The Distinct Capacity of Fyn and Lck to Phosphorylate Sam68 in T Cells Is Essentially Governed by SH3/SH2-Catalytic Domain Linker Interactions

The distinct capacity of Fyn and Lck to phosphorylate Sam68 in T cells is essentially governed by SH3/SH2-catalytic domain linker interactions

Vincent Feuillet1, Monique Semichon1, Audrey Restouin2, Julie Harriague1, Julia Janzen3, Anthony Magee3, Yves Collette2 and Georges Bismuth*,1

1De´partement de Biologie Cellulaire, INSERM U.567, CNRS UMR 8104, Universite´ Rene´ Descartes, Institut Cochin, 75014 Paris, France. Laboratoire labellise´ par la Ligue Nationale contre le Cancer; 2INSERM U119, Institut Paoli Calmette, 13009 Marseille, France; 3Section of Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK

Sam68 phosphorylation correlates with Fyn but not Lck initiation of the T cell activation cascade is still unclear. expression in T cells. This substrate has been used here They are probably not or only partially redundant since to explore the possible basis of the specificity of Fyn studies of mice or T cell lines defective in Lck or Fyn versus Lck. We show that this specificity is not based on have shown a different phenotype in T cell development a spatial segregation of the two kinases, since a chimeric and T cell activation (Appleby et al., 1992; Groves et al., Lck molecule containing the membrane anchoring 1996; Molina et al., 1992; Stein et al., 1992; van Oers et domain of Fyn does not phosphorylate Sam68. More- al., 1996). In addition, an increasing number of proteins over, a Sam68 molecule targeted to the plasma has been found to interact with and/or to be membrane by the farnesylation signal of c-Ha-Ras phosphorylated specifically by Fyn (da Silva et al., remains poorly phosphorylated by Lck. In T cells, Fyn 1997; Feshchenko et al., 1998; Marie-Cardine et al., appears to be the active Src kinase in rafts, but Sam68 is 1997; Marie-Cardine et al., 1998, 1999; Qian et al., 1997; not expressed in rafts, and its distinct phosphorylation by Tsygankov et al., 1996) or by Lck (Takemoto et al., Fyn and Lck is not affected by raft dispersion. The Fyn/ 1995, 1996) in vivo. Furthermore, Lck and Fyn catalytic Lck specificity does not reflect a higher kinase activity of activities in T cells appear to be differently regulated Fyn in general, as both Fyn and Lck are similarly depending on their localization in plasma membrane recognized by an anti-active Src antibody. Both also microdomains (Kabouridis et al., 2000). strongly phosphorylate another Src substrate in vivo. Src PTKs are thought to be regulated mainly by the Mainly, Lck phosphorylates Sam68 when the interaction phosphorylation state of two critical tyrosine residues, between the SH3 domain and the SH2-catalytic domain one within the C-terminal tail (Tyr527 in Src) and the linker is altered in heterologous Src molecules or after other within the kinase domain in the so-called Src mutating key residues in the linker that increase the autophosphorylation motif (Tyr416 in Src) (Cartwright accessibility of the SH3 domain. Thus, the distinct et al., 1987; Cooper et al., 1986; Kmiecik and potential of Fyn and Lck to phosphorylate Sam68 is Shalloway, 1987; Piwnica-Worms et al., 1987). It is likely controlled by the interaction of the kinase SH3 currently believed that the PTKs are in an active state domain with the linker and Sam68, possibly on a when the tyrosine in the Src autophosphorylation site competitive binding basis. is phosphorylated. As for the C-terminal tail tyrosine Oncogene (2002) 21, 7205 – 7213. doi:10.1038/sj.onc. residue its phosphorylation by Csk (Nada et al., 1991; 1205929 Okada et al., 1991) (and/or CHK; not in T cells) (Davidson et al., 1997; Klages et al., 1994) inhibits the Keywords: Fyn; Lck; substrate specificity; T cells; kinase activity, whereas its dephosphorylation by Sam68 tyrosine phosphatases, such as CD45 in T cells (Mustelin et al., 1989, Mustelin and Altman, 1990), has the opposite effect. Structural data from crystal- lization of the Src, Hck, and Lck proteins (Sicheri et Introduction al., 1997; Xu et al., 1997; Yamaguchi and Hendrickson, 1996) together with a large body of mutational T cells express primarily two Src protein tyrosine analyses have illustrated how phosphorylation of this kinases (PTKs), Lck and Fyn, both of which are likely distal tyrosine residue controls the conformation of the fundamental in signal transduction after T cell receptor kinase by interacting with its own SH2 domain. (TcR) ligation. However, their distinct role in the However, they also demonstrated that Src-family kinases are maintained in an inactive and closed conformation by intramolecular binding of the SH3 *Correspondence: G Bismuth; E-mail: [email protected] domain to the linker between the SH2 domain and the Received 26 March 2002; revised 28 July 2002; accepted 1 August catalytic domain (SH2-CD linker). Accordingly, displa- 2002 cements of these two interactions seem to participate in Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7206 the regulation of Src PTKs activity (Alexandropoulos and Baltimore, 1996; Hartley et al., 1999; Moarefi et al., 1997). Whether these interactions are differently regulated between Fyn and Lck is unknown but one can assume that this may also contribute to the different behaviour of the two Src PTKs in T cells. Of concern with this problem is the phosphorylation of Sam68 in T cells. Sam68 is a 68 kDa protein first reported to associate with and to be strongly phosphorylated by Src during mitosis (Fumagalli et al., 1994; Taylor and Shalloway, 1994) and to regulate cell growth (Barlat et al., 1997). Sam68 is actually classified as a member of the GSG family protein (GRP, Sam68, Gld-1) whose function might be to link the nuclear export of mRNA to signaling events (Vernet and Artzt, 1997). The molecule also has several proline-rich regions and a tyrosine-rich C-terminal region allowing it to interact with the SH3 domain of Src PTKs, and, once phosphorylated, with various SH2 domain containing signaling molecules suggesting an adapter function (Jabado et al., 1998; Richard et al., 1995 ). Others and ourselves previously reported that Sam68 was constitutively phosphorylated in various T cell lines (Fusaki et al., 1997; Lang et al., 1999). This phosphorylation was probably linked to the high level expression of Src PTKs in transformed T cells. However, it was found that, for unknown reasons, this constitutive phosphorylation of Sam68 correlates with Fyn levels, but not with Lck expression (Lang et al., 1997, 1999). Investigating this issue, we show in the Figure 1 A Lck molecule containing the membrane targeting do- present work that the inability of Lck to phosphorylate main of Fyn does not phosphorylate Sam68. (a) Jurkat cells were transfected with the cDNA of Lck wild-type (WT), Fyn WT or Sam68 in vivo can be bypassed by altering the the cDNA of a chimera (N-ter Fyn/Lck) in which the coding se- conformation of the kinase through modifications of quence of the 10 first a.a. of Lck was swapped with the corre- SH3/SH2-CD linker interactions. sponding Fyn sequence. Forty-eight hours after transfection, lysates were analysed with anti-phosphotyrosine mAb 4G10, anti-Sam68, anti-Fyn or anti-Lck Abs. Sam68, immunoprecipi- Results tated from the same lysates, was probed with anti-phosphotyrosine monoclonal Ab (mAb) 4G10. (b) Cells, transfected with the cDNA of the N-ter Fyn/Lck chimera, were subjected 48 h after A different localization of Fyn and Lck is not involved in transfection to immunofluorescence with the anti-Lck Ab. Micro- the specific phosphorylation of Sam68 by Fyn in T cells scopic fields of two individual cells are shown Fyn but not Lck fully phosphorylated Sam68 in T cells (Lang et al., 1999). A difference in their localization might be responsible, as Fyn was suggested to be molecule was expressed at the plasma membrane like expressed not only at the cell membrane, like Lck, but wild-type Lck (see Figure 4a for comparison). also in the centromeric region (Ley et al., 1994). Both We next used a complementary approach by PTKs contain at the N-terminus, within the so-called targeting Sam68 at the plasma membrane using its SH4 domain, a glycine residue and cysteine residues cDNA, deleted of the nuclear localization signal, fused that are subjected to myristoylation and palmitoyla- to enhanced green fluorescent protein (GFP) and the tion, respectively, and control their binding to farnesylation signal of c-Ha-Ras (GFP-F). GFP-F membranes and subsequent localization. We thus alone gave a homogeneous membrane pattern in constructed a molecule in which the first 10 a.a. of fluorescence microscopy. Sam68-GFP-F was also Lck were replaced with those of Fyn, and analysed localized at the cell membrane (Figure 2a). The Sam68 phosphorylation induced by this chimeric PTK molecule accumulated in membrane patches in most (Figure 1a). Fyn induces a strong phosphorylation of cells. This phenomenon may be driven by the Sam68. In contrast, like wild-type Lck, the N-ter Fyn/ previously reported propensity of Sam68 to multi- Lck chimera did not trigger any significant phosphor- merize (Chen et al., 1997). Mainly, as with wild-type ylation of the molecule. Note that unidentified proteins Sam68, only Fyn strongly phosphorylated Sam68- around 100 and 110 kDa (see also Figure 3b) were also GFP-F after cotransfection (Figure 2b). These data specifically phosphorylated by Fyn. Figure 1b shows likely exclude that a particular localization of Fyn the labeling with an anti-Lck antibody (Ab) of cells outside the plasma membrane might account for its transfected with the N-ter Fyn/Lck mutant. The specific role.

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7207

Figure 2 Fluorescence microscopy and phosphorylation of a membrane targeted form of Sam68. (a) Jurkat cells were transfected with a vector encoding GFP fused to the farnesylation signal of c-Ha-Ras (GFP-F) or a vector encoding GFP-F fused to Sam68 without its nuclear localization signal (NLS) (Sam68-GFP-F). Fluorescence was analysed on viable cells after a 48 h culture. For each construct, six individual cells are shown. (b) Jurkat cells were transfected with the cDNA of Lck WT or Fyn WT together with the Sam68-GFP-F vector. Forty-eight hours after transfection, lysates were analysed with anti-GFP, anti-Fyn or anti-Lck Abs. Sam68-GFP-F was immunoprecipitated from the same lysates with the Sam68-speciﬁc polyclonal antiserum and probed with anti-phosphotyrosine mAb 4G10

Sam68 phosphorylation in vivo. To explore this, we Sam68 phosphorylation is not dependent on membrane first used an anti-active Src Ab against the phosphory- rafts lated form of the activation loop on residue Y418. This Fyn and Lck are expressed inside and outside lipid rafts loop is conserved between Src and Fyn, and only at the cell membrane. However, while Fyn is active in differs by a single substitution (Q?E) at position 399 both compartments, Lck appears to encounter inhibitory in Lck. Using recombinant Lck, we first verified that conditions in these microdomains (Kabouridis et al., this Ab specifically recognized the active form of the 2000). This might explain the difference between the two molecule in vitro (data not shown). We next probed in Src PTKs to phosphorylate Sam68 if this metabolic blotting experiments cells transfected with Fyn or Lck process occurs in rafts. Thus, we analysed Sam68 with the anti-active Src Ab. Several bands were expression in rafts. Immunoblotting showed Sam68 strongly recognized in both cell extracts (Figure 4a, absent from the low-density fractions, which contain left panel). Immunoprecipitation with the anti-Src rafts, but within the lower-most detergent-soluble pY418 Ab followed by blotting with anti-Fyn or fractions (Figure 3a). Sam68 expression in these fractions anti-Lck Abs were also performed (Figure 4a, right was expectedly low as we reported that the protein was panel). The results suggested that Fyn was only one of not abundant in T cell membranes (Lang et al., 1999). the three major bands recognized in whole cell lysates Blotting with anti-LAT, a protein enriched in T cell rafts, by the anti-active Src Ab. In contrast, most bands was used as a control. We next assessed the effects of raft precipitated in Lck-transfected cells were blotted by the disruption by incubating cells overexpressing Fyn or Lck anti-Lck Ab. To further confirm this substantial with methyl-b-cyclodextrin (MbCD) that depletes phosphorylation of the activation loop in Lck, the membrane cholesterol and destroys rafts (Ostermeyer spatial distribution and levels of the active PTKs in et al., 1999). No significant changes in Sam68 phosphor- Lck- and Fyn-transfected cells were compared by ylation by Fyn were observed in treated cells and Lck fluorescence microscopy (Figure 4b). Fyn- and Lck- remained ineffective in phosphorylating Sam68 (Figure specific Abs strongly labeled the plasma membrane of 3b). Note that we controlled in parallel experiments that cells overexpressing the corresponding kinase with rafts have been destroyed by MbCD treatment since similar intensities. Mainly, we also observed a similar LAT was no more detectable by Western-blot in the low- recognition level and fluorescence distribution at the density fractions (not shown). plasma membrane with the anti-Src pY418 Ab in cells transfected with either PTKs. Note that this last staining was very low in non transfected cells (not Overexpressed Fyn and Lck present similar shown). We concluded from these results that the activity in vivo phosphorylation status on residue Y418 in the The catalytic activity of Src PTKs is controlled in vivo activation loop between Lck and Fyn was very similar. by complex mechanisms not yet completely under- To check more directly the in vivo catalytic activity stood. Hence, speculations can be made about a higher of Lck, the phosphorylation in Jurkat cells of Dok-1 activity of overexpressed Fyn resulting in an increased was also examined. Dok-1 is a privileged substrate of

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7208 (Shen et al., 1999), a required interaction for the molecule to be phosphorylated. In inactive Src PTKs, the SH3 domain binds the region linking the SH2 domain to the catalytic part of the molecule (the so- called SH2-CD linker), which adopts a polyproline type II helix conformation (Xu et al., 1999). However the contacting a.a. in the linker are quite different between Fyn (and Src) on the one hand and Lck on the other ((Williams et al., 1998), see also Discussion). We thus made the hypothesis that the distinct phosphorylation of Sam68 by Fyn and Lck may be explained on a competitive binding basis between its proline-rich motifs and the SH2-CD linker for the SH3 domain of the corresponding kinase. To evaluate this point, different constructs were established in which this intramolecular interaction was altered (Figure 5a). We first generated a chimeric molecule, Fyn/Lck(SH2+CD), in which the C- terminus of Lck, including the SH2 domain and the catalytic domain, was fused to the N-terminus of Fyn. In this kind of molecule, the SH3 domain is unlikely to adapt to the heterologous SH2-CD linker. As shown Figure 5b, the chimera was able to phosphorylate Sam68 as efficiently as wild-type Fyn. We next used a molecule, Src(SH3)Lck, in which the SH3 domain of Src has been substituted by the SH3 domain of Lck. We knew from preliminary experiments that wild-type Src triggered, like Fyn, a strong phosphorylation of Sam68 in Jurkat cells. Clearly, Src (SH3)Lck was as efficient as wild-type Src to phosphorylate Sam68 (Figure 5c), suggesting that the SH3 Lck domain can fully substitute to the one of Src when it is not facing the homologous SH2-CD linker. This assumption was then directly checked with an Figure 3 Sam68 phosphorylation and membrane rafts. (a) Parti- Lck molecule, Lck K232E/P233G, mutated on the tion of Sam68 into rafts. The expression of Sam68 and LAT in the different fractions, partitioned through a sucrose gradient as two essential residues in the SH2-CD linker to contact described in Materials and methods, was analysed by immuno- the SH3 domain. Sam68 was strongly phosphorylated blotting with specific Abs. (b) Raft-disruption by MbCD does by the mutated PTK (Figure 5d). We conclude that not affect Sam68 phosphorylation. Jurkat cells were transfected the interplay between the SH2-CD linker of Lck and with the cDNA of Lck WT or Fyn WT. Forty-eight hours after its SH3 domain is probably essential to explain the transfection, cells were treated with 10 mM MbCD for 15 min at 378C. Cell lysates were then analysed with anti-phosphotyro- lack of ability of the PTK to phosphorylate Sam68 in sine, anti-Sam68, anti-Fyn or anti-Lck Abs. Sam68, immunopre- vivo. cipitated from the same lysates, was probed with anti- phosphotyrosine mAb 4G10 Discussion

the PTK Abl, also phosphorylated by Src in Sam68 is predominantly present in the nucleus of T transformed fibroblasts. Moreover, Dok-1 is a cells and its expression outside this cell compartment is substrate of Lck in T cells (Nemorin and Duplay, low (Lang et al., 1999). Presumably, the protein 2000; Yang et al., 1999). We thus speculated that an shuttles very rapidly outside the nucleus. We reported altered kinase activity of overexpressed Lck in vivo however that its physiological phosphorylation by Fyn would also be apparent with this substrate. Strikingly, likely occurred in a membrane compartment since a Figure 4c shows that, contrary to Sam68, both Fyn Fyn mutant deleted of its N-terminal SH4 membrane and Lck phosphorylated Dok-1. anchoring domain was inactive (Lang et al., 1999). Thus, the different phosphorylation of Sam68 by Fyn and Lck could be explained first by the presence of a Lck is able to phosphorylate Sam68 when the interaction fraction of Fyn in a localization where Lck was between the SH3 domain and the SH2-CD linker is missing. A dissimilar distribution of the two Src PTKs altered at the plasma membrane was also conceivable. In both Sam68 was initially described as a binding partner for cases, differences in the SH4 domain may be Src PTKs SH3 domains through its proline-rich motifs responsible. However, we show here that a chimeric

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7209 A B

Figure 4 Overexpressed Fyn and Lck exhibit similar activity in vivo.(a) Left panel: Jurkat cells were transfected with the cDNA of Lck WT or Fyn WT. Forty-eight hours after transfection, lysates were subjected to Western-blot with anti-Src pY418. Right panel: Cells were transfected with Lck WT or Fyn WT and whole cell lysates or anti-Src pY418 immunoprecipitates were resolved in parallel and blotted with the indicated Abs. (b) Jurkat cells were transfected with the cDNA of Lck WT or Fyn WT. Forty-eight hours after transfection, cells were subjected to immunofluorescence with anti-Lck, anti-Fyn or anti-Src pY418 Abs, as indicated. Shown are histograms giving the distribution of the labeling measured in the transfected cells (n5180) as indicated in Materials and methods. Mean fluorescence are also indicated. In the different insets microscopic fields of two individual cells are shown for each con- dition. (c) Dok-1 is phosphorylated by Lck and Fyn. Jurkat cells were transfected with the cDNA of Lck WT or Fyn WT and cotransfected with the cDNA of Dok-1 as indicated. Forty-eight hours later, Dok-1 tyrosine phosphorylation was analysed by blotting Dok-1 immunoprecipitates with mAb 4G10. Whole cell lysates, resolved in parallel, were blotted with anti-Dok-1, anti-Fyn or anti-Lck Abs

Lck molecule containing the membrane binding while both kinases are strongly active outside rafts domain of Fyn did not increase Sam68 phosphoryla- (Kabouridis et al., 2000). It has been speculated that tion levels, likely excluding this hypothesis. Lck activity was more efficiently turned off by the PTK Nevertheless, the possibility remained of a different Csk, the main Src PTK regulatory molecule recruited catalytic activity of Fyn and Lck at the plasma into rafts by the phospho-adaptor Cbp (Itoh et al., membrane. Of concern with this conjecture is the 2002; Kawabuchi et al., 2000; Torgersen et al., 2001). recent demonstration that the activity of Lck from lipid However, we found that Sam68 was not detectable in rafts in Jurkat T cells is very low, in contrast to Fyn, rafts and that raft disruption did not strongly affect

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7210 A

BCD

Figure 5 The interplay between the SH3 domain and the SH2-CD linker of Lck is important for Sam68 phosphorylation in vivo.(a) A diagram of the different constructs used in the experiment. (b) A chimeric molecule containing the SH2 domain and the catalytic domain of Lck phosphorylates Sam68. Jurkat cells were transfected with the cDNA of Lck WT, Fyn WT or Fyn/Lck(SH2+CD) chimera consisting of the C-terminal part of Lck, including the SH2 domain and the catalytic domain, fused to the N-terminus of Fyn. Forty-eight hours after transfection, lysates were analysed with anti-phosphotyrosine, anti-Sam68, anti-Fyn or anti-Lck Abs. Sam68, immunoprecipitated from the same lysates, was probed with anti-phosphotyrosine mAb 4G10. (c) The SH3 domain of Lck can substitute for the one of Src for Sam68 phosphorylation. Jurkat cells were transfected with the cDNA of Lck WT, Fyn WT, Src WT, or Src(SH3)Lck chimera, a construct in which the SH3 domain of Src has been substituted by the SH3 domain of Lck. Forty- eight hours after transfection, lysates were probed with anti-phosphotyrosine mAb 4G10, anti-Sam68, anti-Fyn, anti-Lck or anti-Src Abs. Sam68 immunoprecipitates were blotted with anti anti-phosphotyrosine mAb 4G10. (d) Mutation of residues K232 and P233 in the Lck SH2-CD linker triggers Sam68 phosphorylation. Jurkat cells were transfected with the cDNA of Lck WT, Fyn WT or Lck K232G/P233E mutant. Forty-eight hours after transfection, lysates were probed with anti-Sam68, anti-Fyn, or anti-Lck Abs. Sam68 was immunoprecipitated from the same lysates and probed with anti anti-phosphotyrosine mAb 4G10

Sam68 phosphorylation by Fyn or Lck. It is recognition of transfected Fyn or Lck molecules, noteworthy that Sam68 fused to the farnesylation suggesting that a significant fraction of the PTKs was signal of c-Ha-Ras remained poorly phosphorylated in an active form. These results cannot be considered by Lck. As the fused Ras sequence is farnesylated, this as a formal proof that they have exactly the same chimera is likely enriched in rafts, as is a full-length c- activity. They nevertheless suggest no large difference. Ha-Ras (Prior et al., 2001), further suggesting that This conclusion was further strengthened by our Sam68 is highly phosphorylated by Fyn and poorly experiments with the adaptor Dok-1. Taken together, phosphorylated by Lck regardless of its localization these data are strongly indicative that the differential inside or outside rafts. To directly investigate the in phosphorylation of Sam68 by Fyn and Lck in T cells is vivo catalytic activity of overexpressed Fyn and Lck not due to a globally decreased ability of Lck to molecules, experiments were also undertaken using an phosphorylate substrates inside the cell membrane. Ab against a phosphopeptide corresponding to the Instead, they suggest that some intrinsic properties of conserved Src activation loop. We found a similar the two PTKs are involved.

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7211 Crystallographic data have shown that in the What final conclusions can be drawn? As mentioned inactive form of Src PTKs, the SH2-CD linker above, probably the most interesting one relies on a interacts with the SH3 domain, maintaining the model where an in vivo competition could take place molecule in a ‘closed’ conformation. It has also been between the SH2-CD linker and Sam68 for the SH3 reported that SH3 domain polyproline ligands are able domain. It must be emphasized that in this case both to displace this interaction in vitro as shown for Nef the relative affinity of Sam68 for the SH3 domain and (Moarefi et al., 1997), Sin (Alexandropoulos and the strength of interaction between the linker and the Baltimore, 1996) and Tip (Hartley et al., 1999). This SH3 domain can be involved. Indeed, we found in process is likely required for substrates like Sam68 to parallel experiments using a mammalian two-hybrid be phosphorylated in vivo. Indeed, both deletion of system a more potent binding in vivo of Sam68 to the proline motif I in human Sam68 (Shen et al., 1999) Fyn-SH3 domain than to its Lck counterpart (not (the protein encodes at least five distinct polyproline shown). However, we cannot exclude that Lck is also sequences numbered from I to V) and III and IV in its hardly unfastened by SH3-binding ligands, restraining murine homologue (Richard et al., 1995) as well as the accessibility of substrates to the SH3 domain. This mutations inactivating the SH3 domain strongly work also raises the question of the respective roles of altered Sam68 phosphorylation by Fyn or Src. Src Fyn and Lck in T cells. Although Fyn has been shown PTKs belong to a very homogeneous family of to be able to substitute for Lck in some experimental proteins. However, they exhibit some significant model (Groves et al., 1996), Sam68 is not the only differences in their primary sequence that have been example of a protein interacting and/or phosphorylated used to distinguish two phylogenic groups, A and B by Fyn specifically. Other molecules such as Pyk2 (Williams et al., 1998). Fyn and Src are in group A (Qian et al., 1997), c-Cbp (Feshchenko et al., 1998; while Lck is in group B. Interestingly, these two Tsygankov et al., 1996), Fyb (da Silva et al., 1997), groups notably differ in the interacting region between SKAP55 (Marie-Cardine et al., 1997), SKAP-HOM the SH3 domain and the SH2-CD linker. Moreover, (Marie-Cardine et al., 1998) also showed a privileged the SH3 domains of the different Src kinases have not relationship with Fyn. Future works will be necessary exactly the same properties (Abrams and Zhao, 1995; to know if in these cases also the interplay between the Hiroaki et al., 1996). Hence, we raised the hypothesis SH3 and the SH2-CD linker is an essential parameter that the low phosphorylation of Sam68 by Lck may be to explain this dissimilar behaviour of the two Src caused by a reduced accessibility of its SH3 domain to PTKs in T cells. Sam68. To explore this possibility we constructed mutant molecules in which the interaction between the SH3 domain and the SH2-CD linker was altered, including a Lck molecule mutated on residues K232 Materials and methods and P233. A Src mutated on the corresponding residues (K249 and P250) has been shown to have a Cell culture and transfection constitutively accessible SH3 domain (Gonfloni et al., Tag Jurkat cells (JTag) were grown in RPMI 1640 medium 1999, 2000). Remarkably, all these mutants strongly supplemented with 10% FCS, 2 mML-glutamine, and 1 mM phosphorylated Sam68. It is now clear that the SH3/ sodium pyruvate. Transient transfections of Jurkat cells were SH2-CD linker interplay not only commands the performed by electroporating (at 320 V, 500 mF) 10 to interaction of Src PTKs with SH3 substrates but also 206106 cells with 20 mg of plasmid DNA. Expression in controls the catalytic activity of the enzyme. Indeed, transfected cells was conducted for 48 h followed by analysis. its disruption is an essential step for the activation of the kinase as the SH3 engagement with the linker Antibodies controls the aC-helix orientation by maintaining the conformation of the N-lobe of the catalytic domain The phosphotyrosine mAb 4G10, the rabbit polyclonal Ab (Gonfloni et al., 1997, 1999, 2000; Moarefi et al., 1997; specific for LAT and the rabbit polyclonal Ab specific for Fyn were from Upstate Biotechnology (UBI, Lake Placid, Williams et al., 1997; Xu et al., 1999). So, although we NY, USA). The rabbit polyclonal Ab specific for Sam68 used have clearly shown that a different PTK activity for immunoprecipitation experiments has already been between the wild-type forms of Fyn and Lck to described (Lang et al., 1997). The rabbit polyclonal Ab phosphorylate Sam68 is not involved, one can make specific for the C-terminal region of Lck, the mouse mAb the hypothesis that the alteration of the SH3/SH2-CD specific for Lck and the polyclonal Ab against the C-terminus linker interplay in our Lck mutants participates in the of Sam68 used for Western blotting experiments were from restoration of Sam68 phosphorylation also by improv- Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Fyn ing the kinase activity. However, while the Lck mAb was from Transduction Laboratories (Lexington, KY, K232E/P233G mutant was much more potent than USA). A rabbit antiserum against the C-terminus of Dok-1 the Fyn/Lck (SH2+CD) chimera to trigger whole (RVGTDKTGVKSEGST) was used for immunoprecipitation. The anti-Dok-1 Ab M-276, purchased from Santa Cruz, tyrosine phosphorylations, it was usually slightly less was used for immunoblotting. The rabbit polyclonal Abs effective in phosphorylating Sam68. This ultimately specific for Src and Src pY418 were from Biosource suggests that Sam68 is a good in vivo substrate of Lck International (Camarillo, CA, USA). The anti-GFP mAb providing that the accessibility of the SH3 domain of was purchased from Clontech Laboratories (Palo Alto, CA, the PTK is increased. USA).

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7212 West Grove, PA, USA) as secondary Abs. Fluorescence Plasmids and constructs microscopy was performed with a Nikon Eclipse TE300 Full lengh cDNAs encoding wild-type murine Fyn and Lck in inverted microscope equipped with fluorescein filters using a pSRa-puro were already described (Lang et al., 1999). The 660 oil objective. Fluorescence images were collected with a cDNA encoding Src (SH3)Lck, in pSGT, was kindly given by cooled CCD camera (CoolSNAPfx, Roper Scientific, Evry, Dr G Superti-Furga (European Molecular Biology Labora- France) and the MetaMorph Imaging software (Universal tory, Heidelberg, Germany). The Fyn(N-ter)/Lck and the Imaging Corporation, West Chester, PA, USA). Quantitative Fyn(SH4-SH3)/Lck chimeric mutants were generated by PCR analysis of fluorescence in cells overexpressing PTKs was into the pMXI and the pSRa-puro vectors, respectively. The performed on 12-bit images, using a threshold to eliminate Lck K232E/P233G mutant was generated with Site-directed the untransfected cells, and by measuring the intensity of the mutagenesis kit, Quickchange (Stratagene, La Jolla, CA, fluorescence using MetaMorph. USA). GFP fused to the farnesylation signal of c-Ha-Ras was amplified by PCR using the EGFP-F vector (Clontech) as a Raft analysis template and inserted into the pEF6/Myc-His B vector (Invitrogen, Groningen, The Netherlands). Sam68 lacking For purification of rafts, cells (26107) were lysed for 40 min its nuclear localization signal (Lang et al., 1999) was next on ice in 700 ml of 0.5% Triton X-100 in MNE buffer (MES inserted 3’ of EGFP-F. The full length cDNA encoding 25 mM, NaCl 150 mM, EDTA 2 mM), dounced 10 times, and human Dok-1 in pBluescript SK-, kindly given by Dr Nick mixed with 700 ml 85% sucrose in MNE buffer after removal Carpino (Cold Spring Harbour Laboratory, New York, NY, of large debris by a 10 min centrifugation at 450 g. After USA), was sub-cloned into pSRa-puro. transfer to a centrifuge tube, the suspension was overlaid with 1.4 ml 30% sucrose in MNE buffer and 700 ml5% sucrose in MNE. After centrifugation for 18 h at 200 000 g, Western blot and immunoprecipitation 280 ml gradient fractions were collected from the top of the Cells lysis, SDS-polyacrylamide gels and immunoblotting gradient and precipitated with trichloroacetic acid before were performed as previously described (Lang et al., 1999). boiling in sample buffer. For treatment with MbCD cells Peroxidase-labeled goat anti-mouse or goat anti-rabbit were incubated with 10 mM MbCD in RPMI HEPES 20 mM antiserum, and an enhanced chemiluminescence system for 15 min at 378C. Cell lysates were prepared for (ECL, Amersham Pharmacia Biotech) were used for blotting. immunoprecicipitation and Western blotting as above. For immunoprecipitations, cell lysates (800 mg) were incubated for 2 h at 48C with 5 mg of protein A-Sepharose beads (Amersham Pharmacia Biotech) previously incubated 2 h at 48C with the appropriate Ab. After four washes, immune Acknowledgments complexes were boiled and analysed by Western blot. We thank Drs A Trautmann, C Randriamampita and V Lang for their critical reading of the manuscript and helpful suggestions. We are grateful to Dr G Superti-Furga Fluorescence analysis for the Src(SH3)Lck construct. This work was supported in For fluorescence microscopy analysis of GFP, cells were part by grants from the Association de la Recherche contre directly observed in their culture medium on a glass coverslip le Cancer and by grants from the Ligue Nationale contre le mounted on 35 mm Petri dishes in a final volume of 100 ml. Cancer. V Feuillet is a recipient of a graduate studentship Immunofluorescence analysis with the anti-active Src Ab or from the Ministe` re de l’Education Nationale et de la Abs against Fyn and Lck were performed on paraformalde- Recherche. M Semichon is supported by Association hyde-fixed cells as previously described (Lang et al., 1999), Claude Bernard. A Restouin is a recipient of a SIDAC- using a FITC-conjugated F(ab’)2 fragment donkey anti-rabbit TION fellowship. J Harriague is a recipient of a Fondation IgG or a rhodamine-conjugated F(ab’)2 fragment goat anti- pour la Recherche Me´ dicale fellowship. J Janzen is mouse IgG (Jackson ImmunoResearch Laboratory, Inc., supported by Leukemia Research Fund grant 9705.

References

Abrams CS and Zhao W. (1995). J. Biol. Chem., 270, 333 – Davidson D, Chow LM and Veillette A. (1997). J. Biol. 339. Chem., 272, 1355 – 1362. Alexandropoulos K and Baltimore D. (1996). Genes Dev., 10, Feshchenko EA, Langdon WY and Tsygankov AY. (1998). 1341 – 1355. J. Biol. Chem., 273, 8323 – 8331. Appleby MW, Gross JA, Cooke MP, Levin SD, Qian X and Fumagalli S, Totty NF, Hsuan JJ and Courtneidge SA. Perlmutter RM. (1992). Cell, 70, 751 – 763. (1994). Nature, 368, 871 – 874. Barlat I, Maurier F, Duchesne M, Guitard E, Tocque B and Fusaki N, Iwamatsu A, Iwashima M and Fujisawa J. (1997). Schweighoffer F. (1997). J. Biol. Chem., 272, 3129 – 3132. J. Biol. Chem., 272, 6214 – 6219. Cartwright CA, Eckhart W, Simon S and Kaplan PL. (1987). Gonfloni S, Frischknecht F, Way M and Superti-Furga G. Cell, 49, 83 – 91. (1999). Nat. Struct. Biol., 6, 760 – 764. Chen T, Damaj BB, Herrera C, Lasko P and Richard S. Gonfloni S, Weijland A, Kretzschmar J and Superti-Furga (1997). Mol. Cell. Biol., 17, 5707 – 5718. G. (2000). Nat. Struct. Biol., 7, 281 – 286. Cooper JA, Gould KL, Cartwright CA and Hunter T. (1986). Gonfloni S, Williams JC, Hattula K, Weijland A, Wierenga Science, 231, 1431 – 1434. RK and Superti-Furga G. (1997). EMBO J., 16, 7261 – da Silva AJ, Li Z, de Vera C, Canto E, Findell P and Rudd 7271. CE. (1997). Proc. Natl. Acad. Sci. USA, 94, 7493 – 7498.

Oncogene Sam68 phosphorylation by Fyn and Lck in T cells V Feuillet et al 7213 Groves T, Smiley P, Cooke MP, Forbush K, Perlmutter RM Okada M, Nada S, Yamanashi Y, Yamamoto T and and Guidos CJ. (1996). Immunity, 5, 417 – 428. Nakagawa H. (1991). J. Biol. Chem., 266, 24249 – 24252. Hartley DA, Hurley TR, Hardwick JS, Lund TC, Medveczky Ostermeyer AG, Beckrich BT, Ivarson KA, Grove KE and PG and Sefton BM. (1999). J. Biol. Chem., 274, 20056 – Brown DA. (1999). J. Biol. Chem., 274, 34459 – 34466. 20059. Piwnica-WormsH,SaundersKB,RobertsTM,SmithAE Hiroaki H, Klaus W and Senn H. (1996). J. Biomol. NMR., 8, and Cheng SH. (1987). Cell, 49, 75 – 82. 105 – 122. Prior IA, Harding A, Yan J, Sluimer J, Parton RG and Itoh K, Sakakibara M, Yamasaki S, Takeuchi A, Arase H, Hancock JF. (2001). Nat. Cell. Biol., 3, 368 – 375. Miyazaki M, Nakajima N, Okada M and Saito T. (2002). Qian D, Lev S, van Oers NS, Dikic I, Schlessinger J and J. Immunol., 168, 541 – 544. Weiss A. (1997). J. Exp. Med., 185, 1253 – 1259. Jabado N, Jauliac S, Pallier A, Bernard F, Fischer A and RichardS,YuD,BlumerKJ,HausladenD,OlszowyMW, Hivroz C. (1998). J. Immunol., 161, 2798 – 2803. Connelly PA and Shaw AS. (1995). Mol. Cell. Biol., 15, Kabouridis PS, Janzen J, Magee AL and Ley SC. (2000). Eur. 186 – 197. J. Immunol., 30, 954 – 963. Shen Z, Batzer A, Koehler JA, Polakis P, Schlessinger J, Kawabuchi M, Satomi Y, Takao T, Shimonishi Y, Nada S, Lydon NB and Moran MF. (1999). Oncogene, 18, 4647 – Nagai K, Tarakhovsky A and Okada M. (2000). Nature, 4653. 404, 999 – 1003. Sicheri F, Moarefi I and Kuriyan J. (1997). Nature, 385, KlagesS,AdamD,ClassK,FargnoliJ,BolenJBand 602 – 609. Penhallow RC. (1994). Proc. Natl. Acad. Sci. USA, 91, Stein PL, Lee HM, Rich S and Soriano P. (1992). Cell, 70, 2597 – 2601. 741 – 750. Kmiecik TE and Shalloway D. (1987). Cell, 49, 65 – 73. Takemoto Y, Furuta M, Li XK, Strong-Sparks WJ and Lang V, Mege D, Semichon M, Gary-Gouy H and Bismuth Hashimoto Y. (1995). EMBO J., 14, 3403 – 3414. G. (1997). Eur. J. Immunol., 27, 3360 – 3367. Takemoto Y, Sato M, Furuta M and Hashimoto Y. (1996). Lang V, Semichon M, Michel F, Brossard C, Gary-Gouy H Int. Immunol., 8, 1699 – 1705. and Bismuth G. (1999). J. Immunol., 162, 7224 – 7232. Taylor SJ and Shalloway D. (1994). Nature, 368, 867 – 871. Ley SC, Marsh M, Bebbington CR, Proudfoot K and Jordan Torgersen KM, Vang T, Abrahamsen H, Yaqub S, Horejsi P. (1994). J. Cell. Biol., 125, 639 – 649. V, Schraven B, Rolstad B, Mustelin T and Tasken K. Marie-Cardine A, Bruyns E, Eckerskorn C, Kirchgessner H, (2001). J. Biol. Chem., 276, 29313 – 29318. Meuer SC and Schraven B. (1997). J. Biol. Chem., 272, Tsygankov AY, Mahajan S, Fincke JE and Bolen JB. (1996). 16077 – 16080. J. Biol. Chem., 271, 27130 – 27137. Marie-Cardine A, Kirchgessner H and Schraven B. (1999). van Oers NS, Lowin-Kropf B, Finlay D, Connolly K and Eur. J. Immunol., 29, 1175 – 1187. Weiss A. (1996). Immunity, 5, 429 – 436. Marie-Cardine A, Verhagen AM, Eckerskorn C and Vernet C and Artzt K. (1997). Trends Genet., 13, 479 – 484. Schraven B. (1998). FEBS Lett., 435, 55 – 60. Williams JC, Weijland A, Gonfloni S, Thompson A, Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee CH, Courtneidge SA, Superti-Furga G and Wierenga RK. Kuriyan J and Miller WT. (1997). Nature, 385, 650 – 653. (1997). J. Mol. Biol., 274, 757 – 775. Molina TJ, Kishihara K, Siderovski DP, van Ewijk W, Williams JC, Wierenga RK and Saraste M. (1998). Trends Narendran A, Timms E, Wakeham A, Paige CJ, Hartmann Biochem. Sci., 23, 179 – 184. KU and Veillette A. (1992). Nature, 357, 161 – 164. Xu W, Doshi A, Lei M, Eck MJ and Harrison SC. (1999). Mustelin T and Altman A. (1990). Oncogene, 5, 809 – 813. Mol. Cell., 3, 629 – 638. Mustelin T, Coggeshall KM and Altman A. (1989). Proc. Xu W, Harrison SC and Eck MJ. (1997). Nature, 385, 595 – Natl. Acad. Sci. USA, 86, 6302 – 6306. 602. Nada S, Okada M, MacAuley A, Cooper JA and Nakagawa Yamaguchi H and Hendrickson WA. (1996). Nature, 384, H. (1991). Nature, 351, 69 – 72. 484 – 489. Nemorin JG and Duplay P. (2000). J. Biol. Chem., 275, Yang WC, Ghiotto M, Barbarat B and Olive D. (1999). J. 14590 – 14597. Biol. Chem., 274, 607 – 617.

Oncogene