α plays a role in cell movement through the primitive streak during gastrulation.

We next generated mutant mice expressing forms of FRS2α that lack either the two Shp2‐binding sites (2F mutant) or the four Grb2‐binding sites (4F mutant) by gene targeting (Fig. 5). Frs2α ^2F/2F embryos exhibit a variety of developmental defects and perinatal death, in contrast to the much milder defects in Frs2α ^4F/4F mice. All Frs2α ^2F/2F embryos were defective in eye development and showed anophthalmia (no eye) or microphthalmia (small eye).^{( 52 )} Consistent with the critical role of FRS2α in FGF signaling, the level of activated ERK in the Frs2α ^2F/2F eye primordium is lower than that of the wild‐type in association with decreased levels of expression of Pax6 and Chx10, molecular markers for lens induction and retinal precursor proliferation, respectively. There are also severe defects in the cerebral cortex's development in Frs2α.^{2F /2F ( 53 )} The Shp2‐binding sites on FRS2α play critical roles in the maintenance of intermediate progenitor cells committed to a neuronal lineage after asymmetrical cell division of neural stem cells. Moreover, the Shp2‐binding sites on FRS2α play an important role in neural stem/progenitor cells proliferation in response to FGF2. The Frs2α ^2F/2F mice have more developmental defects, including lack of carotid body.^{( 54 )}

Fgfr1 mutant mice lacking binding sites for FRS2α and FRS2β die later in embryogenesis than Fgfr1‐null embryos and exhibit defects in neural tube closure and in the development of the tail bud and pharyngeal arches.^{( 55 )} Although FRS2 proteins play major roles in FGF receptor‐mediated developmental processes, these findings indicate that some essential functions of FGF receptor 1 are mediated by FRS2‐independent signaling.

On the other hand, the functions of FRS2βin vivo are still largely unknown, since there is no report yet about FRS2β knock out mice.

xFRS2 is a critical mediator for FGFR‐mediated induction of mesoderm and the accompanying elongation movements.^{( 18 )} In addition, xFRS2 cooporates with Laloo for induction of mesoderm.^{( 17 )}

Relevance of FRS2 proteins to cancer

It is known that FGF ligands and their recepors are overexpressed in a variety of cancers, including of the breast, stomach, prostate, pancreas, bladder, and colon.^{( 19} , ⁵⁶ , ⁵⁷ , ^{58 )} Activating mutations in FGFR3 are observed frequently in bladder and cervical carcinoma and in some cases of multiple myeloma, while activating mutations in FGFR2 are found in gastric carcinoma. Further, several types of fusion proteins involving FGFR1 caused by chromosomal translocation are found in hematologic malignancies. Given that aberrant FGF signaling is important in tumorigenesis based on all these reports, it is reasonable to predict that FRS2 proteins are also involved in tumorigenesis (Fig. 6). Consistent with this idea, expression of the PTB domain of FRS2α inhibits antiestrogen growth of breast carcinoma cells induced by overexpression of FGF1.^{( 59 )}

Somatic rearrangements of the TrkA gene are detected in a fraction of papillary thyroid carcinomas and produce chimeric proteins. All of them contain a portion of TrkA, including tyrosine kinase domain. The resultant thyroid Trk oncoproteins activate FRS2α and FRS2β.^{( 60 )} Gene rearrangements leading to fusion of the kinase domain of RET with heterologous proteins containing dimerization motifs result in constitutively activated RET proteins.^{( 21} , ^{28 )} Such fusion proteins are expressed in some cases of papillary thyroid carcinoma. Germ line point mutation in RET results in inherited multiple endocrine neoplasm types 2α and 2β and familial medullary thyroid carcinoma. FRS2α is tyrosine phosphorylated by ligand‐stimulated and by constitutively activated oncogenic forms of RET, leading to activation of ERK. The NPM‐ALK oncoprotein activates FRS2α and FRS2β and transforms NIH3T3 cells.^{( 32 )}

Expression of FRS2β suppresses EGF‐induced cell transformation and proliferation.^{( 35 )} Moreover, the expression of FRS2β is down‐regulated in lung and brain cancer cell lines. These findings suggest that FRS2β is suppressive for tumorigenesis in which aberrant EGF signaling is involved.

Concluding remarks

From extensive studies since FRS2α cDNA was cloned in 1997, it is now clear that FRS2α acts as a ‘conning center’ in signaling via FGF receptors and plays significant roles in the promotion of signaling via several other RTKs. Thus, it is reasonable to predict that FRS2α is involved in the malignancy of tumors, especially when they are addicted to aberrant FGF signaling. On the other hand, FRS2β appears to have inhibitory effects on tumorigenesis when tumors are addicted to aberrant EGF signaling.

Recently, many more small compounds that inhibit RTKs and humanized antibodies against RTKs have been developed and several of them are already in use in the treatment of cancer. FGF receptors are among the targets of such molecular targeted therapies. However, there are few good diagnostic tools with which to decide which patients should be treated with drugs targeting FGF receptors. Therefore, there is an urgent need to develop diagnostic tools such as biomarkers to predict the effectiveness of such drugs. Taking the characteristic that FRS2α promotes signaling via limited species of RTKs into account, it is worth testing if FRS2α can be used as a biomarker. The EGF receptor is one of the most important targets in cancer treatment. We have evidence suggesting that FRS2β is applicable as a biomarker to predict the effectiveness of such drugs.

One direction of research into FRS2 proteins would be the validation of these proteins as candidates for biomarkers using many cancer tissue samples. Another direction would be continuing research aiming at further elucidation of physiological and pathological functions of this protein family and of molecular mechanisms for them.