Proteomic analyses of signalling complexes associated with receptor tyrosine kinase identify novel members of fibroblast growth factor receptor 3 interactome

Accepted Manuscript Proteomic analyses of signalling complexes associated with receptor tyrosine kinase identify novel members of fibroblast growth factor receptor 3 interactome

Lukas Balek, Pavel Nemec, Peter Konik, Michaela Kunova Bosakova, Miroslav Varecha, Iva Gudernova, Jirina Medalova, Deborah Krakow, Pavel Krejci PII: DOI: Reference:

S0898-6568(17)30271-1 doi:10.1016/j.cellsig.2017.10.003 CLS 9011

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

5 May 2017 13 September 2017 5 October 2017

Please cite this article as: Lukas Balek, Pavel Nemec, Peter Konik, Michaela Kunova Bosakova, Miroslav Varecha, Iva Gudernova, Jirina Medalova, Deborah Krakow, Pavel Krejci , Proteomic analyses of signalling complexes associated with receptor tyrosine kinase identify novel members of fibroblast growth factor receptor 3 interactome. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cls(2017), doi:10.1016/j.cellsig.2017.10.003

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ACCEPTED MANUSCRIPT Proteomic analyses of signalling complexes associated with receptor tyrosine kinase identify novel members of fibroblast growth factor receptor 3 interactome Lukas Balek1, Pavel Nemec1, Peter Konik2, Michaela Kunova Bosakova1, Miroslav Varecha1,3, Iva Gudernova1, Jirina Medalova4, Deborah Krakow5, Pavel Krejci1,3,5*

Department of Biology, Faculty of Medicine, 4Institute of Experimental Biology, Faculty of

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1

Science, Masaryk University, 62500 Brno, Czech Republic

Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia,

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37005 Ceske Budejovice, Czech Republic

International Clinical Research Center, St. Anne's University Hospital, 65691 Brno, Czech

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3

Republic

Department of Human Genetics and Orthopaedic Surgery, University of California Los

Angeles, California, USA, 90095

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*correspondence to: [email protected]

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ACCEPTED MANUSCRIPT Abstract Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag

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immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins

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out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-

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immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine

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kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. Further analyses of FGFR3ICK interaction discovered an unexpected link between the FGF pathway and primary cilia, while elucidation of FGFR3-SHIP2 interaction unraveled a critical role of SHIP2 in

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regulation of canonical FGF-ERK MAP kinase signalling. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via

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proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the 54 known human

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RTKs.

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tyrosine kinase.

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Keywords: Fibroblast growth factor; FGFR3; interactome; signal transduction; receptor

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ACCEPTED MANUSCRIPT 1. Introduction The fibroblast growth factor receptor 3 (FGFR3) transduces extracellular communication signals delivered by fibroblast growth factor (FGF) ligands FGF1,2,8,9,17,18 and 20 [1]. In development, the FGFR3 expression domain is largely restricted to cartilaginous tissues, implicating a role in endochondral ossification [2]. Genetic studies in mice have established FGFR3 as a major physiological negative regulator of growth plate cartilage, which restricts

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skeletal growth via incompletely understood mechanisms involving inhibition of chondrocyte proliferation and differentiation [3,4]. Distinct skeletal dysplasias are caused by germline

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activating mutations in the FGFR3 gene, including hypochondroplasia, achondroplasia,

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thanatophoric dysplasia, SADDAN, and Crouzon syndrome with acanthosis nigricans. Achondroplasia is the most prevalent form of nonlethal human dwarfism, whereas thanatophoric dysplasia represents the most common lethal skeletal dysplasia [5]. Somatic

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activating FGFR3 mutations lead to excessive cell proliferation, which underlies the pathology of several skin disorders (epidermal nevi, seborrheic keratosis, acanthosis

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nigricans) and cancers (multiple myeloma, bladder cancer, seminoma and others) [6]. The mechanism of FGFR activation involves ligand-induced dimerization, followed by trans-phosphorylation within the kinase domains of the FGFR dimers. Well characterized for

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FGFR2, the sequential phosphorylations at tyrosine residues within the activation loop

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(Y656/Y657), C-terminal tail (Y769), kinase insert (Y586/Y588), and juxtamembrane region (Y466) upregulate FGFR2 catalytic activity and provide docking sites that recruit FGFR2 substrates containing phosphotyrosine-binding SH3 and PTB domains [7]. A sequence of

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protein interactions involving adapter proteins, phosphatases, small GTPases, and other signalling mediators is initiated, further propagating the FGF signal to effectors that alter

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gene transcription, i.e. RAS/ERK MAP kinase and PI3K/AKT pathways. Protein networks involved in the alteration of cell shape, migration, proliferation, differentiation and other basic cell phenotypes are also induced [8]. Finally, a combination of transcriptional and epigenetic responses defines the nature of cell response to the FGF stimulus. The full composition of the FGFR interactome (a small proteome that associates with FGFRs) is not known. Several interactors have been characterized, including adapter proteins FRS2, SH2B and GRB14, PI3-kinase regulatory subunit p85, serine/threonine kinases RPS6K, TAK1, CBL ubiquitin ligase, transcriptional regulators STAT and -catenin, and others [9–15]. Unfortunately, the exact biophysical mode of the interaction, derived from solved crystal structure, is only known for one protein, the phospholipase C (PLC [16].

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ACCEPTED MANUSCRIPT Thus, there is limited knowledge concerning the dynamics of protein-protein interactions within the FGFR signalling complexes as well as the underlying molecular mechanisms. Moreover, the list of FGFR interactors is most likely far from complete, as RTKs are usually organized in large, multiprotein complexes. These complexes dynamically alter their composition depending on changes in spatio-temporal organization during the course of signal activation, propagation, and feedback. An example includes a basic mechanism of

simple

sequence

of

interactions

among

eight

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FGFR-mediated activation of the RAS/ERK MAP kinase pathway, involving a relatively proteins

(FRS2/SHP2-GRB2-

SOS/RAS/RAF/MEK/ERK) [17]. However, as many as 150 proteins may participate in the

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regulation of the RAS/ERK module itself [18]. The fact that FGFRs activate ERK and are, at

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the same time, subjects of ERK-mediated negative-feedback phosphorylation [19], implies that many molecules belonging to the ERK pathway may participate in the FGFR-associated

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signalling complexes. Thus, the actual amount of the interacting proteins within the FGFRassociated signalling complexes may be much larger than currently appreciated. There is a need for deeper understanding of the FGFR3 signalling complexes in order

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to design better and more effective FGFR3 therapeutics particularly for skeletal and proliferative disorders such as cancer. Although direct targeting of FGFR3 is an attractive

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mode of therapy of achondroplasia and related disorders, no effective treatment is available yet. Targeting FGFR3 signalling intermediates may yield more promising therapeutic

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approaches compared to direct inhibition of FGFR3 catalytic activity, which is hampered by low specificity and toxicity of the present tyrosine kinase inhibitors [20]. Uncovering the full spectrum of protein-protein interactions within the FGFR3 signalling complexes holds the

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key to understanding the FGFR3 function in hopes of designing future FGFR3 therapeutics. This study was undertaken to identify novel FGFR3 interactors via proteomic and

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phosphoproteomic approaches.

2. Material and methods

2.1. Cell culture, vectors and transfection

293T and NIH3T3 cells were obtained from ATCC (Manassas, VA). RCS cells [21] were obtained from B. de Crombrugghe (MD Anderson Cancer Center, Houston, TX). Cells were propagated in DMEM media, supplemented with 10% FBS and antibiotics (Invitrogen, Carlsbad, CA). FGF2 was from RnD Systems (Minneapolis, MN), heparin was from Sigma4

ACCEPTED MANUSCRIPT Aldrich (St. Louis, MO). Cells were transfected using FuGENE6 reagent according to manufacturer’s protocol (Promega, Madison, WI) or with polyethylenimine (PEI). Vectors expressing C-terminally FLAG- or V5-tagged FGFR3 were described elsewhere [22,23]. Vectors expressing STAM1, SHOX2 and EEF1A1 were obtained from Origene (San Diego, CA). For proteomic experiments, four 15 cm2 tissue culture dishes were seeded with 293T cells at the density of 4.5x106 cells/dish in 25 ml of DMEM media (Sigma), supplemented

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with 10% FBS and antibiotics (Gibco). Cells were transfected ~16 hours after seeding, with solution containing 64 µl of FuGene6 (Promega) mixed 20 µg of plasmid DNA in 1 ml of media, according to manufacturer’s protocol. Cells were grown for 24 hours after

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transfection. For RCS cells, six 15 cm2 dishes were seeded with ~3 x106 of cells each and

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grown overnight before they were treated with 20 ng/ml FGF2 for 30 minutes and harvested. Cells were washed with PBS (Gibco) and lysed for 30 minutes on ice in 2.5 ml/dish of lysis

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buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 0.25% sodium deoxycholate, 2 mM EDTA and 1 mM Na3VO4, supplemented with proteinase inhibitors (Complete Mini protease inhibitors; Roche, Basel, Switzerland). Lysates were cleared by

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centrifugation (10,000g/4°C/1 hour) and V5 (Invitrogen) or FLAG (Sigma) antibody was added to a final concentration of 1 µg (antibody)/1 ml (lysate). Lysates were rotated at 4°C

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with addition of A/G beads (30 µl; Santa Cruz, Dallas, TX) one hour later. In 4G10 (Millipore, Billerica, MA, USA) immunoprecipitations, 100 µl of 4G10 antibody conjugated

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to A/G-beads was used per 10 ml of lysate. Following the overnight incubation at 4oC, the A/G beads were collected by centrifugation (200g/4°C/2 minutes) and washed 3-times with 5 ml of lysis buffer without protease inhibitors and Na3VO4, followed by additional 3 washes

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with 2 ml of lysis buffer without protease inhibitors, Na3VO4, NP-40 and sodium

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deoxycholate. Immunocomplexes were stored at -80°C before MS analysis.

2.2. Immunoprecipitation (IP), western blot (WB) and activity assays For IP, cells were lysed in lysis buffer for 20 minutes on ice. Lysates were cleared by centrifugation and supernatants incubated for 1 hour with antibodies. Immunocomplexes were collected on A/G-agarose (Santa Cruz) in overnight incubation. pTyr IPs were carried out with 100 μl 4G10 antibody conjugated to agarose beads (Millipore). For WBs, cell lysates were resolved by SDS-PAGE, transferred onto a PVDF membrane and visualized by chemiluminiscence (Thermo Scientific, Rockford, IL). Supplementary Table 2 lists antibodies used in the study. FGFR3 kinase assays were performed as described before [20].

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ACCEPTED MANUSCRIPT 200 ng of recombinant FGFR3 was incubated for 30 minutes at 30°C with recombinant p130CAS (SignalChem, Richmond, CA) or immunoprecipitated SHOX2 as a substrate, in 50 l of kinase buffer (60 mM HEPES pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 10 M Na3VO4, 1.2 mM DTT) supplemented with 10 M ATP.

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2.3. MS data acquisition and analysis

The immunoprecipitates were resuspended in 100 mM ammonium bicarbonate pH 8, and

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proteomic grade trypsin (Sigma-Aldrich) was added for overnight digestion at 37oC. Peptides were isolated from the fluid using ZipTip C18 pipette tips (Merck-Millipore, Germany).

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LC/MS analysis was performed on a NanoAcquity UPLC (Waters, Milford, MA) on-line coupled to a MicromassTM Q-TOF PremierTM mass spectrometer (Waters). Peptides were

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separated on a BEH300 C18 analytical column (Waters) with a linearly increasing 3-40% (v/v) gradient of 0.1% (v/v) formic acid in acetonitril. Eluted peptides flowed directly into the

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ESI source. Raw data was acquired in data independent MS^e Identity mode. Peptide spectra and fragment spectra were acquired with 2 ppm and 5 ppm tolerance, respectively. Raw data was then processed by the PLGS2.3 software (Waters; false discovery rate set to 4%) and

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subjected to a database search using species specific (human or rat) protein databases

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downloaded from NCBI and Uniprot websites. Identification of 3 consecutive y- or b-ions was required for a positive peptide match. Protein entries were explored through UniProt ID mapping tool (available online at www.uniprot.org) (June 2015). Further steps (duplicates

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look-up and data merging) were processed in batch using R-script. The workflow outlining protein data analysis is described in Supplementary Figure 1.

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3. Results and Discussion

Two cell lines were used in this study to explore the FGFR3-associated signalling complexes, human embryonal kidney 293T and rat chondrosarcoma (RCS) cells. 293T cells were used in earlier FGFR proteomic studies [24]; RCS cells represent a well-studied model for pathological FGFR signalling [25,26]. The 293T cells were transfected with either wildtype FGFR3 or its activating mutants K650E or K650M, which are responsible for thanatophoric dysplasia (skeletal dysplasia) and cancer [5]. FGFR3 transfection lead to overexpression in 293T cells, but a significant portion of FGFR3 localized to cell membrane, as determined by

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ACCEPTED MANUSCRIPT FGFR3 immunocytochemistry and western blot detection of ~140 kDa fully matured wildtype FGFR3, demonstrating normal biogenesis (Fig. 1A-C). In 293T cells, FGFR3 expression followed by treatment with FGF2 ligand caused phosphorylation of PLC(Y783), STAT1(Y701), STAT3(Y705) and ERK MAP kinase (T202/Y204) (Fig. 1A). RCS cells were stimulated with the FGFR ligand FGF2 to activate endogenously expressed FGFR2 and FGFR3 [27]. In RCS cells, treatment with FGF2 triggered

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phosphorylation of PLC(Y783), ERK(T202/Y204), and LRP6(T1572), a co-receptor for WNT family of morphogens (Fig. 1B) [22]. Both cell types thus responded to FGFR

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activation with the recruitment of known pathways of FGFR signal transduction.

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3.1. Identification of proteins interacting with FGFR3 via proteomic approaches

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FGFR3 was C-terminally tagged with either a V5 or FLAG epitope, expressed in 293T cells, and purified from 293T lysates via V5 or FLAG immunoprecipitation (IP). The immunocomplexes were then analysed by liquid chromatography/tandem mass spectrometry

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(MS). The proteins specifically associated with FGFR3 were obtained by subtracting the proteins found in FLAG or V5 immunocomplexes in cells transfected with an empty vector.

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An alternative approach relied on the isolation of proteins phosphorylated on tyrosine

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residues upon FGFR3 transfection and activation. The 4G10 pan-pTyr antibody was used for these experiments, and proteins phosphorylated by FGFR3 signalling were obtained by subtracting hits in 4G10 IPs obtained from cells expressing an empty plasmid instead of the FGFR3. A total of 26 experiments were carried out in 293T cells transfected with WT or

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K650E/M FGFR3, consisting of six V5, ten FLAG and ten 4G10 IP experiments. In RCS cells, endogenous FGFR signalling was activated by stimulation with FGF2 and proteins

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phosphorylated by FGFRs were obtained by subtracting hits found in FGF2 untreated cells. The analysis was repeated six times. Peptides found on MS spectra were searched against NCBI and Uniprot databases and mapped to 1,861 proteins (1,625 in 293T cells and 236 in RCS cells). The data were subjected to supervised analyses aimed to remove redundant and synonymous hits (Supplementary Fig. 1). We did not expect absolute fidelity of the used immunoprecipitation antibodies [28], and therefore considered interacting proteins as ‘putative interactor’ if they were found in at least 4 out of 26 experiments carried-out in 293T cells. The only exceptions to this criterion were proteins previously known to interact with FGFRs, compiled in a detailed survey of published literature (Supplementary Table 1), and SHOX2, which is 7

ACCEPTED MANUSCRIPT described below. Experiments carried out in 293T cells identified 55 putative interactors (Table 1). In RCS cells, we considered as putative interactors the proteins that were found in at least 2 out of 6 experiments. This way, we identified 36 proteins (Table 2). The proteins identified in these experiments overlapped with 63 known FGFR interactors (Supplementary Table 1) by 32.7% (18/55 proteins) in 293T cells and 41.7% (15/36 proteins) in RCS cells

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(Fig. 2).

3.2. FGFR3 interacts with adapter protein STAM1

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Surprisingly, the least frequent FGFR3 interactors identified by MS were among the

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signalling adapters (Tables 1, 2), which nevertheless represent the first substrates phosphorylated by FGFRs in cells. Five out of 13 known adapters interacting with FGFRs

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(p130CAS, GAB1, GRB2, SHC, NCK2) (Supplementary Table 1) were identified in less than 12% of experiments carried out in 293T cells, while the well-established FGFR adapter, FRS2, was not recovered at all (Table 1). Interestingly, all the adapters identified in MS

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experiments were among the proteins isolated with pTyr IPs but not in FGFR3 tagged IPs, despite the fact that at least FRS2, GAB1, SHC and p130CAS are directly phosphorylated by

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FGFRs and therefore must physically interact [29–31] (Fig. 3). Other direct FGFR3

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interactors such as the p85 subunit of PI3-kinase, PLC1, or direct binders to FRS2 such as SHP2 and SOS1 were also poorly represented among the recovered hits, and isolated only in pTyr IPs but not in FGFR3 tagged IPs. Poor adapter recovery demonstrates a limitation of the MS analyses carried out here, i.e. the omission of bona fide FGFR interactors. This could be

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due to the limited sensitivity of the MS detection or a low affinity of the 4G10 antibody for some phosphorylated motifs [32]. Moreover, some FGFR3-adapter interactions might be

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transient in nature and thus not be amenable to MS due to a limited quantity of given adapter bound on FGFR3 at any time point. Stabilization of complexed proteins, for instance by covalently cross-linking using dithiobis (succinimidyl propionate), could potentially improve the MS recovery of transient interactors, but may introduce artefact [33] . To determine to what extent adapters were omitted in MS analyses, we carried out a survey of FGFR-mediated adapter phosphorylation in 293T or RCS cells. A total of 15 tyrosine phosphosites in 12 different adapters were determined by western blots with specific antibodies (Supplementary Table 2). We found FGFR3-mediated phosphorylation of nine different adapters, i.e. FRS2(Y436), p130CAS(Y249/Y410), SHC(Y239/Y240), IRS1(Y896),

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ACCEPTED MANUSCRIPT GAB1(Y627), CRKL(Y207), SHB(Y246), SSH3BP1(Y435), DOK1(Y398) in 293T cells and five adapters (FRS2, GAB1, CRKL, SHC, p130CAS) in RCS cells (Fig. 3A, B). A comparison of these data with the MS data (Tables 1, 2) revealed that seven and eight adapters were omitted by MS in 293T and RCS cells, respectively. Our data nevertheless demonstrate that FGFRs utilize many different adapters to engage their downstream signalling. Moreover, the data show that adapter recruitment is cell-specific and includes

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adapters not previously implicated in FGFR signalling, such as IRS1 and DOK1 (Tables 1, 2; Supplementary Table 1).

Two additional adapters, STAM1 and HRS were found among the tyrosine

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phosphorylated proteins in 293T cells expressing tagged FGFR3 (Table 1). We confirmed

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STAM1-FGFR3 association by a co-immunoprecipitation in 293T cells (Fig. 4A). STAM1 and HRS form a complex at the endosomal membrane, which interacts with ubiquitinated

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receptor tyrosine kinases and directs their lysosomal degradation [34]. STAM1 and HRS may therefore interact with internalized FGFR3 and affect the balance between its degradation and recycling [35]. Since STAM proteins also regulate ER to Golgi transport [36], FGFR3 may

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associate with STAM1 during its maturation and translocation to the cell membrane.

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3.3. FGFR3 interacts with translation elongation factor eEF1A1

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The identification of STAM1 as an FGFR3 interactor suggests that the proteins recovered by MS analyses in 293T cells may not all belong to signalling complexes associated with active FGFR3 at the cell membrane. This is due to the fact that FGFR3 cDNA was transfected de

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novo into the 293T cells and thus had to be translated, transported via the ER-Golgi network, and glycosylated in the Golgi before reaching the cell membrane. Therefore, the MS recovery

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of ribosomal proteins and factors regulating protein synthesis such as eukaryotic translation elongation factor 1A1 (eEF1A1) (Table 1) may represent non-signalling proteins that were recovered based on our transfection experimental system. However, we did find eEF1A1 and several ribosomal proteins among the tyrosine phosphorylated proteins recovered in FGF2treated RCS cells (Table 2). This suggests an active involvement of FGFR signalling in the regulation of proteosynthesis. The association of eEF1A1 with FGFR3 was confirmed by coimmunoprecipitation in 293T cells (Fig. 4B). FGFs are major mitogens and an anabolic effect of FGFR activation on protein synthesis is not surprising. This may be mediated by increased rRNA transcription, upregulation of factors involved in translational initiation (EIF1a, eIF4E, EIF5, EIF6, eIF49

ACCEPTED MANUSCRIPT BP1), enzymes involved in amino acid and tRNA synthesis, and enhanced recruitment of RNA to polysomes [37,38]. Serine/threonine phosphorylation of translational regulators such as eIF4E and S6-RP was also found with FGFR activation in mouse embryonal fibroblasts and epithelial cells, and this has been previously attributed to actions of ribosomal protein S6kinase (RSK) [39–41], well recognized FGFR target found also among the MS interactors identified here (Tables 1, 2). We demonstrate that FGFR3 interacts with previously

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unreported proteins involved in protein synthesis thus adding to the complexity of FGFmediated regulation of proteosynthesis. As eEF1A1 interacts with several molecules integral

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to the FGFR3 signalling complexes, such as GRB2, SHC, SHP2, CRK and PLC1 [42], future research should elucidate what role the eEF1A1 plays in the FGFR3 signalling Furthermore,

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FGFR3-mediated

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complexes.

phosphorylation affects the major canonical functions of eEF1A1, i.e. the shuttling of

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aminoacyl-tRNAs to the ribosome and organization of cytoskeletal actin [43].

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3.4. FGFR3 interacts with transcriptional regulator SHOX2

Some of the bona fide FGFR3 interactors may have been omitted from the MS data analyses

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because of their low abundance, the transient nature of their interactions, and/or our criteria

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for potential interaction. To test for this, we chose to interrogate the transcriptional regulator SHOX2, which was recovered in only one out of 26 MS experiments in 293T cells (Table 1), but represents an important regulator of postnatal skeletal growth, a major role of physiological FGFR3 function [4,44]. Co-immunoprecipitation experiments proved SHOX2

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association with FGFR3 in 293T cells (Fig. 5A). Moreover, recombinant FGFR3 phosphorylated immunopurified SHOX2 in a cell-free kinase assay, demonstrating that

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SHOX2 is a FGFR3 substrate (Fig. 5B). In humans, two paralog SHOX genes exist (SHOX and SHOX2) that display similar expression patterns during skeletal development [45]. Loss of SHOX function accounts for the short stature phenotypes in Turner syndrome, Leri-Weill dyschondrosteosis, and Langer dysplasia [46]. Shox2 deletion leads to marked limb shortening leading to the absence of the humeri and femora due to diminished Indian hedgehog signalling and downregulation of Runx2 and Runx3 expression in growth plate chondrocytes [47,48]. Ectopic SHOX2 expression leads to premature cell cycle arrest in cultured primary chondrocytes, which are associated with the induction of cell cycle inhibitors p21Cip1 and p27kip1 [49]. These effects

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ACCEPTED MANUSCRIPT closely resemble the cellular manifestations of pathological FGFR3 signalling in chondrocytes [4]. Interestingly, heterozygosity for K385E and R382C missense mutations in the SHOX gene were recently found to be associated with hypochondroplasia, a skeletal dysplasia usually caused by mildly activating FGFR3 mutations [5,50]. In our experiments, SHOX2 immunoprecipitated with the FGFR3-K650M mutant associated with thanatophoric dysplasia, but not with wt FGFR3 (Fig. 5A), opening an attractive possibility that FGFR3

or neomorphic activity in SHOX and/or SHOX2.

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3.5. FGFR3 interacts with SHOX2 and eEF1A1 in NIH3T3 cells

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signalling mediates some of its pathological functions in skeletal dysplasias via an acquired

Figure 1A-C demonstrates that 293T cells mature FGFR3 normally and show FGF-mediated

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activation of usual mediators of FGFR signaling. In addition, the number of interactors identified in 293T cells roughly corresponds to the known amount of FGFR interactors (55 vs. 63 proteins), with 17 overlapping hits (Fig. 2). These findings argue against the existence

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of many false positive hits found in 293T cells. However, the 293T cells produce high levels of transfected protein, creating the possibility that some of the interactions occur only at non-

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physiological levels of FGFR3 expression. We therefore evaluated the SHOX2 and eEF1A1 interaction with FGFR3 in co-immunoprecipitations carried out in NIH3T3 fibroblasts

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expressing significantly lower levels of expressed FGFR3 (Fig. 4E). The experiments show that both SHOX2 and eEF1A1 interact with FGFR3 in NIH3T3 cells (Fig. 4F).

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3.6. FGFR3 interacts with ser/thr kinases ICK, MAK and CCRK

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Among the protein kinases interacting with FGFR3, ephrin receptors and focal adhesion kinase are known to interact with FGFRs [51,52]. Three ser/thr kinases previously unknown to interact with FGFRs were identified, belonging to less understood family of RCK kinases (for v-ros cross-hybridizing kinase) that include intestinal cell kinase (ICK), male germ-cell– associated kinase (MAK) and MAPK/MAK/(MAK-related kinase) MRK-overlapping kinase (MOK) [53]. ICK was found in 10/26 (38%) of 293T MS experiments, while MAK was identified in 12/26 (46%) of experiments. Moreover, the ICK-activating kinase CCRK (CDK20) [54] was identified in 10/26 (38%) experiments (Table 1). In addition, MAK was also found among proteins phosphorylated at tyrosine upon FGF2 treatment of RCS cells (Table 2). A characterization of ICK interaction with FGFR3, detailed elsewhere [55], 11

ACCEPTED MANUSCRIPT demonstrated that the FGFR3-mediated tyrosine phosphorylation of ICK partially inactivates ICK’s ser/thr kinase activity in cells, which is necessary for proper ciliogenesis (formation of fully functional primary cilia). Thus the FGF signalling affects length and the function of primary cilia via its direct action on ICK [55].

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3.7. FGFR3 interacts with SRC-family tyrosine kinases and with inositol phosphatase SHIP2

Other protein kinases recovered in both 293T and RCS cells were members of the SRC family of non-receptor tyrosine kinases BLK, FGR, FYN, HCK, LCK, LYN and YES. The

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SRC kinases were frequent FGFR3-interactors, being identified in 54% of 293T and 50% of

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RCS MS experiments (Tables 2, 3). FGFR activation leads to increased phosphorylation and activation of SRC kinases [56]. The SRCs, in turn, participate in a pleiotropic array of FGF-

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regulated events including cell proliferation, changes in cellular shape, migration, adhesion, and differentiation [57–60]. The mechanism of FGFR-mediated recruitment of SRCs is somewhat unclear, although earlier studies demonstrate direct SRC-FGFR interaction or

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recruitment via FRS2 [56,61,62]. Similarly, it is not clear how SRCs regulate cellular phenotypes mediated by FGF signalling.

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Inositol phosphatase SHIP2 (INPPL1) was one of the most frequent proteins phosphorylated upon FGFR activation in RCS cells, being found in 4/6 (67%) experiments

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(Fig. 2, Table 2). SHIP2 is a negative regulator of insulin signalling [63], but its role in FGFR signalling is unknown. As detailed elsewhere [64], SHIP2 knock-out effectively converted sustained FGF-mediated ERK activation into the transient signal, and rescued the

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pathological cell phenotypes triggered by FGFR-ERK signalling. Mechanistically, SHIP2 recruited SRC kinases to the active FGFRs, which assisted the FGFR-mediated

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phosphorylation and assembly of FRS2 and GAB1 adapter complexes that relay the FGFR signal to ERK pathway [64].

3.8. Receptor tyrosine kinase (RTK) plasmid library for proteomic studies

In this study, we applied two different proteomic methods on two cell models, to study the signalling complexes associated with FGFR3. We found several hitherto unknown FGFR3 interactors, such as STAM1, SHOX2, eEF1A, SHIP2, ICK and others. The list of interactors identified in RCS and 293T cells overlap by ~50% (Fig. 2), with few notable differences, such as the presence of SHIP2, ICK and CDK20 only in the RCS cells. These differences 12

ACCEPTED MANUSCRIPT suggest differential composition of FGFR3-associated signaling complexes between the two cell types, and point to pathways of FGF signaling specific to a given cell type. A precise characterization of these pathways requires, at first, the definition of core components of the FGFR signaling complex. Although some interactors, such as FRS2, GRB2, GAB1 and PLC1 undoubtedly represent the core interactors, the precise composition of basic FGFR signaling complex in not clear, and should be addressed in future research.

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The follow up studies carried out on the ICK and SHIP2 interactions with FGFRs revealed a novel pathway of FGFR signalling in regulation of primary cilia [55], and a

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molecular mechanism underlying the sustained ERK activation by FGFRs [64]. Thus, the unappreciated and important interactors and pathways exist for well-studied RTKs, such as

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FGFR3, and are identifiable via proteomic approaches described here.

A precise characterization of RTK-associated protein complexes is crucial to

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understanding of RTK function. Evidence presented here demonstrates that identification of proteins interacting with one RTK is a lengthy task that is impossible to accomplish in one

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series of experiments. This is due to the dynamic nature of signalling complexes, heterogeneity of complex components among different tissue and cell types, and the transient nature of some interactions. Additionally, the technical drawbacks include intrinsic

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limitations to MS experiments. Reconstructing a detailed interactome of a given RTK

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therefore requires combining different approaches carried out in different cell models to achieve the task. To facilitate such efforts, we prepared a RTK vector library containing 37 out of 54 known human RTKs [65]. Similar to the FGFR3 vectors used for MS experiments here, each RTK was cloned into the pcDNA3.1 plasmid backbone and represented a full

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length human sequence equipped with C-terminal V5/His epitope. For each RTK we also prepared a series of disease-associated mutants selected from published literature, Sanger

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Cosmic, and OMIM databases. The RTK plasmid library is offered to the scientific community on a collaborative basis.

Acknowledgement

Authors wish to apologize for not mentioning those FGFR interactors which could have been omitted in literature surveys conducted here. We thank Lenka Radova for help with MS data analysis. Supported by Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT II LH15231); Ministry of Health of the Czech Republic (15-33232A, 15-

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ACCEPTED MANUSCRIPT 34405A) and the Czech Science Foundation (GA17-09525S). MKB and PN were supported by junior researcher funds from the Faculty of Medicine, Masaryk University.

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FIGURE LEGENDS Figure 1 Activation of FGFR signal transduction in 293T and RCS cells (A) 293T cells were transfected with wildtype (WT) FGFR3 or its activating mutant K650M, grown for 24 hours, treated with 20 ng/ml FGF2 for 1 hour, and analysed for phosphorylation (p) of indicated signalling mediators by western blot. Total levels of each molecule serve as control for phosphorylation, actin serves as loading control. Non-transfected cells or those 21

ACCEPTED MANUSCRIPT transfected with empty plasmid serve as controls of FGFR3 expression. Arrows indicate the fully mature FGFR3 at the cell membrane (top arrow) and the two immature FGFR3 variants (middle and bottom arrow) undergoing glycosylation during the ER-Golgi transport to the cell membrane. Note the defective maturation of FGFR3-K650M described before [66]. (B) 293T cells were transfected with V5-tagged FGFR3, and immunostained using the FGFR3 antibody. Scale bar, 10 µm. Ph2, phase contrast. (C) Z-stacks of single cells showing

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subcellular distribution of expressed FGFR3, which is localized throughout the cells permeabilized (Per.) during the staining. Without cell permeabilization (N-Per.), only a surface signal is detected, demonstrating the presence of transfected FGFR3 at the cell

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membrane. A non-transfected cell is shown as a control. Scale bar, 10 µm. (D) RCS cells

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were treated with 25 ng/ml of FGF2 for indicated times and the activation of FGFR signal

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transduction was detected similar to (A).

Figure 2 The overlap of FGFR3 interactors found in 293T and RCS cells with known FGFR interactors

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The overlap of FGFR3 interactors identified in MS experiments carried out in 293T (55 proteins) or RCS cells (36 proteins), with known FGFR interactors compiled via published

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more paralogs of a given protein were found.

Figure 3 Survey of FGFR-mediated adapter phosphorylation in 293T and RCS cells (A) 293T cells were transfected with wildtype (WT) FGFR3 and its activating mutant

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K650M treated with 20 ng/ml FGF2 for 1 hour, and analysed for phosphorylation (p) of indicated adapters by western blot with specific antibodies. Total levels of each adapter serve

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as loading control, actin serves as loading control for phosphorylated SSH3BP1 and DOK1. Non-transfected cells or those transfected with empty plasmid serve as transfection controls. (B) RCS cells were treated with 20 ng/ml of FGF2 and 1 µg/ml of heparin for indicated times and analysed for adapter phosphorylation. Untreated cells at the beginning and the end of experiment serve as negative controls. (C) Phosphorylation of p130CAS by FGFR3 in a cellfree kinase assay, determined by antibodies recognizing p130CAS phosphorylation at Y165, Y249 and Y410, respectively. Total level of FGFR3 and p130CAS serve as loading controls, sample with ATP omitted serves as a control for kinase assay.

Figure 4 FGFR3 interacts with STAM1, eEF1A1 and SHOX2 22

ACCEPTED MANUSCRIPT (A-C) 293T cells were transfected with V5-tagged wildtype (WT) FGFR3 or FGFR3-K650M together with FLAG-tagged STAM1 (A), translation elongation factor eEF1A1 (B), and transcriptional regulator SHOX2 (C). FGFR3 or its partners were immunoprecipitated (IP) 24 hours later and the immunocomplexes were analysed for FGFR3, STAM1, eEF1A1 and SHOX2 expression by western blot (WB). Cells transfected with GFP-expressing vector serve as IP control. Actin serves as loading control. The data are representative for three

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independent experiments. (D) Phosphorylation of immunoprecipitated SHOX2 by recombinant FGFR3 in a cell-free kinase assay, determined by WB with 4G10 pan-pTyr antibody (pY). Total level of FGFR3 and SHOX2 serve as loading controls, sample with ATP

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omitted serves as a control for FGFR3 activity. (E) Comparison of FGFR3 expression in

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293T and NIH3T3 cells demonstrating the significantly lower expression of transgenic FGFR3 in NIH3T3 cells. Actin serves as loading control. (F) NIH3T3 cells were transfected

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with FGFR3-K650M and FLAG-tagged eEF1A1 or SHOX2. eEF1A1 or SHOX2 were purified by IP 24 hours later and the immunocomplexes were analysed for indicated proteins by western blot (WB). The position of immunoglobulin heavy chain (IgH) is indicated, as is

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the position of a SHOX2 dimer (di.). The data are representative for two independent

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Literature

Receptor tyrosine kinase; signaling 26 (100) Adapter protein; signaling 2 (8) Adapter protein; signaling 1 (4) Adapter protein; signaling 3 (12) Adapter protein; signaling 1 (4) Adapter protein; signaling 1 (4) Adapter protein; signaling 4 (15) Adapter protein; signaling 4 (15) Receptor tyrosine kinase; signaling 4 (15) Non-receptor tyrosine kinase; signaling 14 (54) Non-receptor tyrosine kinase; signaling 2 (8) Tyrosine phosphatase; signaling 1 (4) Serine/threonine kinase; signaling 10 (38) Serine/threonine kinase; signaling 12 (46) Serine/threonine kinase; signaling 11 (42) Serine/threonine kinase; signaling 5 (19) Serine/threonine; signaling 10 (38) Lipid kinase; signaling 3 (12) Phospholipase; signaling 3 (12) Guanine nucleotide exchange factor; signaling 1 (4) Transcription regulator; gene transcription 4 (15) Transcription regulator; gene transcription 1 (4) Transcription regulator; gene transcription 4 (15) Chaperone; protein folding 5 (19) Chaperone; protein folding 26 (100) Chaperone; protein folding 13 (50) Chaperone; protein folding 6 (23) Ubiquitin ligase; protein degradation 1 (4) Ubiquitin ligase; protein degradation 9 (35) Protein degradation 8 (31) ATPase; energy metabolism 8 (31) Carboxypeptidase; protein metabolism 6 (23) Dehydrogenase; energy metabolism 5 (19) Dehydrogenase; energy metabolism 1 (4) Component of ribosome; protein synthesis 11 (42) Component of ribosome; protein synthesis 20 (77) Translation regulator; protein synthesis 14 (54) Translation regulator; protein synthesis 10 (38) RNA binding protein; nucleic acid metabolism 8 (31) RNA binding protein; nucleic acid metabolism 6 (23)

6 0 0 0 0 0 0 0 0 4 0 0 4 4 5 4 4 0 0 0 2 0 2 3 6 5 2 0 1 2 4 1 2 0 4 6 5 5 4 2

10 0 0 0 0 0 0 0 0 4 0 0 1 3 4 0 1 1 0 0 1 1 0 0 10 4 2 1 3 5 2 1 2 0 2 8 4 2 1 2

10 2 1 3 1 1 4 4 4 6 2 1 5 5 2 1 5 2 3 1 1 0 2 2 10 4 2 0 5 1 2 4 1 1 5 6 5 3 3 2

Y Y Y Y Y N N Y Y Y Y N N Y N N Y Y Y N N N N N Y Y Y N Y N N N N N N N N N N

RNA binding protein; nucleic acid metabolism

13 (50)

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6

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RNA binding protein; nucleic acid metabolism Component of cytoskeleton Component of cytoskeleton Component of cytoskeleton Transport; energy metabolism Transport Transport Transport RNA binding factor; transport Motor protein; transport Transport Transport Transport Unknown Integral membrane protein; unknown

5 (19) 2 (8) 6 (23) 4 (15) 5 (19) 10 (38) 4 (15) 6 (23) 8 (31) 8 (31) 3 (12) 8 (31) 4 (15) 6 (23) 3 (12)

1 0 3 1 2 5 0 4 3 3 1 1 1 0 1

1 0 0 2 0 1 0 1 2 3 0 1 0 1 1

3 2 3 1 3 4 4 1 3 2 2 6 3 5 1

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41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

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Fibroblast growth factor receptor 3; FGFR3 Breast cancer antiestrogen resistance protein 1; p130CAS GRB2-associated binding protein 1; GAB1 Growth factor receptor-bound protein 2; GRB2 NCK adaptor protein 2; NCK2 Src homology and collagen containing protein; SHC Signal transducing adapter molecule 1; STAM1 HGF-regulated tyrosine kinase substrate; HRS Ephrin receptor; EPH* (A4, A5, A7, A8, A10) SRC family kinase; SRC* (BLK, FGR, FYN, HCK, LCK, LYN, YES) Focal adhesion kinase; FAK1, FAK2 Tyrosine-protein phosphatase non-receptor type 11; SHP2 Cyclin-dependent kinase 20; CDK20 Male germ cell-associated kinase; MAK Ribosomal protein S6 kinase; RPS6K DNA-dependent protein kinase; DNAPK Intestinal cell kinase; ICK Phosphatidylinositol 3-kinase, p85 subunit; PI3K p85 Phospholipase C PLC1 Son of sevenless homolog 1; SOS1 LIM homeobox protein; LHX* (1, 2, 5) Short stature homeobox protein 2; SHOX2 TATA-binding protein-associated factor; TAF15 Heat shock 40 kDa protein 1; HSP40 Heat shock protein 70; HSP70 Heat shock protein 90; HSP90 Hsp90 co-chaperone Cdc37; CDC37 E3 ubiquitin-protein ligase NEDD4; NEDD4 E3 ubiquitin-protein ligase TRIM; TRIM* (5, 21, 23, 28, 41, 56, 60) Ubiquitin C; UBC AAA domain-containing ATPase; ATAD Carboxypeptidase A; CPA* (2, 3, 4, 6) Hydroxyacyl dehydrogenase; HADH NADH-coenzyme Q reductase; NDUFS6 Ribosomal protein L; RPL* (4, 10, 11, 13, 14, 18, 22-24, 27, 29, 31, 32) Ribosomal protein S; RPS* (2-9, 13-16, 18, 23, 24, 27) Eukaryotic translation elongation factor; eEF* (1A1, 1G, 2) Eukaryotic initiation factor 4; eIF4* (A, B, H, G) DEAD-box protein; DDX* (1, 3, 5, 6, 17, 21, 60) Matrin 3; MATR3 Heterogeneous nuclear ribonucleoprotein; hnRNP* (A, C, D, F, K, L, M, Q, R, U) Polyadenylate binding protein; PABP Cortactin; CTTN Kelch-like protein 22; KLHL22 Keratin 8; KRT8 ATP synthase 5, mitochondrial; ATP5 Solute carrier family member; SLC* (4-6, 25) Clathrin heavy chain; CLTC Exportin; EXP* (1, 5) RNA-binding protein FUS; FUS Kinesin family member; KIF* (1, 14, 26, C2) Karyopherin; KPN* (A, B) Transport protein SEC; SEC* (13, 16, 23, 24, 31) Sorting nexin; SNX* (1, 2, 5, 13) POTE ankyrin domain family member; POTE* (E, F) Reticulon; RTN* (2, 4)

FGFR3 (V5) FGFR3 (FLAG)

Function; process

Bait 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Exp. total (%)

No. Interactor name; symbol

pTyr

Table 1 FGFR3 interactors identified by MS in 293T cells

FGFR3 interactors identified in pull-down experiments in 293T cells expressing V5-tagged FGFR3 (n=6; n, amount of independent experiments), FLAG-tagged FGFR3 (n=10) or in MS analyses of proteins purified with pTyr antibody in cells transfected with FGFR3 (n=10). Cells transfected with wildtype FGFR3, FGFR3-K650M or with empty plasmid constitute one experiment; data from wildtype and K650M FGFR3 pulldowns were added together. Proteins associated with FGFR3 were obtained by subtracting hits in V5, FLAG or pY pulldowns from cells expressing empty plasmid from those expressing FGFR3. One hit means the appearance of given protein in one experiment (one FGFR3 transfection). When paralog proteins were found in the same experiment, they were given value 1 irrespectively of their actual amount, to prevent over interpretation of the data. *indicates the paralogs which are listed in parentheses. Whether the given protein is known FGFR interactor is also indicated (Yes/No), based on survey of published literature (Supplementary Table 1).

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Receptor tyrosine kinase; signaling Adapter protein; signaling Adapter protein; signaling Adapter protein; signaling Adapter protein; signaling Adapter protein; signaling Adapter protein; signaling Adapter protein; signaling Adapter protein; signaling Receptor tyrosine kinase; signaling Non-receptor tyrosine kinase; signaling Non-receptor tyrosine kinase; signaling Non-receptor tyrosine kinase; signaling Tyrosine phosphatase; signaling Serine/threonine kinase; signaling Serine/threonine kinase; signaling Lipid kinase; signaling Phospholipase; signaling Inositol phosphatase; signaling GTPase activating protein; signal transduction Transcription regulator; gene transcription Chaperone; protein folding Chaperone; protein folding Chaperone; protein folding Dehydrogenase; energy metabolism Component of ribosome; protein synthesis Component of ribosome; protein synthesis Translation regulator; protein synthesis DNA binding protein RNA binding protein; nucleic acid metabolism RNA binding protein; nucleic acid metabolism RNA binding protein; nucleic acid metabolism Component of cytoskeleton Component of cytoskeleton Cytoskeletal associated protein; cell adhesion Transport Transport

293T cells

Function; process

Fibroblast growth factor receptor; FGFR Docking protein 1; DOK1 Fibroblast growth factor receptor substrate 2; FRS2 GRB2-associated binding protein 1; GAB1 Growth factor receptor-bound protein 2; GRB2 NCK adaptor protein 2; NCK2 Breast cancer antiestrogen resistance protein 1; p130CAS Adapter protein with PH and SH2 domains; SH2B2 Src homology and collagen containing protein; SHC Ephrin receptor; EPH* (A2, B1, B2, B3) Focal adhesion kinase; FAK1, FAK2 SRC family kinase; SRC* (HCK, FYN, LYN, FGR) Activated Cdc42-associated kinase 1; TNK2 Tyrosine-protein phosphatase non-receptor type 11; SHP2 Male germ cell-associated kinase; MAK Ribosomal protein S6 kinase; RPS6K Phosphatidylinositol 3-kinase, p85 subunit; PI3K p85 Phospholipase C1; PLC1 SH2 domain-containing inositol phosphatase 2; SHIP2 ARF GTPase-activating protein; GIT* (1, 2) LIM homeobox protein 5; LHX5 Heat shock 40 kDa protein 1; HSP40 Heat shock protein 70; HSP70 Heat shock protein 90; HSP90 Glyceraldehyde-3-phosphate dehydrogenase; GAPDH Ribosomal protein L; RPL* (7, 9, 13, 23) Ribosomal protein S; RPS* (3, 13, 14, 15) Eukaryotic translation elongation factor 1 A1; eEF1A1 Barrier-to-autointegration factor; BANF1 DEAD-box protein; DDX* (3x, 21) Heterogeneous nuclear ribonucleoproteins; hnRNP* (A2, A3, U) Nucleolin; NCL Kelch-like protein 22; KLHL22 Pleckstrin homology domain containing family H2; PLEKHH2 Paxillin; PXN Solute carrier family member; SLC* (4, 25) Sorting nexin; SNX18

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No. Interactor name; symbol Bait 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Exp. total (%)

Table 2 Tyrosine phosphorylated proteins identified by MS in RCS cells

6 (100) 2 (33) 3 (50) 1 (16) 1 (16) 3 (50) 3 (50) 1 (16) 3 (50) 2 (33) 3 (50) 3 (50) 4 (67) 4 (67) 1 (16) 2 (33) 1 (16) 4 (67) 4 (67) 3 (50) 1 (16) 1 (16) 4 (67) 2 (33) 4 (67) 2 (33) 2 (33) 2 (33) 2 (33) 3 (50) 4 (67) 2 (33) 3 (50) 2 (33) 2 (33) 2 (33) 3 (50)

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N Y Y Y Y Y Y Y Y Y Y N Y N Y Y Y Y N N N N Y N N N N N N N N N N N N N

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FGFR interactors purified with phosphotyrosine antibody in RCS cells treated with FGF2 (25 ng/ml) and heparin (1 g/ml) for 1 hour. Cells treated with FGF2 and untreated controls constitute one experiment; data are compilation of 6 independent experiments (n=6). Proteins specifically phosphorylated by FGFRs were obtained by subtracting hits recovered in FGF2-naïve cells in each experiment. One hit means the appearance of given protein in one experiment. When paralog proteins were found in the same experiment, all of them were given value 1 irrespectively of their actual amount, to prevent over interpretation of the data. *indicates the paralogs which are listed in parentheses. Whether the given protein was found in 293T MS experiments (Table 1) or is known FGFR interactor based on a published literature (Supplementary Table 1) is also indicated (Yes/No).

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Highlights Proteomic analyses identify 46 novel members of FGFR3 interactome FGFR3 interacts with translation regulator eEF1A1 and transcription factor SHOX2 FGFR3 interacts with serine/threonine kinase ICK to regulate primary cilia function FGFR3 interacts with SHIP2 to trigger sustained activation of ERK MAK kinase

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