Tr-Kit Promotes the Formation of a Multimolecular Complex Composed by Fyn, Plcc1 and Sam68

Oncogene (2003) 22, 8707–8715 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc tr-kit promotes the formation of a multimolecular complex composed by Fyn, PLCc1 and Sam68

Maria Paola Paronetto1, Julian P Venables2, David J Elliott2, Raffaele Geremia1, Pellegrino Rossi1 and Claudio Sette*,1

1Department of Public Health and Cell Biology, Section of Anatomy, University of Rome ‘Tor Vergata’, Rome, Italy; 2Institute for Human Genetics, University of Newcastle Upon Tyne, Newcastle, UK

tr-kit is a truncated form of the tyrosine kinase receptor c- (Blume-Jensen et al., 2000). In addition, an alternative kit expressed in the haploid phase of spermatogenesis. mRNA transcribed from a haploid-specific intronic Upon microinjection, tr-kit triggers metaphase-to-ana- promoter, which encodes for the truncated protein tr- phase transition in mouse eggs by the sequential activation kit, is active in postmeiotic haploid cells and the protein of Fyn and PLCc1.Here, we show that tr-kit promotes is expressed in spermatids and mature spermatozoa the interaction of several tyrosine-phosphorylated proteins (Rossi et al., 1992; Albanesi et al., 1996). tr-kit contains with the SH3 domain of PLCc1.Western blot analysis only the phosphotransferase domain and the carbox- indicates that one of these proteins is Sam68, an RNA- yterminal tail of c-kit, and it is catalytically inactive binding protein that is known to interact with and be because it lacks the ATP binding site (Rossi et al., 1992). phosphorylated by Src-like kinases in mitosis.tr-kit A human tr-kit homolog has been described in promotes the association of Sam68 with PLCc1 and Fyn hematopoietic and gastrointestinal cancer cells (Toyota in a multimolecular complex, as demonstrated by co- et al., 1994; Takaoka et al., 1997), but the role played by immunoprecipitation of the phosphorylated forms of these this protein in cell transformation has not yet been proteins using antibodies directed to anyone of the addressed. On the other hand, microinjection of partners of the complex.Expression of tr-kit potentiates recombinant murine tr-kit into metaphase-arrested the interaction of endogenous Sam68 also with the SH3 oocytes triggers cell cycle resumption through the domain of Fyn.Furthermore, the subcellular localization sequential activation of the Src-like kinase Fyn and of Sam68 is affected by tr-kit through activation of Fyn in PLCg1 (Sette et al., 1998, 2002). tr-kit function in the live cells.Lastly, we show that interaction with the SH3 oocyte requires Ca2 þ mobilization from intracellular domain of Fyn triggers the release of Sam68 from bound stores and it is blocked by PLC or Src-kinase inhibitors RNA.Thus, our data suggest that tr-kit promotes the (Sette et al., 1997, 2002). We have also shown that tr-kit formation of a multimolecular complex composed of Fyn, binds to the SH2 domain of Fyn and causes the relief of PLCc1 and Sam68, which allows phosphorylation of the intramolecular inhibition that maintains Src-like PLCc1 by Fyn, and may modulate RNA metabolism. kinases in a low-activity state, thereby promoting Oncogene (2003) 22, 8707–8715. doi:10.1038/sj.onc.1207016 tyrosine phosphorylation and activation of PLCg1 (Sette et al., 2002). Interestingly, coinjection of the Keywords: tyrosine kinase; SH3; RNA; adaptor SH3 domain of PLCg1 (Sette et al., 1998), and to a lesser proteins; signal transduction extent of the SH3 domain of Fyn (Sette et al., 2002), inhibited tr-kit-induced cell cycle resumption, suggesting that protein–protein interactions through SH3 domains are crucial in the pathway that leads to the stimulation of PLCg1 activity and oocyte activation. However, the Introduction relevant partners that interact with the SH3 domains of PLCg1 and Fyn were not identified. The c-kit gene encodes for a tyrosine kinase receptor SH3-mediated interactions play a role in the regula- that plays a role in the establishment, maintenance and tion of the subcellular localization of proteins, activa- function of three cell lineages: hematopoietic cells, tion of enzymes, and recruitment of signaling pathway melanocytes and germ cells (Besmer et al., 1993). In components by adaptor proteins (Pawson, 1994). A the postnatal testis, the c-kit receptor is expressed in scaffold molecule for SH3-containing signaling proteins mouse spermatogonia (Sorrentino et al., 1991), where it is the RNA-binding protein Sam68 (Src associated in is required for both cell proliferation and survival mitosis), a member of the STAR family of proteins (signal transduction and activation of RNA metabolism) (Vernet and Artzt, 1997). Sam68 contains polypro- *Correspondence: C Sette, Dipartimento di Sanita` Pubblica e Biologia Cellulare, Universita` di Roma ‘Tor Vergata’, Via Montpellier 1, 00133, line sequences that mediate the interaction with the SH3 Rome, Italy; E-mail: [email protected] domains of Fyn, PLCg1, Grb2 and PI3K (Richard et al., Received 14 May 2003; revised 14 July 2003; accepted 15 July 2003 1995). Sam68 also contains numerous tyrosines in its tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8708 carboxyl-terminal domain and it is the main substrate deletion causes germ cell tumors in C. elegans (Lee and for Src-like kinases in mitosis (Taylor and Shalloway, Schedl, 2001). 1994); it has been demonstrated that the interaction with We have previously shown that tr-kit stimulates Fyn- the SH3 domain of Fyn is necessary for Fyn-dependent mediated phosphorylation of PLCg1 and of a p70 tyrosine phosphorylation of Sam68 (Shen et al., 1999). protein that co-immunoprecipitates with PLCg1 (Sette Once phosphorylated in the carboxyterminal region, et al., 2002). Here, we demonstrate that the p70 protein Sam68 recruits the SH2 domains of PLCg1, Grb2 and is Sam68, and that tr-kit stimulates its interaction with Src-kinases themselves (Richard et al., 1995). Since these Fyn and PLCg1 via their SH3 domains. Our data signaling proteins already interact with Sam68 through suggest that tr-kit promotes the formation of a multi- their SH3 domains, SH2-binding may reinforce this molecular complex composed by Sam68, Fyn and interaction and/or modulate a conformational modifica- PLCg1, which facilitates the phosphorylation and tion of the bound proteins. On the other hand, activation of PLCg1 and might influence some aspects interaction between the SH3 of Fyn and Sam68 of RNA metabolism. stimulates the activity of Fyn in mitosis (Oneyama et al., 2002). Much less is known on the functional relevance of the interaction between PLCg1 and Materials and methods Sam68. The polyproline sequence of Sam68 that binds to the SH3 domain of PLCg1 does not appear Cell culture and transfections to overlap with that required for binding of Fyn Hek293 cells were maintained in Dulbecco’s minimal essential (Richard et al., 1995; Shen et al., 1999), therefore the medium supplemented with 10% fetal bovine serum (FBS) possibility exists that PLCg1 and Fyn bind to a single (Gibco BRL). CaPO4 transfections were performed with 10 mg molecule of Sam68. of the appropriate plasmid DNA. Plasmids pCR–PLCg1, In addition to its role as scaffold in Src-kinases signal pCMV5–tr-kit, pCMV5–tr-kitY161F, pCMV5–Fyn and transduction, Sam68 plays a function in RNA metabo- pCMV5–FynY528F for eukaryotic expression have been de- lism. Sam68 is a member of the STAR family of RNA- scribed previously (Sette et al., 1997, 1998, 2002); pCDNA4– binding proteins, which are characterized by an hnRNP Sam68–GFP was constructed by subcloning human Sam68 K homology (KH) RNA-binding domain embedded in upstream of GFP in the EcoRI and SalI restriction sites of conserved flanking regions (Vernet and Artzt, 1997). pCDNA4. pCMV5–tr-kit–GFP was constructed by subcloning GFP in frame downstream of tr-kit in pCMV5–tr-kit using the The STAR family includes several proteins such as XbaI and BamHI sites. At 24–48 h after transfection, cells were mammalian SLM1, SLM2 and Quaking I, the Caenor- harvested in homogenization buffer (50m M HEPES, pH 7.5, habditis elegans homolog GLD1 and Drosophila How 10m M b-glycerophosphate, 2 mM EGTA, 15 mM MgCl2, (Vernet and Artzt, 1997). Like Sam68, all STAR 0.1 mM sodium orthovanadate, 1 mM DTT, 10 mg/ml leupeptin proteins are involved in the control of RNA metabolism and 10 mg/ml pepstatin, 10 mg/ml aprotinin, 1 mM PMSF), during cell proliferation and/or differentiation (Ven- homogenized in a glass homogenizer, or lysed by adding 1% ables et al., 1999; Matter et al., 2002; Zhang et al., 2003). Triton X-100 and 120 mM NaCl to the homogenization buffer Sam68 is preferentially concentrated in the nucleus (lysis buffer). Lysates were centrifugated 10 min at 10 000 g at where it binds to heterogeneous RNA and is implicated 41C and soluble extracts were used for further analysis. in RNA export and processing (Li et al., 2002). Membrane-bound Fyn is required for phosphorylation Expression and purification of GST fusion proteins and nuclear localization of Sam68 in T cells (Lang et al., Plasmids (pGEX-) containing GST–PLCgSH3 and GST– 1999), suggesting that the protein continuously shuttles FynSH3 (Sette et al., 2002) were transformed into the BL21 in and out of the nucleus, possibly mediating RNA Escherichia coli strain, grown at 301C in an LB medium to an export. Furthermore, post-translational modification of OD600 ¼ 0.6, and induced with 0.5 mM isopropyl-b-thiogalac- Sam68 affects its ability to bind RNA: phosphorylation topyranoside (IPTG, Sigma-Aldrich) for 3 h to produce GST of Sam68 by Src-kinases (Wang et al., 1995) or direct fusion proteins. Recombinant proteins were purified from association of the SH3 domain of Fyn (Taylor et al., bacterial lysates on glutathione–agarose (Sigma-Aldrich) as 1995), interferes with the ability of the protein to bind described (Sette et al., 1998). Purified proteins were analysed by SDS–PAGE and stained with Coomassie blue to test purity RNA. Arginine methylation has recently been shown to and integrity. affect localization of Sam68 and RNA export (Cote et al., 2003). In addition, threonine phosphorylation of Sam68 by the MAPKs Erk1/2 modulates processing of Glutathione–agarose pull-down assays target mRNAs (Matter et al., 2002). Thus, Sam68 Cell extracts (500 mg of total proteins) were added to 2–4 mgof localization and function are regulated at the post- GST fusion proteins adsorbed on glutathione–agarose (Sigma) translational level in multiple ways and this protein may in 250 ml (final volume) of homogenization buffer supplemen- 0 represent a direct link between signaling events and ted with 0.05% BSA for 90 incubation at 41C under shaking. control of expression of target genes. In line with such Beads were washed three times with homogenization buffer, eluted in 15 mM reduced glutathione in 50m M Tris-HCl, pH crucial function, it has been reported that cells lacking 8.0, diluted in SDS–sample buffer (62.5 mM Tris-HCl, pH 6.8, Sam68 undergo neoplastic transformation and can 10% glycerol, 2% (w/v) SDS, 0.7 M 2-mercaptoethanol, and develop tumors if injected into nude mice (Liu et al., 0.0025% (w/v) bromophenol blue) and proteins were resolved 2000). Interestingly, a tumor suppressor role has also on a 10% SDS–PAGE gel for subsequent Western blot been proposed for the Sam68 homolog GLD1, whose analysis.

Oncogene tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8709 Immunoprecipitation assay 2002). Hoechst staining and direct fluorescence of tr-kit–GFP were performed as described above for GFP-Sam68. Images Cell extracts (500 mg of total proteins) prepared in lysis buffer were collected with an RT-slider Spot camera (Diagnostic containing a cocktail of protease inhibitors (see above) were Instruments, Inc.) and digitally recorded using imaging soft- incubated with 1 mg of the specific antibody for 2 h at 41C ware (IAS 2000) and Photoshop (Adobe Systems, Inc., under constant shaking. Protein A-Sepharose or protein G- Mountain View, CA, USA). Sepharose (Sigma-Aldrich) were preadsorbed with 0.05% BSA before the incubation with the immunocomplexes and added to the extracts together with the antibody. In some experiments, when background nonspecific precipitation was ob- Results served, cell extracts were precleared for 1 h on protein A- Sepharose beads before using them for immunoprecipitation. tr-kit stimulates phosphorylation of PLCg1-bound Sam68 The Sepharose beads were washed three times with homogenization buffer or lysis buffer. Proteins adsorbed to the We have recently demonstrated that tr-kit activates the antibody–beads complex were eluted in SDS–sample buffer for soluble tyrosine kinase Fyn and causes a strong Western blot analysis. stimulation in tyrosine phosphorylation of PLCg1and of a protein of approximately 70kDa that co-immuno- Poly-U-Sepharose pull-down assay precipitates with PLCg1 (Sette et al., 2002). Since Sam68 For the competition of the GST–FynSH3 with RNA binding is a substrate for Src-like kinases (Taylor and Shallo- of Sam68, Hek293 cell extracts were precleared with Sepharose way, 1994) and it is known to interact with PLCg1 beads (Sigma) and then incubated for 600 at 41C under (Richard et al., 1995), we asked whether phosphoryla- constant shaking with poly-U-Sepharose (Pharmacia) pread- tion of Sam68 is modulated by Fyn and tr-kit. Hek293 sorbed with 0.05% BSA. The beads were washed three times cells were transfected with PLCg1 alone or together with with lysis buffer and then divided in four tubes for additional Fyn and tr-kit (Figure 1a), PLCg1 was immunoprecipi- three washes with: homogenization buffer (HB) alone; HB tated from soluble cell extracts (Figure 1a), and supplemented with 1 mg/ml of GST; or of GST–FynSH3, or of immunoprecipitates were probed with an a-phosphotyr- GST–PLCg1SH3. Eluted proteins were collected for each wash osine antibody or an a-Sam68 antibody (Figure 1b). As and processed for Western blot analysis. After another wash et al with HB, proteins still bound to the poly-U-Sepharose beads previously observed (Sette ., 2002), coexpression of were eluted in sample buffer, resolved on a 10% SDS–PAGE tr-kit and Fyn with PLCg1 induced tyrosine phosphor- gel, and analysed in Western blot analysis using the a-Sam68 antibody.

Western blot analysis Proteins were separated on 10% SDS–PAGE gels and transferred to polyvinylidene ﬂuoride Immobilon-P membranes (Millipore) using a semidry blotting apparatus (Bio- Rad). Western analysis was carried out as previously reported (Sette et al., 2002). First antibody (1 : 1000 dilution) overnight at 41C: rabbit a-Fyn (SC-16); rabbit a-PLCg1 (SC-426); rabbit a-Sam68 (C-20); mouse a-phosphotyrosine (PY20) (all from Santa Cruz Biotechnology); rabbit a-c-kit (Sette et al., 1997). Secondary anti-mouse or anti-rabbit IgGs conjugated to horseradish peroxidase (Amersham) were incubated with the membranes for 1 h at room temperature at a 1 : 10 000 dilution in PBS containing 0.1% Tween 20. Immunostained bands were detected by chemiluminescent method (Santa Cruz Biotech- nology).

Immunofluorescence analysis Hek293 cells were transfected with Sam68–GFP alone or together with tr-kit, Fyn or FynY528F. At 15 h after transfec- Figure 1 tr-kit and Fyn promote association and phosphorylation tions, Hoechst dye (0.5 mg/ml, Sigma) was added to the culture of Sam68 and PLCg1. (a) Western blot analysis of Hek293 cells 0 for 15 , cells were rinsed briefly with fresh medium and transfected with PLCg1 alone (first lane) or together with Fyn and analysed immediately for the GFP fluorescence with an tr-kit (second lane). Cell extracts were immunoprecipitated with the Olympus invertoscope by using a Â 40objective. For analysis a-PLCg1 antibody and samples were analysed in Western blot of Fyn and tr-kit localization, Hek293 cells were transfected using either the same antibody (a, lower panel) or the a- phosphotyrosine antibody (b, left panel) or the a-Sam68 antibody with Fyn, FynY528F and tr-kit–GFP and fixed 15 h after transfection using 4% paraformaldehyde (10min at 4 1C), (b, right panel). (c) The same experiment was repeated after those permeabilized with 0.1% Triton X-100 in PBS (10 min at 41C) samples were either mock-depleted with preimmune IgGs (0.5 mg) or Sam68-depleted with a-Sam68 IgGs (0.5 mg). The upper panel and blocked for 1 h with 5% BSA and 1% goat serum. shows the immunoprecipitation of PLCg1, the middle panel shows Immunofluorescence analysis was performed with a-Fyn tyrosine-phosphorylated proteins immunoprecipitated by the a- (1 : 400 dilution; 2 h at room temperature) and secondary PLCg1 antibody, and the lower panel shows co-precipitating rhodamine-conjugated anti-rabbit IgGs antibody (1 : 400; 1 h Sam68. These experiments were performed three times with similar at room temperature) as previously described (Sette et al., results

Oncogene tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8710 ylation of two major proteins: the p120kDa band, electrophoretic mobility, which is indicative of activa- which corresponds to immunoprecipitated PLCg1, and a tion of the kinase (Sette et al., 2002). 70kDa phosphotyrosine band that co-immunoprecipitates with PLCg1 (Figure 1b). When samples were tr-kit stimulates Fyn-dependent phosphorylation of probed with the a-Sam68 antibody, it was observed that Sam68 this phosphoprotein comigrates with Sam68 and that Sam68–PLCg1 association was induced by coexpression To determine which are the predominant tyrosine- of Fyn and tr-kit with PLCg1 (Figure 1b). Furthermore, phosphorylated proteins induced by the coexpression if cell extracts were immunodepleted of Sam68 before of Fyn and tr-kit, cell extracts were immunoprecipitated immunoprecipitation with a-PLCg1, both the p70 using the a-phosphotyrosine antibody and analysed in phosphoprotein and Sam68 were no more detected Western blot using either the same or the a-Sam68 (Figure 1c). Mock depletion with preimmune IgGs had antibody. In the absence of Fyn, no detectable tyrosine- no effect. phosphorylated protein was immunoprecipitated from cell extracts (Figure 3a; asterisks indicate the bands of the immunoglobulins used for immunoprecipitation). tr-kit stimulates association of PLCg1, Fyn and Sam68 in Fyn induced tyrosine phosphorylation of ﬁve proteins of a complex approximately 120, 100, 70, 40 and 30 kDa, of which the Thus, tr-kit promotes the interaction of PLCg1 with 70kDa one was by far the most intense (Figure 3a,c). both Fyn (Sette et al., 2002) and Sam68 (Figure 1). To Interestingly, tr-kit, but not the tr-kitY161F mutant, determine if Sam68, PLCg1 and Fyn formed a complex which is unable to interact with and activate Fyn (Sette upon tr-kit expression, we performed co-immunoprecipitation studies using the other two antibodies. When cell extracts were immunoprecipitated with a-Fyn, both Sam68 and PLCg1 were co-precipitated only from cell extracts expressing tr-kit (Figure 2a). Antiphosphotyr- osine staining of these immunoprecipitates showed that tr-kit expression stimulated phosphorylation of four major polypeptides: a 120kDa band corresponding to PLCg1, a 70kDa band corresponding to Sam68, a p55 band corresponding to Fyn and an unknown p40 phosphoprotein. Similarly, when cell extracts were immunoprecipitated with the a-Sam68 antibody, we observed that both Fyn and PLCg1 were co-immunoprecipitated and that tr-kit stimulated both the association of the two enzymes with Sam68 and tyrosine phosphorylation of Sam68 (Figure 2b). Furthermore, we observed that Fyn co-immunoprecipitated with Sam68 from tr-kit-expressing cells displayed a slower

Figure 3 tr-kit and Fyn promote the tyrosine phosphorylation of five major proteins in Hek293 cells. Hek293 cells were transfected with PLCg1, Fyn, tr-kit and tr-kitY161F in various combinations as listed in the figure. Cell extracts were immunoprecipitated with the a-phosphotyrosine antibody and samples were analysed in Western blot using the same antibody (a) or the a-Sam68 antibody (b). The arrows in (a) point to the 120, 100, 70, 40 and 30 kDa proteins that are heavily phosphorylated when cells express Fyn and wild-type tr-kit; asterisks mark the migration of the heavy and light chains of immunoglobulin, which are detected by the secondary antibody. (b) Figure 2 tr-kit stimulates the association of PLCg1 and Fyn with The arrow points to the migration of Sam68 and shows that it Sam68. (a) Hek293 cells were transfected with no DNA, with corresponds to p70in (a). In the lower panels ( c and d), Hek293 PLCg1 and Fyn7tr-kit as indicated in the figure, cell extracts were cells were transfected with Fyn and tr-kit in various combinations. immunoprecipitated with the a-Fyn antibody and samples were Cell extracts were precleared for 1 h on protein A-Sepharose beads analysed in Western blot with the antibody described on the left and then immunoprecipitated as described above. Western blot side of each panel. (b) Samples as in (a) were immunoprecipitated analysis of the immunoprecipitated proteins was carried out using with the a-Sam68 antibody and analysed in Western blot with the either the a-phosphotyrosine antibody (c) or the a-Sam68 antibody a-Sam68 antibody, a-phosphotyrosine antibody, a-PLCg1 anti- (d). The blot in panel c was exposed for shorter time than in panel a body, or a-Fyn antibody as described on the left side of each panel. to avoid saturation of the p70band. This experiment was These experiments were performed three times with similar results performed three times with similar results

Oncogene tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8711 et al., 2002), further increased tyrosine phosphorylation and tr-kit. The size of four of these proteins corre- of all these bands. We have previously shown that the sponded to the 100, 70, 40 and 30 kDa phosphoprotein 120kDa phosphoprotein band corresponds to PLC g1 bands observed in Figure 3a, whereas the p55 band (Sette et al., 2002). Now we found that the a-Sam68 could have been masked by the comigrating band of the antibody recognized the p70band immunoprecipitated heavy chains of the immunoglobulin. As expected, the by the a-phosphotyrosine, indicating that the major 120kDa phosphoprotein band corresponding to PLC g1 substrate of tr-kit-activated Fyn is Sam68. We found was not bound to GST–PLCg1SH3. A similar pattern of that, although Sam68 was already precipitated from phosphoproteins interacted with GST–PLCg1SH3 also extracts of non-transfected cells or overexpressing only when Fyn was expressed alone or together with tr- PLCg1, coexpression of tr-kit and Fyn increased kitY161F, but the intensity of such bands was much the amount of phospho-Sam68 immunoprecipitated. fainter (Figure 4c). When the samples bound to GST– The background immunoprecipitation of nonpho- PLCg1SH3 were probed with the a-Sam68 antibody, we sphorylated Sam68 observed in Figure 3b was almost observed that tr-kit, but not tr-kitY161F, promoted the completely eliminated by preclearing the cell extracts on interaction of Sam68 with the SH3 of PLCg1 (Figure 4d), protein A-Sepharose beads before the immunoprecipita- further indicating that p70is indeed Sam68 and that tion with the a-phosphotyrosine antibody (Figure 3c,d). activation of Fyn by tr-kit is required for efficient interaction with the SH3 domain of PLCg1. tr-kit stimulates the association of Sam68 with the SH3 domain of PLCg1 tr-kit stimulates the interaction of Sam68 with the SH3 domain of Fyn tr-kit triggers metaphase exit in ovulated mouse oocytes by the sequential activation of Fyn and PLCg1 (Sette To understand if tr-kit influences the ability of Sam68 to et al., 2002). Remarkably, microinjection of an excess of bind directly to the SH3 domain of Fyn, GST–FynSH3 the SH3 domain of PLCg1 inhibited cell cycle resump- fusion protein or GST alone (Figure 5b) were incubated tion, indicating that this domain plays a crucial role in with cell extracts derived from cells expressing tr-kit or PLCg1 function in mouse oocytes (Sette et al., 1998). tr-kitY161F (Figure 5a). Although binding of Sam68 to Since Sam68 is known to interact with the SH3 domain the SH3 domain of Fyn was already detected in extracts of PLCg1, we investigated the effect of tr-kit and Fyn on from nontransfected cells, expression of tr-kit strongly such interaction. Cell extracts from Hek293 expressing increased this interaction (approximately four fold), Fyn and tr-kit in various combinations (Figure 4a) were whereas tr-kitY161F, which is unable to activate Fyn incubated with glutathione–agarose beads preadsorbed and to stimulate phosphorylation of Sam68, did to either GST or GST–PLCg1SH3 (Figure 4b). Bound not exert any effect (Figure 5c,d). This experiment proteins were then analysed by Western blot using a- indicates that tr-kit induces a conformational modifica- phosphotyrosine (Figure 4c) or a-Sam68 antibodies tion of Sam68, possibly exposing the proline-rich (Figure 4d). Five phosphotyrosine bands associated sequences as a consequence of tyrosine phosphorylation, specifically with GST-PLCg1SH3 in the presence of Fyn and promotes the interaction of Sam68 with the SH3 domain of Fyn.

Interaction of Sam68 with the SH3 of Fyn causes release of bound RNA in vitro It has been reported that an excess of the SH3 of Fyn (Taylor et al., 1995) or Fyn-dependent tyrosine phosphorylation of Sam68 (Wang et al., 1995) interferes with its ability to bind to RNA, as monitored by pull-down experiments using poly-U-Sepharose beads. Since tr-kit promotes both phosphorylation of Sam68 by Fyn and interaction with the SH3 domain of the kinase, it likely affects the ability of Sam68 to bind to RNA. To test whether direct interaction with the SH3 domain of Fyn is capable to displace Sam68 when it is already bound to RNA, Sam68 was bound to poly-U-Sepharose and then the adsorbed beads were incubated with 1 mg/ml of Figure 4 tr-kit and Fyn induce the interaction of the SH3 domain of PLCg1 with phosphoproteins. Hek293 cells were transfected purified GST–FynSH3 or GST–PLCg1SH3, or GST in with Fyn either alone or with tr-kit or with tr-kitY161F.(a) Western three sequential elution of 10min each. Indeed, we blot analysis of the corresponding cell extracts. GST or GST– found that the SH3 domain of the kinase was able to FynSH3 were purified onto GSH–agarose beads (b) and used in displace Sam68 from the polyU-beads efficiently, pull-down assays with cell extracts from samples described in (a). whereas GST and GST–PLCg1SH3 were much less Adsorbed proteins were eluted with 15 mM GSH and detected either in Coomassie blue for the GST proteins (b) or using either efficient (Figure 6). Thus, our data suggest that the a-phosphotyrosine antibody (c) or the a-Sam68 antibody (d). interaction with Fyn is incompatible with the binding This experiment was performed three times with similar results of Sam68 to RNA.

Oncogene tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8712

Figure 5 tr-kit promotes the interaction of Sam68 with the SH3 domain of Fyn. Pull-down assay of cell extracts from Hek293 cells transfected with no DNA, tr-kit or tr-kitY161F (a) using either GST or GST–FynSH3 bound to GSH–agarose beads. After washes, proteins adsorbed to beads were eluted using 15 mM GSH and detected either in Coomassie blue for the GST proteins (b) or analysed in Western blot using the a-Sam68 antibody (c). Densitometric analysis of the intensity of the Sam68 band detected in panel c is shown in panel d: data were obtained from three experiments that gave similar results

was followed by fluorescence microscopy. Although Sam68–GFP was initially localized mainly to the nucleus of transfected cells (5–10h after transfection), we observed that after 15 h from the transfection most of the protein was localized to perinuclear membranes and formed a ring around the nucleus (Figure 7a). The cytoplasmic localization of Sam68 upon its accumulation suggested that a limiting factor was required for its default nuclear localization. Indeed, we observed that cotransfection of Fyn leads to an increased number of cells with nuclear staining and a redistribution of the cytoplasmic Sam68–GFP in dots at the level of the perinuclear membranes (Figure 7a). Interestingly, when Fyn was activated by cotransfection with tr-kit, Sam68– GFP was localized in the nucleus of most cells (Figure 7a) and displayed a punctuate pattern that has Figure 6 Interaction with the SH3 of Fyn displaces Sam68 from been previously described for this protein (Chen et al., RNA. Extracts from Hek293 cells (lane 1) were used for pull-down assays with poly-U-Sepharose beads. After washes, Sam68 bound 1999). This augmented localization in the nucleus was to poly-U-Sepharose was eluted using either 1 mg/ml purified GST also confirmed by Western blot analysis (data not (lanes 5–6) or GST–PLCg1SH3 (lanes 7–8), or GST–FynSH3 shown). Activation of Fyn by tr-kit was required for the (lanes 9–10). The proteins retained on beads after elution with nuclear localization of Sam68–GFP, since it was also either GST (lane 2) or GST–PLCg1SH3 (lane 3), or GST–FynSH3 observed in cells that coexpressed the constitutively (lane 4) were eluted in sample buffer. Samples were analysed in Western blot using the a-Sam68 antibody. This experiment was active FynY528 (Figure 7a). performed two times with similar results We next investigated the localization of overexpressed Fyn and tr-kit in Hek293 cells. Immunofluorescence analysis shows that Fyn, besides being localized to the Translocation of Sam68 is modulated by Fyn plasma membrane, is accumulated in perinuclear Sam68 localizes in the nucleus (Vernet and Artzt, 1997), structures resembling the localization of overexpressed whereas Fyn is associated to the intracellular mem- GFP–Sam68 (Figure 7b), indicating that the two branes and the centrosome (Ley et al., 1994). Since we proteins can actually interact as we hypothesized above. observed that activation of Fyn by tr-kit leads to a Interestingly, when cells were cotransfected with dramatic increase in tyrosine phosphorylation of Sam68, Fyn and tr-kit–GFP, both proteins were found to we asked if Sam68 could exit the nucleus to interact with colocalize to a discrete perinuclear dot, suggesting that Fyn. To this end, a Sam68–GFP chimeric protein was tr-kit induces Fyn relocalization. Moreover, a similar expressed either alone or in combination with constructs localization of Fyn was observed when cells encoding Fyn, tr-kit, or the constitutively active were transfected with the constitutively active FynY528F. The localization of Sam68–GFP in live cells FynY528F mutant (Figure 7b), suggesting that the open

Oncogene tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8713 this report, we show that tr-kit promotes phosphorylation of Sam68 by Fyn and the recruitment of the SH3 domains of Fyn and PLCg1 by Sam68. Our results suggest that Sam68 acts as a platform to favor the interaction between Fyn and PLCg1 and to promote phosphorylation and activation of PLCg1, which are triggered by tr-kit (Sette et al., 1998, 2002). We have demonstrated that tr-kit stimulates the association of Sam68 with PLCg1 and Fyn via their SH3 domains. Moreover, since in the presence of tr-kit the three proteins are immunoprecipitated together independently of the antibody used, our results indicate that tr-kit promotes a multimolecular complex. Since this complex is composed of the phosphorylated forms of the three proteins, we suggest that tr-kit promotes phosphorylation of both PLCg1 and Sam68 by activated Fyn through the clustering of the three proteins in a complex (Figure 8). Microinjection of tr-kit into metaphase-arrested oocytes triggers cell cycle resumption via the sequential activation of Fyn and PLCg1 (Sette et al., 2002). Moreover, expression of tr-kit in heterologous transfected cells triggered activation of the same pathway as in mouse oocytes (Sette et al., 1998, 2002), thereby providing a useful tool to investigate the signal transduction pathway triggered by tr-kit at the bio- chemical level. In these previous reports, two observa- tions were left unanswered: (a) tr-kit action in oocytes was suppressed by an excess of the SH3 domain of PLCg1, suggesting the involvement of this domain in the pathway, even though a direct interaction between tr-kit Figure 7 Active Fyn is required for accumulation of Sam68 in the and PLCg1 was not observed; and (b) tr-kit promoted nucleus. (a) Hek293 cells were transfected with Sam68–GFP either tyrosine phosphorylation of PLCg1 and of an unknown alone or in combination with Fyn, Fyn and tr-kit, or FynY528F.At 15 h after transfection, 0.5 mg/ml Hoechst 33342 was added to the protein of approximately 70kDa that co-immunopreci- culture for 10min, then cells were rinsed with fresh medium and pitated with it. The results herein presented provide an analysed by fluorescence microscopy. The left column shows answer to those questions. We have now shown that this localization of Sam68–GFP in live cells, the central column shows DNA staining, and the right column shows the computational p70phosphoprotein is the RNA-binding protein Sam68 merge of the previous two images. (b) Indirect immunofluorescence and that tr-kit promotes its association with the SH3 analysis of Fyn in Hek293 cells. Cells were transfected with Fyn, domain of PLCg1. Thus, it is possible that an interaction FynY528F and tr-kit-GFP as indicated in the figure and fixed 15 h between Sam68 and the SH3 domain of PLCg1is after transfection. Immunofluorescence analysis was performed required to bring it close to Fyn and to allow with a-Fyn as described under Materials and methods. Direct fluorescence of tr-kit–GFP was also acquired to test the colocaliza- phosphorylation and activation of PLCg1. In support tion of the two proteins. Hoechst staining and computational of such a scaffold role for Sam68, we also show that tr- merges were performed as described in (a). Acquisition of images kit enhances the association of Sam68 with the SH3 was performed as described under Materials and methods. This domain of Fyn and that Fyn co-immunoprecipitates experiment was performed four times with similar results with the phosphorylated forms of PLCg1 and Sam68 conformation of Fyn triggered by either interaction with tr-kit or the Y528F mutation (Sette et al., 2002) stimulates translocation of the kinase to this discrete cellular structure.

Discussion Figure 8 Hypothetical model of the multimolecular complex induced by tr-kit. Activation of Fyn by tr-kit exposes the SH2 and tr-kit activates the Src-like kinase Fyn by interacting SH3 domains of the kinase; activated Fyn interacts with Sam68 via with its SH2 domain directly. This interaction relieves the SH3 domain and phosphorylates Sam68 in the perinuclear compartment; interaction of Sam68 with the SH3 domain of the intramolecular inhibition exerted by the binding of PLCg1 facilitates phosphorylation of PLCg1; interaction with Fyn the SH2 domain to the phosphorylated tyrosine 528 in and phosphorylation of Sam68 triggered by tr-kit cause the release the carboxyterminal tail of Fyn (Sette et al., 2002). In of Sam68-bound RNA, which becomes accessible to ribosomes

Oncogene tr-kit promotes protein–protein interactions with Sam68 MP Paronetto et al 8714 only in cells expressing tr-kit. Interestingly, Sam68 the nucleus. Interestingly, we also observed that activa- contains six proline-rich sequences, with which it docks tion of Fyn by either tr-kit or the Y528F mutation, several signaling proteins and its role as a scaffold has which both allow an open conformation of the kinase been proposed in the signal transduction events with the SH2 and SH3 domains exposed (Sette et al., that lead to T lymphocytes activation (Fusaki et al., 2002), causes a relocalization of Fyn to a discrete 1997). Since we have detected Sam68 in mouse oocytes perinuclear dot. Remarkably, we observed that tr-kit– (MP Paronetto & C Sette, unpublished observation), it GFP also accumulates in this perinuclear structure and is likely that Sam68 is also part of the pathway that colocalizes with Fyn in Hek293 cells. This result further leads to tr-kit-induced oocyte activation. In this confirms the functional interaction between tr-kit and scenario, an excess of GST–PLCg1SH3 may interfere Fyn in vivo, and suggests that interaction of the SH2- with tr-kit action in the oocyte (Sette et al., 1998) by SH3 domains of the kinase with cellular targets binding to Sam68 and competing for the association regulates its intracellular distribution. with the endogenous PLCg1, thereby preventing Sam68 binds to RNA in the nucleus of cells, and it phosphorylation and activation of the endogenous takes part in RNA export (Li et al., 2002) and splicing PLCg1 by Fyn. (Matter et al., 2002). Tyrosine phosphorylation of It has been proposed that membrane localization of Sam68 by Src-like kinases interferes with RNA binding, Fyn is required for the tyrosine phosphorylation of as judged by its ability to bind to poly-U-Sepharose Sam68 in T lymphocytes (Lang et al., 1999). In addition, beads (Wang et al., 1995). Furthermore, Taylor et al. since Sam68 recruits cytoplasmic signaling proteins in T (1995) have reported that the binding of Sam68 to RNA cells (Fusaki et al., 1997), it is conceivable that it has to is impaired in the presence of an excess of the SH3 exit the nucleus transiently. Indeed, we have observed domain of Fyn. Herein, we have extended this observa- that upon its accumulation after transfection, most of tion and shown that a GST–FynSH3 can displace Sam68–GFP is associated to perinuclear membranes, Sam68 even when it is already bound to poly-U- most likely the endoplasmic reticulum, suggesting that Sepharose beads. The effect of the SH3 domain of Fyn an endogenous activity limits the translocation of the is specific, since in the same experiment both GST and protein into the nucleus. Our data indicate that Fyn is GST–PLCg1SH3 do not exert the same effect. Thus, the the limiting factor for the localization of overexpressed interaction between Fyn and Sam68 induced by tr-kit Sam68–GFP, because cotransfection of the kinase and might trigger the release of the transported RNA near tr-kit causes an increase in the number of cells with the membrane-bound ribosomes (Figure 8). Since nuclear Sam68–GFP. Moreover, when Fyn is activated activation of an Src-like kinase is a conserved feature by the activating Y528F mutation, Sam68–GFP is also at fertilization across species (Runft et al., 2002) and localized in the nucleus, demonstrating that Fyn activity stored mRNAs become actively translated at fertiliza- is the limiting factor when Sam68 is overexpressed in tion to overcome the block in genome transcription cells. Interestingly, when only Fyn was coexpressed, during the first embryonic cycles (Oh et al., 2000), it is Sam68–GFP could also be localized to discrete cyto- possible that interaction of an Src-kinase with Sam68 is plasmic dots, which resemble the intracellular mem- part of the mechanism that renders these maternal branes where Fyn is localized (Ley et al., 1994). It is mRNAs accessible for translation. possible that under these conditions the extra-nuclear Since a tr-kit protein is aberrantly expressed in localization of Sam68–GFP highlights the sites of its hematopoietic and gastrointestinal cancer cells (Toyota interaction with Fyn. Indeed, we found that also Fyn et al., 1994; Takaoka et al., 1997) and in primary localizes to similar perinuclear structures and to the prostatic tumors (Paronetto et al., in preparation), and plasma membrane in Hek293 cells. Thus, our results hyperactivation of Src-kinases leads to a transformed suggest that Sam68 interacts with Fyn in the cytoplasm phenotype in many cell types (Thomas and Brugge, and that such interaction is required to promote 1997), our studies may be useful to elucidate cell signaling events, like PLCg1 activation, and transloca- transformation pathways in which activation of Src- tion of Sam68 in the nucleus. Sam68 has a nuclear kinases is involved. localization signal in the C-terminus, just downstream of the many tyrosine residues that are phosphorylated by Acknowledgements Src-like kinases (Ishidate et al., 1997). It is possible that We thank Dr Marco Barchi for preparing some tr-kit recruitment of Fyn and tyrosine phosphorylation of expression vectors. This work was supported by grants from Sam68 exposes the C-terminal nuclear localization the Ministero Italiano Universita` e Ricerca (MIUR) cofin 2002 signal and favors the translocation of the protein to and Agenzia Spaziale Italiana (ASI).

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