EGF Regulates the Interaction of Tks4 with Src through Its SH2 and

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EGF regulates the interaction of Tks4 with Src through its SH2 and SH3 domains Metta Dulk, Balint Szeder, Gabor Glatz, Balazs Mero, Kitti Koprivanacz, Gyongyi Kudlik, Virag Vas, Szabolcs Sipeki, Anna Cserkaszky, László Radnai, and Laszlo Buday Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00084 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Biochemistry Dülk et al., Interaction of Tks4 with Src

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EGF regulates the interaction of Tks4 with Src through its SH2 and SH3 domains

Metta Dülka, Bálint Szedera, Gábor Glatzb, Balázs L. Merőa, Kitti Koprivanacza, Gyöngyi Kudlika, Virág Vasa, Szabolcs Sipekic, Anna Cserkaszkya, László Radnaia, d,*, and László Budaya, c, *

a

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of

Sciences, Budapest, Hungary, bDepartment of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary, cDepartment of Medical Chemistry, Semmelweis University Medical School, Budapest, Hungary, dCurrent Address: Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida

*

Corresponding authors:

László Buday and László Radnai Research Centre for Natural Sciences Hungarian Academy of Sciences Institute of Enzymology Magyar tudósok körútja 2. Budapest 1117, Hungary E-Mail: [email protected] and [email protected]

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Biochemistry

Page 2 of 35 Dülk et al., Interaction of Tks4 with Src

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ABSTRACT The non-receptor tyrosine kinase Src is a central component of the EGF signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues: 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues: 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to synergistic enhancement of binding strength and a highly stable, “bidentate”-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least three hours in living cells. We conclude that the interaction of Tks4 with Src may result in the long-term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation.

Highlights We have elucidated the molecular mechanism by which Src kinase interacts with the scaffold protein Tks4. We have identified the regions of closely adjacent consensus SH2 and SH3 domain binding motifs in Tks4 scaffold protein, and showed that they are necessary and sufficient for high affinity interaction with Src, but only upon the phosphorylation of Tyr508 in Tks4. We have performed the missing biochemical analyses to understand the mechanism of the interaction between Tks4 and Src after EGFR activation. Keywords: EGF, Src, Tks4, SH2 and SH3 domains, tyrosine phosphorylation

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INTRODUCTION Epidermal growth factor receptor (EGFR) is one of the most studied receptor tyrosine kinases that controls fundamental cellular processes such as cell proliferation, differentiation, adhesion, survival, and migration1. Binding of EGF to its receptor at the plasma membrane generates dimerization of EGFR, which results in the autophosphorylation and activation of EGFR2. The activated receptor takes part in several signaling pathways which may modulate the actin cytoskeleton, such as activation of phospholipase Cγ1 and Rho GTPases3,4,5. Tyrosine kinases of the Src family and a variety of Src substrates are also identified as members in transmitting signals downstream of EGFR and other receptors6,7. The protein product of the SH3PXD2B gene is known as Tks4, which plays an important role in the formation of functional podosomes and invadopodia of normal and tumor cells, respectively8. It is required for the production of reactive oxygen species (ROS) by tumor cells9,10, moreover, it is involved in EGF signaling and regulate actin cytoskeleton via Src and EGFR11,12,13. Recently, our group established a novel function for Tks4 in the regulation of mesenchymal stromal cell differentiation14. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome, which is a genetic disorder associated with skeletal defects, craniofacial anomalies including macrocornea, brachycephaly, large anterior fontanels, hypertelorism, anteverted nostrils, thoracolumbar kyphosis, short hands, cardiovascular abnormalities and reduced lipoid tissue14,15,16. The majority of FTHS patients die in infancy or in early childhood due to cardiovascular symptoms or respiratory infections. The analysis of patients identified 4 different intragenic mutations, and one complete deletion of SH3PXD2B on chromosome 5q35.115.

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Previously, Bushman et al. was unable to detect the association between Src and Tks4 in 3T3 cell line8. In contrast, our group demonstrated that EGF treatment of COS7 cells induced the complex formation of Tks4 with Src using co-immunoprecipitation, though the nature of the interaction was not clear17. In this study, we proved that there is a direct physical interaction between phosphorylated Tks4 and Src. By the means of in vitro approaches, we showed that the interaction requires both the SH2 and SH3 domains of Src. In the structure of Tks4, the proline-rich region PSRPLPDAP (residues 466-474) and the tyrosine-phosphorylated SH2 binding motif pYEEI (Residues 508-511) are responsible for the interaction with the SH3 and SH2 domains of Src, respectively. This bidentate-type interaction displays enhanced affinity showing that the intrinsically unstructured linker region (residues 475-507) between the SH3 and SH2 binding motifs of Tks4 was evolved to have a length matching the distance between the binding sites of the SH3 and SH2 domains of Src. In vivo data confirms that mutation of the proline-rich region and changing Tyr508 to phenylalanine in the sequence of Tks4 result in the complete loss of interaction with Src. Therefore, our findings reveal the detailed mechanism by which Src kinase interacts with its substrate Tks4 upon EGF stimulation.

EXPERIMENTAL PROCEDURES Prediction of potential Src binding motifs of Tks4 First, we analyzed the sequence of Tks4 to predict the potential SH3 and SH2 binding linear motifs that could be responsible for the interaction with Src. Disorder tendency was predicted by IUPred18. Homologous Tks4 sequences were downloaded from the HOGENOM19 database. A multiple alignment and a sequence logo representation of the alignment was generated by MUSCLE20and Weblogo 3.421, respectively. Known phosphorylation sites of human Tks4 were assigned according to the PhosphoSitePlus22 database. Linear motifs within unstructured regions were predicted by using the ELM23 prediction service.

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DNA Constructs, Protein Expression, Purification and Phosphorylation Subcloning of the full-length human Tks4 (UniProt ID A1X283) into the mammalian expression vector pcDNA3.1/TOPO-V5-His was described previously11. The mammalian expression vector pCMV6 harboring the full-length coding region of human Src (UniProt ID P12931) was purchased from OriGene Technologies, Inc. These plasmids were used as templates for the polymerase chain reactions to generate all of the constructs for this study. Tks4 and Src constructs were subcloned into a modified pET vector allowing the expression of the recombinant proteins with an N-terminal hexahistidine tag followed by a TEV protease cleavage site. The Tks4 constructs contained the following regions: Tks4-PRRN-TCM (466516), Tks4-TCM-PRRC (505-537). Moreover, the original Tks4-PRRN-TCM construct was changed

to

Tks4-PRRN-(SG)10-TCM

by

insertion

of

20

residues

(SGSGSGSGSGSGSGSGSGSG) between 481Gly and 482Leu. Src constructs contained the following regions: SH3 domain (86-144), SH2 domain (145-249), SH3-SH2 domains (86-249). To generate glutathione-S-transferase-(GST-) fused fragments, segments of Tks4 were subcloned into a pGEX4T-1 vector. Tks4-GST constructs contained the following regions: Tks4-TCM (505-516), Tks4-TCMY508F (505-Y508F-516), Tks4-PRRN-TCM (466-516), Tks4PRRN-TCMY508F (466-Y508F-516). For cell-based assays, mutant V5-tagged Tks4 constructs were generated by QuickChange Lightning Site-Directed Mutagenesis Kit (210518, Agilent Technologies) using the original pcDNA3.1/TOPO-V5-His vector harboring the wild type Tks4 gene as a template. The SH3 and SH2 domain binding motifs PSRPLPDAPH and SAGYEEISDPDM were changed to PSASASDAPH and SAGFEEISDPDM, respectively. All constructs were verified by DNA sequencing. Proteins were expressed in Escherichia coli Rosetta (DE3) pLysS (Novagen) cells using standard techniques24. His-tagged proteins were purified on Ni2+-affinity columns (Novagen).

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The GST fusion proteins were used for pull-down experiments. The His-tagged proteins were digested by TEV protease. The SH3, SH2, and SH3-SH2 domains of Src were further purified on HiTrapQ or HiTrapSP ion-exchange columns (Amersham). Tks4 fragments were further purified by reverse phase chromatography on a C8 HPLC column. The identity of each Tks4 fragment was verified by mass spectrometry. Tks4 fragments were phosphorylated overnight or one hour by the kinase domain of EphB1 in kinase buffer (20 mM TRIS, 150 mM NaCl, 1 mM TCEP, 10 mM MgCl2, 0.01% Triton-X 100, pH=7.6) at room temperature. Expression and purification of EphB1 kinase was described earlier25. Substrates were added to the kinase in 50-fold molar excess. The phosphorylation was initiated by the addition of 100 mM ice-cold ATP solution to the reaction mixtures to reach a final 5-fold molar excess of ATP over the molar amount of Tks4 fragments present. For the fluorescence polarization-based experiments, the phosphorylated Tks4 fragments were purified on a Proteomix SAX-NP3 HPLC column (Sepax Technologies). The molecular weight of the phosphorylated Tks4 fragments was verified by mass spectrometry and the concentration was determined using UV spectroscopy by measuring the absorbance at 280 nm, using calculated molar extinction coefficients26. For phosphotyrosine, the extinction coefficient of 460 M-1cm-1 was used27.

GST pull-down experiments For GST pull-down experiments, 25 µl aliquots of gluthation-Sepharose beads were equilibrated with phosphate buffered saline (PBS). After removing excess PBS, the beads were added to 1 ml of Escherichia coli cell lysates containing various GST-Tks4 fragments. After 30 minutes of incubation at room temperature, the beads were washed four times with PBS, and then equilibrated with kinase buffer. The bound GST-tagged Tks4 fragments were phosphorylated at room temperature using EphB1 kinase (as described above). After one hour

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of phosphorylation, beads were washed four times with PBS to remove the kinase. Src fragments (SH2, SH3, and SH2-SH3 domains) were added to the beads at a final concentration of 100 µM. After 30 min of incubation at room temperature, GST beads were pelleted by centrifugation and washed four times with PBS. Retained proteins were eluted with SDS loading buffer. Samples were subjected to 15% SDS-PAGE and stained with Coomassie protein dye. As controls, purified Src fragments in SDS buffer were loaded onto the gels.

Fluorescence polarization For fluorescence polarization (FP)-based binding affinity measurements, two reporter peptides with

N-terminal

carboxy-fluorescein

(Fl)

labeling

Fl-G(pY)EEIA-NH228

and

Fl-

SLARRPLPPLP-NH229 were obtained from GenScript. The labeled peptides were dissolved in 20 mM TRIS pH 8.0, 100 mM NaCl, 0.05% Brij35P, 2 mM DTT. An increase in the FP signal indicating complex formation between Src fragments and the labeled peptide used was monitored as a function of increasing concentration of the purified Src fragment with a Cytation 3 Cell imaging Multi-Mode Reader (BioTek Instruments) in 384-well plates (Figure S2). The resulting binding isotherms of these direct titrations were fitted to a quadratic binding equation using the OriginPro7 software (OriginLab Corporation) and the determined binding constants of the labeled peptides were used in the analysis of competitive titration data of nonlabelled Tks4 fragments. The affinity of the non-labeled Tks4 fragments to Src was measured in competitive titration experiments as follows: 50 nM labeled reporter peptide was mixed with Src fragments at a concentration to achieve ~80% complex formation. Subsequently, increasing amounts of a non-labeled Tks4 fragment was added and the FP signal was monitored as described above for direct titration experiments. The dissociation constant (Kd) for each Src/Tks4 interaction was determined by fitting the data to a competitive binding model30.

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Titration experiments were carried out in triplicates and the average FP signal was used for fitting the data with OriginPro7.

Cell Lines, Transfection and Stimulation COS7 cells were purchased from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 units/ml), and streptomycin (50 µg/ml). Cells were transiently transfected with X-tremeGENE HP DNA transfection reagent (Sigma-Aldrich) according to the manufacturer’s instructions. For stimulation, cells were serum-starved overnight and stimulated with EGF at 50 ng/ml for 10 min. After EGF stimulation, cells culture medium was replaced with EGF- and serum-free medium, then the cells were harvested at the indicated time point.

Antibodies, Co-Immunoprecipitation Antibodies against the V5 epitope (R960-25) and Src (2109) were purchased from Invitrogen and Cell Signaling Technologies, respectively. Antibody against Src-pY418 (44-660G) was obtained from ThermoScientific. Antibodies against pTyr (4G10/05-321) and α-tubulin (T6199) were ordered from Sigma-Aldrich. Cells were washed with PBS and lysed in ice-cold 30 mM Tris buffer (pH 7.5), containing 100 mM NaCl, 1% Triton X-100, 10 mM NaF, 1 mM EGTA, 1 mM Na3VO4, 2 mM p-nitrophenyl-phosphate, 10 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride (PMSF) and 25 µg/ml each of Pepstatin A, trypsin inhibitor, and aprotinin. Lysates were clarified by centrifugation at 20000 g for 10 min at 4°C. For immunoprecipitation, 1 ml cell lysates were added to 25 µl anti-V5-Agarose in PBS and incubated for 1 hour at 4°C, washed four times in ice-cold PBS containing 1% Triton-X, and then boiled in sample buffer (4-fold, 0.2 M Tris, 0.277 M SDS, 40% (V/V) glycerol, 0.588 M β -mercaptoethanol, 0.05 M EDTA, 1.19 mM bromophenol blue in distilled water) for 3 min.

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Equal amounts of samples were subjected to SDS-PAGE using 10% running gels, respectively. The proteins were transferred to PDVF membranes. Membranes were blocked and incubated for 60 min with the appropriate primary antibodies at room temperature. After several washing steps, membranes were incubated for 30 min with a horseradish peroxidase-conjugated secondary antibody (GE Healthcare) and washed again. Reacting antigens were visualized with the enhanced chemiluminescence (ECL) detection reagents (Amersham Life Sciences Limited).

Confocal Microscopy, Duolink Proximity Ligation Assay (DPLA) In situ interactions were detected by the proximity ligation assay Duolink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101-1KT, Sigma-Aldrich). The DPLA probe anti-rabbit plus binds to the Src or Src-pY418 primary antibody, whereas the DPLA probe anti-mouse minus binds to the V5 primary antibody against Tks4. The DPLA secondary antibodies generate a signal only when the two DPLA probes have already been bound, indicating the interaction of Src and Tks4. COS7 cells plated on 12-well removable silicone cultivation chambers were transiently transfected with different Tks4 constructs and subsequently serum-starved overnight. Cells were pre-treated with 50 ng/ml EGF for 10 min. After treatment, cells were fixed in 4 % paraformaldehyde-PBS for 15 min on ice, permeabilized using 0.1 % Triton X-100 dissolved in PBS for 5 min, and blocked with Duolink In Situ Blocking Solution for 30 min at 37 °C. AntiV5 mouse, anti-Src or Src-pY418 rabbit antibody was applied in 1:200, 1:100 or 1:100 dilution for 60 or 120 min, respectively. Upon addition of primary antibodies, the Duolink fluorescence method was employed according to the manufacturer’s instructions. The secondary antibodies were provided as conjugates to oligonucleotides that were able to form a closed circle via base pairing and ligation using Duolink ligation solution when the antibodies were in close proximity (at a distance of less than 40 nm)31. The detection of the signal was conducted by rolling circle amplification using DNA polymerase and fluorescently labelled nucleotides

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hybridized into the amplification products. Each resulting bright fluorescent dot represents an individual interaction event. Nuclei were visualized by DAPI staining. Images of fixed samples were acquired on a Zeiss LSM710 inverted confocal microscope with 40× objective (Carl Zeiss). Images were processed using ZEN software (Carl Zeiss). The total number of PLA foci were determined using the FindFoci32 plugin of ImageJ. In each case the number of foci were counted in a random set of images containing 200 nuclei in total. Subsequently, the total number of foci was divided by the total number of nuclei yielding the foci/nuclei ratio.

RESULTS Sequence analysis of Tks4 Alignment of all available Tks4 sequences from the HOGENOM database19 revealed several conserved linear motifs within the unstructured regions of the protein (see Figure S1 for a sequence logo representing the full alignment). We used the ELM prediction service23 to map the potential binding sites of the Src SH3 and SH2 domains in the disordered regions of Tks4. It has been well documented that the SH2 domains of several Src family kinases (e.g. Fyn, Lck, and Src) prefer to bind pYEEI motifs in the partner sequences33. The ELM server predicted only one Src SH2 binding motif (residues: 508-511, YEEI) in Tks4. Moreover, this tyrosine (Y508) is the only known tyrosine phosphorylation site within the unstructured regions of Tks4 based on the PhosphoSitePlus database22 and literature data8,34. Therefore we hypothesized that this SH2 binding motif in the sequence of Tks4 might be responsible for the regulated interaction with Src17 (Figure 1A). Substrates of Src kinases often interact with the Src SH3 domain as well35,7, therefore, we further analyzed the Tks4 sequence to identify potential SH3 binding motifs. We found a potential binding motif (residues: 466-474, PSRPLPDAP) also in the disordered region of Tks4, which has very similar sequence to a known (Kd ≈ 3.7 µM) ligand of the Src SH3 domain (RPLPPLP)29. Moreover, this particular sequence is located in

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close proximity (33 residues distance) to the SH2 binding motif, potentially allowing a bidentate-type interaction and synergistic enhancement of binding. Based on literature data, the spacing of the potential Src binding sites in Tks4 is consistent with the linker-length of some other tandem interaction partners of Src: cortactin36,37, AFAP-11038, p130Cas39, FAK40, Sam6841, EFS42 (Figure 1B). Therefore, we hypothesized that these motifs together may be responsible for the interaction between Src and Tks4. We also identified another conserved proline rich region (residues: 520-525, PSLPPR) located towards the C-terminus from the SH2 binding motif (Figure 1A). The distance between the two motifs is even shorter in this case (8 residues). Therefore, the ability of the potential SH2 binding motif and both predicted SH3 binding motifs to bind to the corresponding domains of Src was assayed experimentally.

In vitro association of Src SH2 and SH3 domains with Tks4 fragments fused to GST proteins To prove that the predicted motifs PRRN and pTCM identified in the structure of Tks4 can bind to the SH2 and SH3 domains of Src, different constructs of Tks4 and Src were prepared and tested in GST pull-down experiments. Tks4 fragments (Tks4-TCM, aa 505-516; Tks4-PRRNTCM, aa 466-516) were expressed as GST fusion proteins in E. coli, bound to GSH-agarose beads and then phosphorylated in vitro (Tks4-pTCM, Tks4-PRRN-pTCM). Subsequently, the beads were incubated with recombinantly expressed and purified domains of Src (SH3, SH2, SH3-SH2), as described in the Experimental procedures. The phosphorylated Tks4 fragments (Tks4-pTCM and Tks4-PRRN-pTCM) were able to bind the SH2 domain (Figure 2A and B) as well as the tandem SH3-SH2 domains of Src (Figure 2A and B). In contrast, the non-phosphorylated Tks4 fragments (GST-Tks4-TCM and GST-Tks4-PRRN-TCM) showed no detectable binding in either case (Figure 2A and B). We could not detect any interaction between the SH3 domain of Src and GST-Tks4-PRRN-TCM or GST-Tks4-PRRN-pTCM under

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these circumstances (Figure 2B). As the affinity of the SH3 domain toward the Tks4 prolinerich region is relatively low (see below), extensive dissociation might have occurred during the washing steps preventing us to detect these interactions in pull-down experiments. Interestingly, the binding of GST-Tks4-PRRN-pTCM protein to the tandem SH3-SH2 domains of Src was markedly stronger than to the SH2 domain alone, suggesting a synergistic binding (arrows in Figure 2B). The tyrosine to phenylalanine (Y508F) mutant Tks4 constructs showed no detectable binding to any of the Src fragments (Figure 2C and D), underlying that the stable interaction between the two proteins is regulated by the phosphorylation of Tyr508 and that the pTCM region of Tks4 and the SH2 domain of Src are the key structural elements providing the most significant contribution to the total binding affinity between Tks4 and Src.

In vitro association of Src SH2 and SH3 domains with Tks4 fragments assayed by fluorescence polarization experiments Based on the results of sequence analysis and pull down experiments, we hypothesized that the two Src binding sequences of Tks4 may allow for Tks4 a synergistic enhanced binding to Src. To quantitate the binding affinities between Src and Tks4 fragments, we performed a fluorescence polarization (FP) based protein-protein interaction assay. The Src SH2 and SH3 specific fluorescein-labeled reporter peptides Fl-G(pY)EEIA-NH228 and Fl-SLARRPLPPLPNH229, respectively, were used to measure competitive binding of two different unlabeled Tks4 constructs: Tks4-PRRN-TCM and Tks4-PRRN-pTCM. Using Fl-SLARRPLPPLP-NH2 as reporter peptide to Src-SH3, we observed a weak interaction between the PRRN of Tks4 and the SH3 domain of Src (Kd ≈ 30 µM) regardless of phosphorylation status (Figure 3A). The non-phosphorylated fragment Tks4-PRRN-TCM showed a very similar binding constant (Kd ≈ 20 µM) to Src-SH3-SH2 indicating that the linker sequence between Tks4-PRRN and Tks4TCM or the non-phosphorylated Tks4-TCM does not contribute to the binding significantly.

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However, we observed a three orders of magnitude stronger binding (Kd < 30 nM) due to the presence of the phosphate group in Tks4-PRRN-pTCM (Figure 3B and E). Using Fl-G(pY)EEIA-NH2 as a reporter peptide to Src-SH2, Tks4-PRRN-pTCM showed strong binding (Kd ≈ 50 nM) to the SH2 domain of Src (Figure 3C), however, the interaction with SrcSH3-SH2 was remarkably stronger (Kd ≈ 3 nM) (Figure 3D and E). We could not detect any interaction in the same experiments if the phosphate group was not present (Figure 3D and E) as the absence of a suitable SH2 ligand prevents the competitive replacement of the reporter peptide from the SH2 domain of Src. Based on these results, the binding constant for the interaction between Src-SH3 and Tks4PRRN is around 30 µM. The Tks4-TCM needs to be phosphorylated to interact with Src-SH2 with a binding constant of ~50 nM. Although we performed two experiments showing the affinity of Tks4-PRRN-pTCM to Src-SH3-SH2 (upper panels of Figure3B and Figure 3D), dissociation constants falling into the nanomolar range can be more accurately determined if the Kd of the reporter peptide is also in the same range (Figure S2). Therefore, we believe that the correct dissociation constant for the binding of Tks4-PRRN-pTCM to Src-SH3-SH2 is ~ 3 nM. The fact that the binding affinity indicates much stronger interaction in the case of Src-SH3SH2 and Tks4-PRRN-pTCM (Kd ≈ 3 nM) compared to the interactions of individual domains and ligands (Kd ≈ 30 µM for SH3 and Kd ≈ 50 nM for SH2) clearly shows that there is a significant synergism between the binding of Tks4-PRRN to Src-SH3 and Tks4-pTCM to SrcSH2. It is important to note that these results are consistent with the results of GST pull-down experiments, suggesting that the PRRN-pTCM region of Tks4 is responsible for the interaction with the SH3 and SH2 domains of Src. High affinity tandem interactions require an optimal spacing of the binding sites43. Based on our measurements the Src binding sites of Tks4 are approximately in the right positions relative to each other and show an enhanced affinity

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through the bidentate binding effect. As expected, artificial elongation of the linker region in Tks4 by 20 residues results in a reduction of the binding affinity (wild-type linker: Kd= 3 nM, insertion: Kd= 6 nM, Figure S3). Similar experiments demonstrate that the other potential SH3 ligand PRRC (residues: 520-525) located towards the C-terminus of Tks4 from pTCM cannot bind to Src-SH3 (Figure S4).

Cell-based assays to assess the molecular background of the interaction between Tks4 and Src kinase Previously, we reported that upon EGF treatment of COS7 cells, Src kinase associates with its substrate Tks4 in an inducible manner17. However, those experiments revealed neither if there was a direct physical interaction between Tks4 and Src nor the domains or linear motifs responsible for their association. To provide in vivo evidence for the above proposed mechanism of interaction, COS7 cells were transiently transfected with either wild type V5Tks4 or with a V5-Tks4 construct in which both the proline-rich region and the tyrosinecontaining motif were mutated (V5-Tks4PRRN-TCMmut), as described in the Experimental Procedures. Following overnight serum starvation, cells were stimulated with EGF or left untreated. Tks4 proteins were then immunoprecipitated with anti-V5 antibody and subjected to anti-Src and anti-V5 immunoblotting. In agreement with our results described earlier17, wild type Tks4 shows a detectable interaction with Src in response to EGF treatment (Figure 4A). However, the mutant Tks4 construct (V5-Tks4PRRN-TCMmut) could not precipitate Src even upon EGF stimulus (Figure 4A). Moreover we detected the tyrosine-phosphorylation of V5-Tks4wt and V5-Tks4PRRN-TCMmut with anti-pTyr antibody in response to EGF treatment. The stimulation resulted in robust phosphorylation of wild type Tks4, in contrast, the phosphorylation was significantly reduced when the Src-binding motifs were mutated in Tks4 (Figure 4B). These results suggest that the PRRN-pTCM region is the key determinant of the interaction between

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the two proteins and the phosphorylation of Tks4 in vivo. Moreover, the strong reduction in tyrosine phosphorylation of Tks4 when PRRN and TCM were mutated suggests that the absence of the direct physical interaction between Tks4 and Src reduces the phosphorylation at the other two tyrosine phosphorylation sites in Tks4 as well (Tyr25 and Tyr373), most likely due to the reduction of the effective concentration of the substrate around the activated kinase domain of Src.

To confirm that the Src-Tks4 interaction can occur in situ in living cells, we performed a Duolink proximity ligation assay. This approach generates fluorescent spots if the target proteins are located in a radius less than 40 nm. COS7 cells were transiently transfected with wild type V5-Tks4 or with the V5-Tks4PRRN-TCMmut constructs. Following overnight serum starvation, cells were stimulated with EGF or left untreated. Subsequently, cells were incubated with monoclonal mouse anti-V5 and polyclonal rabbit anti-Src antibodies followed by the Duolink fluorescence detection according to the manufacturer’s instructions. Upon EGF stimulation, the number of red fluorescent spots increased significantly compared to the control (non-stimulated) cells (Figure. 5A, B and E). In contrast, there was no change in the number of red fluorescent spots in response to EGF stimulation in the case of Tks4 mutant construct transfected cells (Figure 5C and 5D). Taken together, the series of the above experiments strongly suggest that upon EGF stimulation, Src forms a complex with its substrate Tks4 within the cells, and the interaction of the two proteins is likely mediated by the SH2 and SH3 domains of Src.

Time course of the association of Src with Tks4 in EGF-treated cells To measure the time course of the complex formation between activated Src and Tks4, V5 epitope-tagged Tks4 was transiently expressed in COS7 cells. The cells were stimulated with

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EGF for 10 minutes, then cells culture medium was replaced with EGF- and serum-free medium, finally, cells were harvested at the indicated time point (Figure 6A). Tks4 proteins were then immunoprecipitated with anti-V5 antibody and the samples were subjected to antiSrc immunoblotting. To ensure the equal loading of the immunoprecipitated samples, PDVF membranes were reprobed with anti-V5 antibody. Furthermore, cell lysates were subjected to anti-tubulin immunoblots. Figure 6A and B demonstrate that the interaction reaches its maximum around 30 minutes and lasts at least for 180 minutes. Next, to monitor the activation status of Src upon formation of the complex with Tks4, a phosphospecific antibody against pTyr418 of Src, a known indicator of Src activation35, was applied in the proximity ligation assay. As Figure 6C shows, tyrosine-phosphorylated Src in its active form can associate with Tks4 in response to EGF stimulation. Our results show that Src becomes activated and the kinase associates stably with its substrate Tks4 in the EGF signaling cascade.

DISCUSSION It has been described earlier that the Frank-ter Haar syndrome protein Tks4 contributes to EGF signaling. Upon EGF treatment of COS7 cells, Src kinase phosphorylates Tks4, although the association of Tks4 to the EGFR/Src complex was contradictory8,17. Moreover, the nature and biochemical properties of the interaction remained elusive17. In this study, we aimed to prove if there is a direct physical interaction between these proteins, to identify the exact contact site/sites both on Tks4 and Src, to show that the identified regions are relevant in vivo and to follow the time course of the interaction in living cells.

Detailed sequence analysis revealed a single, phosphorylated tyrosine-containing, SH2 domainspecific linear motif (Tks4-TCM) and seven potential SH3-binding proline-rich motifs in the sequence of Tks4. A single motif located N-terminally from TCM (Tks4-PRRN) showed

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remarkable similarity with a known binding motif of Src-SH329, while the TCM had 100% sequence identity with a known binding motif of Src-SH233. Therefore, we hypothesized that there is indeed a direct interaction between Tks4 and Src mediated by these motifs. By a series of in vitro experiments with recombinant protein fragments, we proved that the predicted proline-rich region (PSRPLPDAP, residues: 466-474) can bind the Src-SH3, while the phosphotyrosine-containing motif (pYEEI, residues: 508-511) can interact with Src-SH2. Our fluorescence polarization experiments demonstrated clearly that the bidentate-type interaction between Src and Tks4 displays a synergistic enhancement of binding strength, similarly to an avidity effect43. This is only possible if the distance between the individual binding motifs is matching the distance between the SH3 and SH2 domains of Src. If the linker is shorter, the two motifs can either interact with the appropriate domains in two different molecules or compete for binding within the same molecule as both of them are inaccessible for both of the domains simultaneously44. In this case, no synergistic binding enhancement can be observed, instead, oligo-, and multimers of the associated proteins may form (depending on the concentrations and affinities). If the linker is too long, the two motifs behave as if they were independent. The binding of the first does not restrict the random movement of the second holding it in close proximity to the appropriate binding site on the other domain. In this scenario, only slight or no enhancement in binding affinities can be observed43. The time course of the complex formation between Src and its substrate was already measured in the case of other Src binding partners such as MVP (major vault protein) and RACK1 (receptor of activated protein C kinase 1) upon EGF or PDGF treatment45,46. It was observed that the association of Src with its substrates through its SH2 domain has a maximum after few minutes and it returns to the basal level (15 and 5 min). Here we observed a very strong enhancement in binding affinities based on the dissociation constants (Src-SH3/Tks4-PRRN: ~30 µM, Src-SH2/Tks4-pTCM: ~50 nM, Src-SH3-SH2/Tks4-PRRN-pTCM: ~3nM), clearly

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demonstrating that these proteins have been evolved to form highly stable complexes upon phosphorylation of Tks4. This high affinity interaction may be important to make pTyr508 inaccessible for phosphatases over prolonged time periods, therefore, supports the long-lasting presence of the activated Src/Tks4 complex in EGF-treated cells. Src kinases are known to be activated by binding of appropriate ligands to their SH2 and/or SH3 domains47,35. For example, Src is activated by SH2 domain-mediated interaction with the autophosphorylated EGF and PDGF receptors13,48,49. A number of Src substrates also contain binding motifs for both the SH3 and SH2 domains of Src, including p110/AFAP150, and p130CAS50,39,51, and focal adhesion kinase (FAK)40,52 (other examples on Figure 1B). Our findings are therefore in line with the above mentioned examples demonstrating that the Src/Tks4 interaction requires both the SH3 and SH2 domains of Src. Interestingly, tandem interaction of Src with its previously identified substrates (e.g. FAK) does not appear to confer a synergistic bidentate-type binding effect53,35, however, the ‘anti-cooperativity’ reported by the authors might be explained by the entropic penalty associated with the restricted movement of the linker region connecting the binding motifs in the Src-bound state. This entropic penalty might influence all similar bidentate-type interactions resulting in a reduced free energy change compared to the sum of free energy changes determined for the individual motif-domain interactions (in other words, weaker binding as expected for an ideal linker). An SH3 binding motif usually supports weak and transient interaction with the kinase that is necessary for substrate recognition6. An SH2 ligand in a substrate, especially in tandem arrangement with an SH3 ligand, serves as a high affinity binding site for the kinase keeping the SH2 and SH3 domains in complex and preventing them from participating in the intramolecular interactions necessary for kinase autoinhibition, thus providing and preserving kinase activity for prolonged time periods6. This way, the activated kinase can processively phosphorylate multiple sites within its interaction partner or within any associated protein participating in the same

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complex7. The phenomenon of processive phosphorylation has been established for a number of Src substrates. This is referred to as a mechanism by which Src remains bound to the substrate while catalyzing multiple rounds of tyrosine phosphorylation. It has been shown that binding of C-terminal proline-rich region of p130CAS to Src SH3 domain is prerequisite of the subsequent phosphorylation of p130CAS on several tyrosine residues54. In addition, use of phosphospecific antibodies has demonstrated that mutation of tyrosine 421 to phenylalanine in the sequence of cortactin prevented the phosphorylation of tyrosine 46655,56. Tks4 has been reported to be phosphorylated on tyrosines 25, 373, and 508 by Src kinase8. Therefore, it is likely that Src binds Tks4 through its SH2 and SH3 domains following phosphorylation of Y508 and then tyrosine phosphorylation could occur on the remaining tyrosine residues 25 and 373. These residues are located in the PX and third SH3 domains of Tks4, respectively. Therefore, they are more likely to be worse substrates of Src compared to Y508 that is located in an intrinsically unstructured region. Though it is tempting to speculate that these phosphorylation events may regulate the lipid binding of the PX domain or the protein ligand binding of the third SH3 domain, we have no information about the potential role of these post translational modifications yet.

In our previous work, we showed that the association of EGFR with Tks4 is not direct but requires the presence of Src kinase17. The activated EGFR is not capable of phosphorylating Tks4 directly, instead, activated Src is responsible for the tyrosine-phosphorylation of Tks417. It is important to note that upon autophosphorylation and activation of the EGF receptor, Src can bind to the receptor through its SH2 domain and becomes activated13. What can be the mechanism by which EGFR-associated and activated Src subsequently interacts with Tks4 via its SH2 and SH3 domains? Based on our findings and the literature the following model could be proposed. In quiescent cells Tks4 is predominantly localized in the cytosol. Upon EGF

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stimulation, dimerized and autophosphorylated EGF receptor recruits and activates Src tyrosine kinase, primarily through phosphotyrosine/SH2 domain interaction. The activated Src interacts with Tks4 via its free SH3 domain. Upon interaction, Src can phosphorylate Tks4 on Y508, then, the SH2 domain of Src may be released from the EGFR and could bind the pTCM sequence in Tks4 (Figure 7) as it is in tandem arrangement with Tks4-PRRN. The very strong, synergistic bidentate-type interaction makes it more likely that Src prefers Tks4 and not the EGFR as a binding partner. However, the activated receptor may bind another molecule of Src later, thus re-starting the cycle and amplifying the signal. Although, it has been demonstrated in the case of a number of Src substrates that their prolinerich region can bind to the SH3 domain of Src family kinases, thereby stabilizing their active conformation, the weak (Kd ~30 µM) interaction between Tks4-PRRN and Src-SH3 seems to be insufficient by itself to promote kinase activation. For example, a proline-rich motif of Caskbinding protein (Cbp) can disrupt the SH3 domain-mediated intramolecular interactions of several Src family kinases leading to the initiation of the kinase activation57. Binding of the HIV-1 protein Nef to the SH3 domain of the Src family kinase Hck can also activate the kinase58,59. In contrast, the phosphorylation of Y508 in Tks4 is the prerequisite for strong Src binding. The PRRN region in non-phosphorylated Tks4 seems to interact only with the free SH3 domain of Src bound to EGFR and not capable of opening the autoinhibited conformation of Src by itself. (In that case, solely the presence of Tks4 could activate Src.) However, the strong, bidentate-type interaction of Src with its phosphorylated substrate results in the long-term stabilization of the kinase in an active conformation allowing further substrate phosphorylation. It is important to note that we cannot exclude the presence of other proteins in vivo that may further stabilize, regulate or modify the above discussed interactions. Nevertheless, further experiments will be required in the future to understand the Src kinase activation with Tks4 in EGF signaling pathway.

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SUPPORTING INFORMATION Additional figures for sequence analysis of Tks4 and fluorescence polarization experiments. ACKNOWLEDGEMENTS The work was supported by a grant from the National Research, Development and Innovation Fund of Hungary (K 124045 and FIEK_16-1-2016-0005) and the MedinProt Program of the Hungarian Academy of Sciences (LB). LR and VV were supported by a post-doctoral fellowship program by the Hungarian Academy of Sciences. The work of VV was supported by a János Bolyai Research Scholarship of the Hungarian Academy of Sciences. We thank Dávid Szüts (Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences) and László Nyitray (Department of Biochemistry, ELTE Eötvös Loránd University) for careful reading of the manuscript. CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article. AUTHOR CONTRIBUTIONS STATEMENT MD conceived the project, carried out the experiments, analyzed data and wrote the paper. LR, VV and LB supervised the research and prepared the manuscript. BS, BLM, and AC did confocal microscopy and prepared figures. KK and GK contributed by carrying out cell-based experiments. SS designed and contributed to the preparation of Figure 4 and 6. GG assisted in the fluorescence polarization experiments. All authors analyzed the results and approved the final version of the manuscript. ORCID Metta Dülk: 0000-0001-9364-2587 Bálint Szeder: 0000-0003-3264-3900 Gábor Glatz: 0000-0003-1474-6746 21


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Balázs L. Merő: 0000-0001-9517-6460 Kitti Koprivanacz: 0000-0003-1576-1040 Gyöngyi Kudlik: 0000-0002-2210-7410 Virág Vas: 0000-0001-7249-6816 Szabolcs Sipeki: 0000-0002-9678-6743 Anna Cserkaszky: 0000-0002-7053-7618 László Radnai: 0000-0001-6843-3740 László Buday: 0000-0003-3518-5757

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W. T. (1997) Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature 385, 650–653. (59) LaFevre-Bernt, M., Sicheri, F., Pico, A., Porter, M., Kuriyan, J., and Miller, W. T. (1998) Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue. J. Biol. Chem. 273, 32129–32134.

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FIGURE LEGENDS

Figure-1. Sequence analysis and the potential Src-binding motifs of Tks4. A, Schematic representation of the domain architecture and known Src phosphorylation sites of human Tks4. The PX domain, SH3 domains and intrinsically unstructured linker regions are blue, cyan and grey, respectively. The known tyrosine phosphorylation sites Y25, Y373 and Y508 are located in the PX domain, third SH3 domain and the linker region connecting the third and fourth SH3 domains, respectively. Weblogo representation of a sequence alignment of homologous Tks4 sequences show three conserved linear motifs (highlighted by red double arrows) within the long unstructured region of Tks4 connecting the third and fourth SH3 domains. Besides the tyrosine phosphorylated pYEEI motif, pTCM (phosphotyrosinecontaining motif, residues 508-511), no other potential SH2 binding motifs could be identified in the sequence. The highlighted conserved motif, PRRN (residues: 466-474) has a remarkable similarity to a known binding sequence of the SH3 domain of Src. There is another potential proline rich SH3 binding motif (PRRC) located C-terminally to pTCM. The sequence logo was generated based on alignment of full length Tks4 sequences obtained from the HOGENOM database. Potential binding motifs within the intrinsically unstructured regions of Tks4 were predicted using ELM and PhosphoSitePlus. B, Several proteins, interacting with Src SH3 and SH2 domains, were collected to demonstrate the optimal length of linker region between the binding sites; based on literature data.

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Figure-2. In vitro association of different Src and GST fusion Tks4 fragments. A, GST-Tks4-TCM (1) and its phosphorylated form, GST-Tks4-pTCM (2), were used for precipitation of Src SH2 and SH3-SH2 domains. B, GST-Tks4-PRRN-TCM (3) and its phosphorylated form, GST-Tks4-PRRN-pTCM (4), were used to pull-down SH3, SH2 and SH3-SH2 domains of Src. C, and D, Y508F mutants of Tks4 fragments Tks4-GST-TCMY508F (5), kinase treated GST-Tks4-TCMY508F (6) and GST-Tks4-PRRN-TCMY508F (7), kinase treated GST-Tks4-PRRN-TCMY508F (8), were negative controls for the phosphorylation of GST fusion constructs. Arrows, in the case of Tks4 fragments 2 and 4, indicate the position of successfully precipitated SH2 and SH3-SH2 domains of Src. The SH3, SH2 and SH3-SH2 domain constructs of Src, as indicated, were used as loading controls. Gels were run simultaneously under the same experimental conditions.

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Figure-3. Competitive titration experiments monitoring the binding affinity of different Tks4 fragments to SH2 and/or SH3 domains of Src. A and B, competitive FP measurements for different Src domains, SH3 (A) and SH3-SH2 (B) by using the SH3 domain specific fluorescein-labeled reporter peptide Fl-SLARRPLPPLPNH2. C and D, competitive experiments for SH2 (C) and SH3-SH2 (D) domains of Src by using the SH2 domain specific fluorescein-labeled reporter peptide Fl-G(pY)EEIA-NH2. E, summary of binding affinities between different Src domains and Tks4 constructs. The upper and lower panels on A, B, C and D, show competitive titrations using unlabeled Tks4-PRRNpTCM and Tks4-PRRN-TCM fragments, respectively. Error bars represent the standard deviation of data based on three independent measurements. Triplicates were independently prepared samples that were assayed at the same time. On panel E, the dissociation constants are shown with the standard error of fitting.

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Figure-4. The Tks4 PRRN-TCM mutant could not associate with Src and become phosphorylated upon EGF treatment of cells. COS7 cells were transiently transfected with V5-Tks4wt or V5-Tks4PRRN-TCMmut. After serumstarvation, cells were stimulated with EGF (lanes 3 and 5 on panel A, 2 and 4 on panel B) or left untreated (lanes 2 and 4 on panel A, 1 and 3 on panel B). Tks4 proteins were immunoprecipitated with anti-V5 antibody and analyzed by anti-Src, anti-pTyr, anti-tubulin and anti-V5 antibodies. The first lane (control) on panel A represents non-specific binding of proteins to V5-beads from lysates of non-transfected cells. Gels were run simultaneously under the same experimental conditions.

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Figure-5. The EGF-dependent association of Tks4 with Src was demonstrated using proximity ligation assay in COS7 cells. COS7 cells were transiently transfected with V5-Tks4wt (A, B) or V5-Tks4PRRN-TCMmut (C, D) and serum-starved overnight. The cells were treated with EGF (lower panels, B, D), or left untreated (upper panels, A, C) then Duolink proximity ligation assay (DPLA) was performed using rabbit anti-Src antibody, and a mouse anti-V5 antibody. Nuclei (blue) were visualized by DAPI staining in the mounting medium of DPLA. The association of Tks4 and Src is indicated by red dots. E, PLA foci were counted in V5-Tks4wt and V5-Tks4PRRN-TCMmut cells using random sets of images containing 200-200 nuclei in total from each sample. Bar graphs represent an average foci/nuclei ratio (±S.D.) of three samples (independent experiments). *Statistical differences at P

EGF Regulates the Interaction of Tks4 with Src through Its SH2 and

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