Oncogene (2009) 28, 937–949 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE XB130, a tissue-specific adaptor protein that couples the RET/PTC oncogenic kinase to PI 3-kinase pathway

M Lodyga1,4, V De Falco2,4, X-h Bai1, A Kapus3, RM Melillo2, M Santoro2 and M Liu1,3

1Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada; 2Dipartimento di Biologia e Patologia Cellulare e Molecolare, Instituto di Endocrinologia ed Oncologia Sperimentale del CNR ‘G. Salvatore’, Naples, Italy and 3Department of Surgery, University of Toronto, Toronto, Ontario, Canada

XB130 is a recently cloned 130 kDa-adaptor protein and et al., 1993; Lodyga et al., 2002; Han et al., 2004), we Src kinase substrate, structurally similar to -filament- cloned a novel 130 kDa protein, referred to as XB130 associated protein. Here we show that XB130 is pre- (Xu et al., 2007). The human xb130 gene is localized on dominantly expressed in the thyroid. Given that XB130 is a 10q25.3, and encodes an 818 amino-acid thyroid-specific tyrosine kinase substrate, we asked protein that shares 35% similarity with AFAP. The whether it is targeted by RET/PTC, a genetically overall structure of XB130 is highly suggestive of a rearranged, constitutively active, thyroid-specific tyrosine complex adaptor protein as it is comprised of various kinase that plays a pathogenic role in papillary thyroid protein–protein and lipid–protein interaction motifs. cancer. RET/PTC induced robust tyrosine phosphory- Specifically, the N-terminal region of XB130 includes lation of XB130, which promoted its subsequent associa- several potential tyrosine phosphorylation sites and tion with the p85a subunit of phosphatidylinositol 3-kinase proline-rich sequences that might interact with SH2- (PI 3-kinase). We identified tyrosine 54 of XB130 as the and SH3-domain-containing proteins, respectively. The major target of RET/PTC-mediated phosphorylation and middle portion harbors two pleckstrin homology a criticalbinding site for the SH2 domains of p85 a. domains that may target proteins to cellular membranes Importantly, downregulation of XB130 in TPC1 papillary through interactions with specific phospholipids thyroid cancer cells, harboring the RET/PTC1 kinase, (Haslam et al., 1993; Harlan et al., 1994), whereas the strongly reduced Akt activity without altering ERK1/2 C-terminal region contains a coiled-coil domain, which phosphorylation, and concomitantly inhibited cell-cycle might be involved in protein oligomerization and DNA progression and survivalin suspension. In conclusion, binding. Our previous studies have indeed implicated XB130 is a novelsubstrate of the RET/PTC kinase that XB130 as a likely substrate and regulator of tyrosine links RET/PTC signaling to PI 3-kinase activation, and kinase-mediated signaling (Xu et al., 2007). Notably, thereby plays an important role in sustaining proliferation XB130 was shown to associate with c-Src, and the and survivalof thyroid tumor cells. overexpression of these proteins in COS-7 cells resulted Oncogene (2009) 28, 937–949; doi:10.1038/onc.2008.447; in increased XB130 tyrosine phosphorylation, together published online 8 December 2008 with enhanced Src activity. We found that XB130 expression is restricted to Keywords: adaptor protein; protein tyrosine phospho- thyroid follicular cells, whereas most other tissues rylation; thyroid cancer; RET/PTC; phosphatidylinositol (exceptthespleen) showed only marginal expression. 3-kinase; PKB/AKT We asked whether XB130 might be an adaptor particularly involved in thyroid-specific tyrosine kinase pathways. Dysregulation in tyrosine kinase signaling is an essential feature in the development of thyroid cancer. RET/PTC rearrangements are a genetic hall- Introduction mark of papillary thyroid cancer (PTC), the most prevalent type of thyroid cancer in human (Grieco During our recent studies aimed at the characterization et al., 1990). These rearrangements lead to the formation of the actin filament-associated protein (AFAP; Flynn of chimeric and constitutively active versions of the proto-oncogene RET tyrosine kinase (Takahashi et al., Correspondence: Dr M Liu, University Health Network Toronto 1988; Arighi et al., 2005). RET/PTC exhibits transform- General Hospital, Room: TMDT 2-814, 101 College Street, Toronto, Ontario, Canada M5G 1L7. ing ability, affecting differentiation, mitogenic and E-mail: [email protected] or Dr M Santoro, Dipartimento di metastatic potential (Santoro et al., 1994b; Mai et al., Biologia e Patologia Cellulare e Molecolare, Universita Federico II di 2001; Knauf et al., 2003). Indeed, transgenic mice with Napoli, via S Pansini 5, 80131 Naples, Italy. thyroid-targeted expression of RET/PTC1 develop E-mail: [email protected] carcinomas resembling those seen in human PTC 4These authors contributed equally to this work. Received 13 May 2008; revised 1 October 2008; accepted 1 November (Santoro et al., 1996), suggesting that RET/PTC plays 2008; published online 8 December 2008 a disease-inducing role in thyroid cancer. XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 938 Secondary to RET/PTC rearrangements, the Results C-terminal tyrosine kinase domain of RET fuses with the end of one of several unrelated genes (Grieco et al., Thyroid specific expression of XB130 1990). RET/PTC1 (the H4-RET fusion) and RET/PTC3 We have demonstrated that XB130 qualifies as (the NCOA4-RET fusion) are the most prevalent a multidomain adaptor protein (Figure 1a), and, in variants. This rearrangement has three major conse- a heterologous expression system, can function as a quences: the kinase becomes constitutively active as the substrate and regulator of Src tyrosine kinase (Xu et al., fusion partner provides a domain capable of sponta- 2007). We determined XB130’s tissue distribution using neous dimerization and autophosphorylation; the chi- northern blot analysis. We found a single transcript of mera loses its intramembrane localization and thus approximately 4 kb, which was abundant in the thyroid resides in the cytosol; and finally, the tissue expression and spleen, but was absent or barely detectable in other pattern changes, as the RET fusion partners are tissues (Figure 1b). Immunohistochemistry staining of able to drive the expression of the rearranged RET thyroid tissue with a specific monoclonal antibody kinase in the follicular epithelial cells of the thyroid verified the presence of XB130 protein in thyroid (Arighi et al., 2005). follicular cells (Figure 1c). No staining was detected RET activation results in receptor oligomerization, when the primary antibody was replaced with isotype- autophosphorylation and the subsequent recruitment of specific IgG2a (Figure 1d). The expression of XB130 proteins containing phosphotyrosine-binding domains. was also found in human papillary thyroid carcinoma; Many of these (for example, Grb 7/10, Src, PLCg, Grb2) approximately half of PTC samples stained, contained bind to individual phosphotyrosine residues, whereas levels of expression comparable to that of normal other signaling molecules share tyrosine 1062 (Y1062) as thyroid tissue (Figure 1e), whereas the remaining their common docking site (Hayashi et al., 2000; Arighi contained weaker staining (Figure 1f). These findings et al., 2005). When phosphorylated, Y1062 acts as the establish XB130 as a thyroid protein, highly expressed in binding site for several protein tyrosine binding (PTB)- normal follicular cells and in follicular-cell-derived containing proteins, including Shc, IRS1/2, FRS2 and tumors. DOK1/4/5. Binding to Shc and FRS2 mediates recruit- ment of Grb2/SOS complexes, which in turn stimulate the Ras/RAF/ERK pathway. Y1062 also mediates RET XB130 is a substrate of RET/PTC binding to Gab (Grb2-associated binding protein) Having verified that XB130 is a thyroid-specific adaptor family adaptors (Gab1 and Gab2) and IRS1/2, large and potential tyrosine kinase substrate expressed in docking proteins recruiting multiple SH2 or SH3 papillary thyroid carcinoma, we asked whether it might domain-containing proteins, including phosphatidylino- be targeted by RET/PTC (Figure 2a). We transiently sitol 3-kinase (PI 3-kinase; Hayashi et al., 2000; Arighi transfected HEK293 cells with myc-tagged XB130 along et al., 2005). Both the RAS/RAF/MAPK and the PI 3- with the most common forms of the RET kinase kinase/Akt pathways play a central role in RET/PTC rearrangement, RET/PTC1 (Pierotti et al., 1992) and signaling and contribute to the ensuing transformation RET/PTC3 (Santoro et al., 1994a). Immunoprecipita- (Santoro et al., 1993; Miyagi et al., 2004; Jung et al., tion of XB130 followed by anti-phosphotyrosine im- 2005; Melillo et al., 2005). Microarray studies in human munoblotting revealed that each form of RET/PTC thyroid cancer indicate that RET/PTC rearrangement is induced strong tyrosine phosphorylation of XB130 associated with expression of genes downstream of PI 3- (Figure 2b). This effectwas dependenton thekinase kinase (Giordano et al., 2005). Further, in FRTL-5 rat activity of RET/PTC as the cotransfection with a thyroid cells overexpression of RET/PTC3 was sug- kinase-dead (KÀ) mutant containing the K284M point gested to preferentially activate the PI 3-kinase over the mutation (K758M in full-length RET; Melillo et al., MAPK pathway (Miyagi et al., 2004). The p85 2001) in RET/PTC3 failed to invoke XB130 phospho- regulatory subunit of PI 3-kinase contains two SH2 rylation (Figure 2c). Coexpression of XB130 and domains (Escobedo et al., 1991), which recognize RET/PTC3 in HEK293 cells did notaffecttyro- phosphotyrosine residues in the contexts of a pYXXM sine phosphorylation of RET/PTC3 (Supplementary motif. As RET does not have such sites, its Figure 1). interaction with PI 3-kinase is mediated by docking We next asked whether autophosphorylation sites of proteins, of which Gab1 (recruited to RET in complex RET/PTC mightbe involved in RET/PTC-induced with Grb2, Shc and ShcC; Besset et al., 2000; Maeda XB130 tyrosine phosphorylation. We cotransfected a et al., 2004; De Falco et al., 2005), IRS-1 (Melillo et al., RET/PTC3 (4YF) mutant in which four identified 2001) and IRS-2 (Miyagi et al., 2004) have been autophosphorylation sites (corresponding to residues implicated. Y826, Y1015, Y1029, Y1062 in the wild-type RET) had Here we show that RET/PTC induces phosphoryla- been mutated to phenylalanine. These substitutions did tion of XB130, predominantly targeting tyrosine 54 not decrease the RET/PTC-triggered tyrosine phosphor- within its YXXM motif. The RET/PTC-induced phos- ylation of XB130 (Figure 2d). This observation implies phorylation results in association between XB130 and that although these tyrosines are key docking sites for a p85 subunit of PI 3-kinase, and this interaction plays a variety of direct substrates or adaptors, they are not major role in the subsequent Akt activation, cell required for XB130 phosphorylation. To verify whether proliferation and survival. RET/PTC and XB130 can form a protein complex, we

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 939

Figure 1 Expression of XB130 in thyroid and papillary thyroid cancer. (a) Schematic representation of the XB130 protein structure. (b) XB130 mRNA is highly expressed in human thyroid and spleen as detected by Northern blotting with XB130 cDNA fragment encompassing nucleotides 811–3750. One major transcript of approximately 4 kb was observed. (c) XB130 protein is abundantly expressed in human thyroid follicular cells as revealed by immunohistostaining with an anti-XB130 monoclonal antibody. (d) Mouse IgG2a was used as a negative control for staining. (e and f) Over 100 papillary thyroid carcinoma samples were stained for XB130 expression. Approximately 50% of samples showed expression comparable to that of normal thyroid tissue, remaining samples possessed relatively weaker staining in the tumor vs the surrounding thyroid tissue, as indicated by the arrow in panel f. coexpressed myc-tagged RET/PTC3 with His-tagged PTC3, we coexpressed Src kinase dead mutant, as a XB130 in HEK293 cells. XB130 can coprecipitate with dominant negative inhibitor of Src PTK (Iavarone et al., RET/PTC; moreover the interaction between these two 2006), along with XB130 and RET/PTC3 in HEK293 proteins critically depends on the kinase activity of cells. The presence of Src KÀ did notreduce RET/ RET/PTC because the kinase-dead version fails to PTC3-induced tyrosine phosphorylation of XB130, nor phosphorylate and form a complex with XB130 did itaffectsignaling downstreamof RET/PTC, Akt (Figure 2e). The interaction was further confirmed by phosphorylation (Supplementary Figures 2a and b). the reverse immunoprecipitation of XB130 with anti-His Further XB130 tyrosine phosphorylation was detected antibody and immunoblotting for RET/PTC with anti- when we coexpressed XB130 along with RET/PTC3 in myc antibody (data not shown). SYF mouse embryonic fibroblasts (Supplementary To determine the role of Src in the tyrosine Figure 3), where three major Src family members (Src, phosphorylation of XB130 in the presence of RET/ Yes and Fyn) have been genetically knocked out.

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 940 Tyrosine phosphorylation of XB130 was further en- the tyrosine phosphorylation of XB130 it is not hanced when Src was reintroduced into these cells. necessary for RET/PTC-mediated tyrosine phosphory- These observations imply that although Src may affect lation of XB130.

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 941 RET/PTC-induced phosphorylation targets Y54 probed for the presence of p85. A small amount of p85 in XB130 was coprecipitated with XB130 in HEK293 cells, and The N-terminal region of XB130 contains several the presence of RET/PTC robustly enhanced the putative tyrosine phosphorylation sites, and our pre- association between p85 and XB130 (Figure 4a). p85 vious studies have shown that this region serves as a contains two SH2 domains, either or both of them can target for Src overexpression-induced phosphorylation. serve as potential target(s) for XB130 recruitment. To To determine whether the N terminus of XB130 is gain insight into the domain requirement of this required for RET/PTC-dependentphosphorylation,and association, we performed a pull-down assay, using N- to identify critical target residues, we utilized an terminal or C-terminal GST-SH2 fusion protein of p85 N-terminal deletion mutant XB130DN (deletion of as bait. The binding assay was carried out with cell residues 2–168), as well as several pointmutationsof extracts derived from HEK293 cells transiently trans- key tyrosines present in this region. Specifically we fected with myc-tagged XB130 alone or in combination substituted tyrosines 54 (DEEYIYMNKV), 124 with RET/PTC. Figure 4b shows that XB130 was able (PEGYYEEAEP) or 161 (SAPYQWPSPE) with phenylalanine to bind to both the N-terminal and the C-terminal SH2 to preclude phosphorylation of these sites (Figure 3a). domain of p85. Importantly, the binding was greatly HEK293 cells were transfected with full-length or potentiated in the presence of RET/PTC. To determine mutant XB130 constructs either alone or in combination the affinity of XB130 to the a-ortheb-isoform of p85, with RET/PTC. Cell lysates were immunoprecipitated we transfected HEK 293 cells with XB130 and/or RET/ with XB130 antibody followed by immunoblotting PTC3. Both a- and b-p85 were detected by direct using anti-phosphotyrosine and anti-XB130 antibodies. western blotting in cell lysates. However, XB130 Figure 3b indicates that deletion of the N-terminal preferentially binds to p85a, butnottop85 b (Supple- region almostcompletelyabolished theRET/PTC- mentary Figure 4). provoked tyrosine phosphorylation of XB130. Experi- Next we checked whether the XB130–p85 interaction ments conducted to probe candidate tyrosine residues is indeed dependent on the phosphorylation status of within this region revealed that point mutations Y124F XB130. We performed a pull-down assay with SH2 and Y161F did notreduce thelevel of RET/PTC- domains of p85 and cell extracts derived from HEK293 triggered XB130 tyrosine phosphorylation, as compared cells transiently cotransfected with RET/PTC and to wild type. In contrast, the Y54F mutant displayed an XB130 mutants (DN, Y54F, Y124F, Y161F). As shown approximately 50% decrease in its tyrosine phosphor- in Figure 4c, the N terminus of XB130 is essential for the ylation, indicating that Y54 is a major although not interaction between XB130 and p85, as in the absence of exclusive target of RET/PTC-mediated tyrosine phos- this region the interaction is almost completely lost. In phorylation (Figure 3b). agreement with our results showing a 50% decrease in RET/PTC-mediated tyrosine phosphorylation of the Y54F XB130 mutant compared to wild type, this XB130 associates with p85 in a RET/PTC-regulated mutant exhibited significant reduction in the interaction manner with both the N- and C-terminal SH2 domain of p85 in The phosphorylated YXXM motif is a specific binding RET/PTC expressing cells. Mutations of Y124F and site for the SH2 domain of the p85 regulatory subunit of Y161F did not noticeably impact the interaction PI 3-kinase (Backer et al., 1992; Carpenter et al., 1993; (Figure 4c). To substantiate the importance of Y54 in Rordorf-Nikolic et al., 1995). As XB130 tyrosine 54 the XB130–p85 interaction, we synthesized two resides in a context of YIYM sequence, which fits the 17-amino-acid peptides based on the sequence surround- required profile, XB130 is a candidate-binding partner ing Y54: one harbors the nonphosphorylated tyrosine of p85. To address this possibility we carried out binding residue (p54), whereas the other contains a correspond- experiments both in vivo and in vitro. HEK293 cells were ing pY (pp54), mimicking the Y54-phosphorylated transiently transfected with myc-tagged XB130 alone or motif within XB130. As shown in Figure 4d, the in combination with RET/PTC, XB130 was immuno- phosphopeptide (pp54) inhibited the interaction precipitated through the tag, and the precipitates were between the SH2 domain of p85 and XB130 in

Figure 2 XB130 is a substrate of RET/PTC kinase. (a) Schematic representation of RET and RET/PTC proteins. SP, signal peptide; EC, extracellular ligand-binding domain; Cys, cysteine-rich region; TM, transmembrane domain; JX, juxtamembrane domain; TK, tyrosine kinase domain. The RET/PTC oncoproteins contain tyrosine kinase domain (TK) of RET and a heterologous domain (X). The catalytic lysine (K758) and major RET autophosphorylation sites (Y826, Y1015, Y1029, Y1062) are indicated on the wild-type version of RET. (b) Tyrosine phosphorylation of XB130 is induced by RET/PTC1 or RET/PTC3. HEK293 cells were cotransfected with cDNAs encoding myc-tagged XB130 and RET/PTC1 or RET/PTC3. Immunoprecipitation of XB130 using a myc antibody was performed followed by western blotting for XB130 with anti-myc and anti-pY antibodies. Total lysates were developed with RET antibody to monitor the transfection level of the RET/PTC (RET/PTC1: 50 kDa; RET/PTC3: 70 kDa). (c) Tyrosine phosphorylation of XB130 depends on RET/PTC kinase activity. XB130 was expressed alone, or coexpressed with RET/PTC3 or a kinase dead (KÀ) mutant. (d) Tyrosine phosphorylation of XB130 does not depend on RET/PTC autophosphorylation sites (Y352, Y541, Y555, Y588 corresponding to Y826, Y1015, Y1029 and Y1062 in full-length RET, respectively). (e) XB130 and RET/PTC3 can form a protein complex. HEK293 cells were transfected with cDNAs encoding His-tagged XB130 alone or in combination with myc-tagged RET/ PTC3 (or its KÀ mutant). Cell lysates were immunoprecipitated with anti-myc antibody and blotted for anti-His, anti-pY or anti-myc antibodies. The fast migration of PTC3KÀ is most likely due to less tyrosine phosphorylation of the protein.

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 942

Figure 3 Identification of RET/PTC-mediated XB130 phosphorylation site(s). (a) Schematic representation of the XB130 mutations. DN, lacking N-terminal amino-acid residues 2–168; Y54F, Y124F and Y161F, carrying the tyrosine (Y) to phenylalanine (F) changes of the indicated tyrosines. (b) Full-length XB130 or its mutants described above were transfected in HEK293 in the presence or absence of RET/PTC3. Cell lysates were immunoprecipitated with anti-XB130 antibody and immunoblotted with anti-phosphotyrosine antibody. Total protein lysates were probed with anti-XB130 and anti-RET antibodies.

a concentration-dependent manner, fully abolishing the used TPC1 thyroid papillary carcinoma cells carrying binding at10 mM. In contrast the nonphosphorylated the RET/PTC1 rearrangement, and treated them with a (p54) peptide failed to affect the association even at specific RET kinase inhibitor, ZD6474 (Carlomagno 50 mM. Collectively these data demonstrates that RET/ et al., 2002), to suppress intrinsic RET/PTC activity. PTC mediates phosphorylation of XB130 at Y54, and Although in untreated TPC1 cells, p85 was constitu- this phosphorylation is required for the efficient inter- tively associated with XB130, ZD6474-treated cells action between XB130 and the p85 subunit of PI failed to associate with endogenous XB130, tested by 3-kinase. coimmunoprecipitation (Supplementary Figure 5a), or Next we examined whether the RET/PTC-dependent either the N-terminal or C-terminal SH2 fusion proteins phosphorylation of XB130 impacts the potential inter- of p85, as verified by GST pull-down assay (Supple- action between XB130 and p85 in a natural setting, that mentary Figure 5b). Following 24 h of ZD6474 treat- is, where these proteins are endogenously expressed. We mentwe observed reduced level of XB130 protein in

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 943

Figure 4 RET/PTC3 enhanced XB130 and p85 interaction. (a) Myc-tagged XB130 was transfected in the presence or absence of RET/ PTC3 in HEK293 cells. Cell lysates were immunoprecipitated with anti-myc and immunoblotted with anti-myc or anti-p85 antibody. (b) Cell lysates collected from HEK293 cells transfected with myc-tagged XB130 and/or RET/PTC3 were incubated with glutathione- Sepharose-immobilized GST-p85 N-terminal or C-terminal SH2 fusion protein. The binding of XB130 to the SH2 domains of p85 was assessed by western blotting using anti-myc antibody. XB130 protein levels in total cell lysates are shown for normalization. (c) Full-length or mutant XB130 cDNA were expressed in the presence or absence of RET/PTC3 in HEK293 cells. Cell lysates were incubated with glutathione-Sepharose-immobilized GST-p85 N-terminal or C-terminal SH2 fusion protein. The samples collected were then processed by western blotting using anti-XB130 antibody. Total cell lysates were analysed for levels of expression of XB130 and RET/PTC3. (d) Cell lysates from HEK293 cells transfected with XB130 and RET/PTC3 were incubated with glutathione-Sepharose- immobilized GST-N-terminal SH2 domain of p85, in the presence of increasing amounts of synthesized peptides designed surrounding Tyrosine 54 of XB130. The phosphorylated peptide (ppY54), but not the nonphosphorylated peptide (p54) reduced binding of XB130 to p85.

TPC1 cells (data not shown). To avoid any confounding strate that RET/PTC-mediated XB130 tyrosine factors that might arise from changes in the endogenous phosphorylation generates binding sites that promote XB130 levels, we expressed the myc-tagged XB130 the interaction between XB130 and p85 in thyroid construct in TPC1 cells before the RET kinase inhibitor tumor cells. treatment. The levels of XB130 protein phosphorylation and its binding to p85 were measured at 3, 6, 12 and 24 h Akt is a downstream target of the RET/PTC-XB130 after treatment with the inhibitor. The efficiency of signaling pathway RET/PTC inhibition by ZD6474 was followed at the To assess the functional significance of XB130 in the same time points by measuring the autophosphorylation RET/PTC-PI 3-kinase pathway, we evaluated the of RET/PTC on residue Y905, which is essential activation of the serine-threonine kinase Akt, a down- for its kinase activity (Iwashita et al., 1996). The results stream target of PI 3-kinase. We measured Akt show that XB130 achieved high basal phosphorylation phosphorylation on Ser 473, which is a sensitive when introduced into the TPC1 cells. Pretreatment of indicator of Akt activity (Alessi et al., 1996). In the cells with ZD6474 caused a large decrease in the HEK293 cells, XB130 or RET/PTC alone led to a phosphorylation of XB130, an effect which correlated moderate increase in Akt phosphorylation, whereas with the inhibition of RET/PTC phosphorylation coexpression of these two proteins exerted a strong (Supplementary Figure 5c). In addition, ZD6474 treat- synergy with regards to Akt phosphorylation. Impor- ment reduced the interaction of XB130 with p85 by tantly, this induction of Akt phosphorylation was approximately 45%. Collectively, these data demon- critically dependent on intact, phosphorylatable

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 944

Figure 5 Akt activation is downstream of RET/PTC-XB130 signaling pathway via a mechanism independent of residue Y1062 of RET/PTC3. (a) HEK293 cells were transfected with XB130, XB130DN, RET/PTC3 alone or in combination. Cell lysates were analysed for phosphorylated Akt (pAKT S473) and total Akt with appropriate antibodies. (b) HEK293 cells were cotransfected with XB130 and RET/PTC3 and incubated either in the absence or presence of 25 mM LY294002. Cell lysates were immunoblotted for total and phosphorylated form of Akt. (c) HEK293 cells were cotransfected with XB130, or XB130 plus RET/PTC3 or 4YF mutant. Cell lysates were incubated with GST-p85 N-terminal or C-terminal SH2 fusion protein. The binding of XB130 to the SH2 domains was determined by western blotting using anti-myc antibody. (d) HEK293 cells were transfected as indicated. Western blot was performed to detect the impact of XB130 on Akt and ERK phosphorylation in the presence of the intact RET/PTC3, or its 4YF or the kinase dead mutant. The changes in phosphorylation were detected with specific Akt and ERK antibodies.

XB130, as it was absent on introduction of the of XB130 (Figure 2d). In agreement with this result, XB130DN mutant (Figure 5a). To substantiate that 4YF mutant was equally proficient at promoting the XB130-RET/PTC promoted phosphorylation of binding between XB130 and either SH2 domain of p85 Akt was indeed mediated by PI 3-kinase, we pretreated (Figure 5c). Accordingly, 4YF mutant was as effective XB130-RET/PTC3 cotransfected HEK293 cells with as the intact RET/PTC3 at promoting the RET/PTC3- LY294002, a PI 3-kinase inhibitor, which abolished XB130-enhanced Aktphosphorylation,however itwas RET/PTC3-XB130-facilitated Akt phosphorylation incapable of inducing ERK phosphorylation (Figure 5b). (Figure 5d). This indicates that XB130 represents an Tyrosine 1062 is a crucial RET binding site for the alternative route available to RET/PTC to stimulate PI activation of various signaling pathways (Hayashi et al., 3-kinase/Aktin a Y1062-indpendentmanner. Moreover, 2000; Arighi et al., 2005). However, RET/PTC3 4YF as XB130 related signaling affects Akt phosphorylation mutation, which includes Y1062F (Figure 2a) was as but not ERK, the XB130-dependent pathway is selective effective as the intact RET/PTC3 at the phosphorylation in terms of downstream consequences.

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 945 To corroborate this effect on natural XB130 expres- XB130 siRNA treated group (Figure 7b), indicating that sors, TPC1 cells were treated for 48 h with two XB130 downregulation of XB130 accelerates the apoptotic siRNAs (A and B) designed againstindependent process. segments of XB130. These tumor cells showed sub- stantial Akt phosphorylation (on Ser 473) under basal conditions. Treatment with either XB130 siRNA Discussion strongly reduced this basal Akt phosphorylation, as compared to nonspecific siRNA, whereas the total Akt Here we show that XB130 is preferentially expressed in levels remained unaffected (Figure 6a). The second the thyroid tissue, moreover, its expression has been siRNA (B) proved to be a more potent inhibitor of Akt detected in papillary thyroid carcinoma, and in TPC1 phosphorylation, in agreement with the observation that cells that carry the RET/PTC1 rearrangement. We also it caused a stronger (near complete) reduction in XB130 show that XB130 is a novel substrate of the thyroid- expression. In addition, this inhibition was specific to specific RET/PTC tyrosine kinase. Accordingly, coex- the Akt pathway as ERK phosphorylation was not pression of XB130 and active (but not kinase dead) affected. Together these observations indicate that RET/PTC in HEK293 cells leads to significant XB130 XB130 can function as a specific mediator in the RET/ phosphorylation. Further, inhibitor blockade of RET PTC/PI 3-kinase/Aktpathway. kinase abolishes XB130 phosphorylation in TPC1 cells, in which both RET/PTC1 and XB130 are endogenously expressed. Downregulation of XB130 in thyroid papillary carcinoma Although the kinase activity of RET/PTC is clearly cells inhibits cell-cycle progression and survival indispensable for the RET/PTC-mediated XB130 phos- PI 3-kinase/Akt pathway plays a central role in RET/ phorylation, the exact mechanism of this process PTC signaling and is involved in cell proliferation and remains elusive. RET/PTC tyrosines (Y826, Y1015, survival. To determine the role of XB130 in cell Y1029 and Y1062), which are known to dock several proliferation, TPC1 and HEK293 cells were transfected intracellular mediators, are not essential for XB130 with nonspecific siRNA or XB130 siRNA mix (A þ B). phosphorylation. XB130 forms a detectable complex Cells were harvested 48 h after transfection, stained with with RET/PTC. The interaction between RET/PTC and propidium iodide for DNA content and processed its established binding partners has been proposed to be through flow cytometry. Figure 6b shows that XB130 mediated by either the SH2 or PTB domains. As XB130 siRNA increased the proportion of TPC1 cells accumu- does not contain such domains, the interaction between lated in the G0/G1 phase of the cell cycle. However in XB130 and RET/PTC is likely mediated by an HEK293 cells, which are devoid of detectable endogen- alternative mechanism and/or possibly by intermediate ous XB130 expression, such effectwas notobserved. To binding partners. substantiate this growth-arresting effect, and to directly We found that Y54 in XB130 is a major target assess the impact of XB130 on the proliferative potential that accounts for approximately 50% of RET/PTC- of these tumor cells, we performed 5-bromodeoxyur- dependentphosphorylation.The sequence surrounding idine (BrdU) incorporation experiments. Transfection Y54 corresponds to a binding site for the SH2 domains with the XB130 siRNAs significantly decreased BrdU of the p85 regulatory subunit of PI 3-kinase (Backer uptake in TPC1 cells (Figure 6c). Treatment of TPC1 et al., 1992; Carpenter et al., 1993; Rordorf-Nikolic cells with siRNA did not induce obvious morphological et al., 1995). Accordingly, both the N- and the C- changes (Supplementary Figure 6). As TPC1 cells carry terminal SH2 domains of p85 bind XB130 in pull-down RET/PTC, these data suggest that XB130 is an assays, and this binding is greatly enhanced by RET/ important mediator of RET/PTC-enhanced cell prolif- PTC. Moreover, pharmacological inhibition of RET/ eration. PTC disrupts the binding between XB130 and p85 in Growing cells on polyHEMA-coated surface renders RET/PTC1-positive TPC1 cells. Cotransfection of anchorage-dependent cells unable to attach to the plates, XB130 with RET/PTC increases the basal Akt phos- and consequently causing them to enter an apoptotic phorylation on Ser473 in HEK293 cells, whereas siRNA pathway, Anoikis. To determine the role of XB130 in downregulation of XB130 in TPC1 cells reduces their cell survival, TPC1 cells transfected with nonspecific or constitutively increased Akt activity. Together these XB130 siRNA were harvested, counted and grown on observations indicate that XB130 can function as a polyHEMA for 24 h. Cells were collected, double specific mediator in the RET/PTC/PI 3-kinase/Akt stained with Annexin-V-flourescein and propidium pathway. iodide, then processed by flow cytometry. The cell Several mechanisms have been elucidated that serve as viability is defined as: viable cells (Annexin-V and PI- a bridge between the activated native RET receptor or negative), early (Annexin-V-positive, PI-ne- the constitutively activated RET/PTC and the P13- gative), late apoptosis (double positive for Annexin-V kinase pathway. As the p85 regulatory subunit of PI 3- and PI; Figure 7a). Although the overall apoptosis kinase does not bind to RET directly, adaptor protein (early plus late) did not yield significant difference serve as links that relay the downstream signaling between XB130 and nonspecific siRNA-treated group, cascade. RET residues Y1062 (common to both RET- there was a significantly lower percentage of cells in the 9 and RET-51 splicing variants) and Y1096 (specific for early phase and higher in the late phase of apoptosis in the RET-51 form) provide binding sites for these

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 946

Figure 6 Downregulation of XB130 contributes to the reduced growth of TPC1 cells. (a) TPC1 cells were transfected with two specific XB130 siRNAs (A, B; 75 nM each). Nonspecific siRNA was used as a control. Cells were serum starved overnight and treated with medium containing 10% fetal bovine serum (FBS) for 15 min. Cell lysates were examined for XB130, phosphorylated Akt (Ser473), total Akt, phosphorylated ERK and total ERK. (b) Cell-cycle progression of TPC1 and HEK293 (control) cells. TPC1 cells were transfected with 75 nM of XB130 siRNA mix (a and b), or nonspecific siRNA as control. Cells were serum starved overnight and then treated with medium containing 10% FBS for 2 h. Cells were then harvested and DNA was stained with propidium iodide and analysed for the cell cycle by flow cytometry. (c) Cells were treated with siRNA as described above. TPC1 cells were transfected with 75 nM of the mix of XB130 siRNA, A and B, or nonspecific siRNA as control. Following overnight serum starvation cells were incubated with medium containing 10% FBS and 10 mM of 5-bromodeoxyuridine (BrdU) for 2 h. Cells were then stained with an anti-BrdU antibody. Hoechst was used as a counterstain for nuclei. The percentage of BrdU incorporation was quantified from 10 randomly photographed fields in a blinded fashion.

adaptors. In particular, phosphorylated Y1062 binds 2005). On the other hand, Grb2 binds to Y1096 directly several docking proteins (such as, Shc) that can recruit and in turn recruits Gab/p85 into its complex (Besset p85 directly or through the help of additional cofactors et al., 2000). XB130 coupling to RET/PTC was (that is, Grb2, Gab1; Hayashi et al., 2000; Arighi et al., independent of either Y1062 or Y1096 (as this residue

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 947

Figure 7 XB130 downregulation accelerates aniokis-induced apoptosis in TPC1 cells. (a) TPC1 cells treated with nonspecific or XB130 siRNA were plated on poly-HEMA coated 60 mm dishes and incubated in the adhesion compromised environment for 24 h, followed by Annexin-V-flourescein/PI double staining. Apoptosis was evaluated by flow cytometry, and representative 2D plot images are shown. (b) Statistical analysis of experiment described in (a). Results are summarized from three independent experiments. was lacking from the RET/PTC constructs used in this in these critical functions. Downregulation of XB130 study). Accordingly, cotransfection of XB130 with the leads to the accumulation of TPC1 cells in the G0 phase RET/PTC 4YF mutant was able to selectively rescue the of the cell cycle, and reduces proliferation as revealed by activation of the Akt pathway, without affecting the the lower BrdU incorporation. The proliferative poten- ERK pathway. Thus, our findings imply that XB130 tial of TPC1 cells has been attributed to the RET/PTC may represent a new mechanism alternative to Y1062 rearrangement. In fact, chemical inhibition of RET/ and Y1096 that couples RET/PTC to PI 3-kinase and PTC1 (Carlomagno et al., 2002; Mariggio et al., 2007) or selectively operates in thyrocytes. Such redundancy in RET/PTC1 silencing by RNAi (de Martimprey et al., the mechanism of PI 3-kinase activation might reflect 2008) blunted TPC1 cell proliferation. Moreover, our fine-tuning and versatility in the regulation, because the data show that anoikis challenged TPC1 cells are more assembly of particular signaling complexes may deter- predisposed to cell death on XB130 downregulation. mine the activation of one or several signaling cascades, Thus, XB130 could be an important intermediate in the and may influence the intensity and duration of the propagation of PI 3-kinase signaling, which contributes response, as well as the specificity for the activation of to abnormal cell proliferation and survival in thyroid one pathway over the other. cancer cells sustained by RET/PTC rearrangement. The PI 3-kinase/Akt pathway plays a central role in Although XB130 is highly expressed in thyroid and mediating cell growth and survival (Hemmings, 1997; thyroid tumors, it has been found in other cell types Kaplan and Miller, 1997; Marte and Downward, 1997). (data not shown), hence the interaction between XB130 Our data provide direct evidence that XB130 is involved and p85 subunit of PI 3-kinase may also contribute to

Oncogene XB130, a novel adaptor in RET/PTC signaling M Lodyga et al 948 in cells containing endogenous washing the beads, proteins were separated by SDS– XB130 expression. polyacrylamide gel electrophoresis and probed with indicated antibodies. Two peptides were synthesized based on XB130 amino- acid sequence surrounding Tyrosine 54 (YXXM motif): Materials and methods KSSSSDEEYIYMNKVTI (p54) and KSSSSDEEpYIYM NKVTI (pp54), by Neosystem Laboratoire (Strasbourg, Portion of the Materials and methods is presented as France). GST-pull-down experimentwas performed as Supplementary materials, including northern blot analysis, described above. immunohistochemical staining, cell lines, protein studies, siRNA transfection, flow cytometry and BrdU incorporation. Anoikis assay Plasmids Apoptosis in TPC1 cells treated with control or XB130 siRNA RET constructs used in this study were cloned into pCDNA3.1 was induced by inhibition of contact with the extracellular (Invitrogen, San Diego, CA, USA) containing either a myc tag matrix. Briefly, 60 mm dishes were coated with 1 ml of 10 mg/ or pBABE (no tag) vectors. They encode the short RET spliced ml of poly-HEMA (poly(2-hydroxyethylmethacrylate)) in 95% form (RET-9) as described elsewhere (Castellone et al., 2003). ethanol and allowed to dry. This process was then repeated. RET/PTC1 and RET/PTC3 constructs encode the H4-RET The dishes were washed with phophate-suffered saline and and RFG (Ele1/ARA70/Ncoa4)-RET chimeric oncogenes, stored dry until further use. TPC1 cells grown for 24 h in the respectively. RET/PTC3(KÀ) is a kinase-dead mutant, carry- presence of siRNA were detached using trypsin/EDTA ing the substitution of the catalytic lysine (residue 758 in full- solution and plated onto the polyHEMA-coated wells at 6 length RET) with a methionine. RET/PTC3(4YF), in which 1 Â 10 cells per dish. After 24 h cells were harvested from tyrosines corresponding to Y826, Y1015, Y1029 and Y1062 in polyHEMA-coated wells, centrifuged and resuspended. Apop- full-length RET were mutated to phenylalanine residues, was totic cells were evaluated with Cytomix FC500 flow cytometry generated by site-directed mutagenesis using a QuikChange system, using the Annexin-V-fluorescein and propidium iodine mutagenesis kit (Stratagene, La Jolla, CA, USA; Melillo et al., (PI) double staining method according to manufacturer’s 2001). Expression vectors for XB130 and its mutants instructions (Roche Molecular Diagnostics, Basel, Switzer- (XB130DN, 2–168 amino-acid deletion), XB130(Y124F) and land). Cells were gated according to the staining, viable cells XB130(Y161F) were cloned into a pCDNA3 backbone (negative for both Annexin-V and PI), Early Apoptosis containing a His tag as described previously (Xu et al., (Annexin-V-positive only), and Late Apoptosis (double 2007). In addition, full-length XB130 was subcloned into positive for Annexin-V and PI). pCDNA3.1 vector containing a myc tag. XB130(Y54F) point mutation was generated by site-directed mutagenesis using the Acknowledgements QuikChange mutagenesis kit (Stratagene). The mutations were confirmed by DNA sequencing. We are grateful to Dr B Han and Dr D Winer for technical assistance. We thank AJ Ryan, AstraZeneca for the ZD6474 Binding assays inhibitor. We also thank Dr S Asa and JM Hershman for GST-N-SH2 p85 and GST-C-SH2 p85 domains (Pelicci et al., human thyroid cell lines, Dr G Pelicci for GST-fusion proteins 2002) were a kind giftof G Pelicci (Milan, Italy).Recombinant and Dr M Chiariello for the Src mutant. This work was proteins were produced in Escherichia coli and purified on supported by operating grants (MOP-13270, MOP-42546) glutathione-conjugated sepharose by standard procedure (GE from Canadian Institutes of Health Research, a grant from the Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). For Italian Association for Cancer Research, MIUR, Alleanza pull-down assays, 20 mg of the recombinant protein bound to contro iL Cancro and the European Union Contract FP6- sepharose were incubated with cell lysates for 2 h at 4 1C. After 36495 (GENRISK-T).

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