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A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX domain‐containing protein, and Src substrate

A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX... The EMBO Journal Vol.17 No.15 pp.4346–4357, 1998 A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX domain-containing protein, and Src substrate 1,2 3 1 Kalff et al., 1992). In normal cells, Src family kinases act Peter Lock , Clare L.Abram , Toby Gibson 1,3,4 in signal transduction cascades. Typically they transduce and Sara A.Courtneidge signals from cell surface receptors, of both the kinase European Molecular Biology Laboratory, 69012 Heidelberg, Germany and non-kinase type (Erpel and Courtneidge, 1995), and and SUGEN Inc., 351 Galveston Drive, Redwood City, CA 94063, mediate the activation of a variety of pathways, including USA those involving the mitogen-activated protein (MAP) kin- Present address: The Ludwig Institute for Cancer Research, ases (Dikic et al., 1996; Luttrell et al., 1996; Sadoshima Royal Melbourne Hospital, Parkville 3052, Australia and Izumo, 1996), the phosphatidylinositol 3-kinase (PI Corresponding author 3-K) (Cantley et al., 1991), and Myc (Barone and e-mail: [email protected] Courtneidge, 1995). P.Lock and C.L.Abram contributed equally to this work Over the years, many putative substrates of Src have been identified by a variety of methods, including direct We describe a method for identifying tyrosine kinase analysis of candidate proteins such as tensin (Davis et al., substrates using anti-phosphotyrosine antibodies to 1991) and paxillin (Turner and Miller, 1994), the use of screen tyrosine-phosphorylated cDNA expression lib- phosphotyrosine antibodies for proteins such as actin raries. Several potential Src substrates were identified filament associated protein (AFAP-110; Flynn et al., 1993), ctn including Fish, which has five SH3 domains and a cortactin (Wu et al., 1991), p120 (Reynolds et al., 1992), Cas dok recently discovered phox homology (PX) domain. Fish p130 (Sakai et al., 1994) and p62 (Carpino et al., is tyrosine-phosphorylated in Src-transformed fibro- 1997; Yamanashi and Baltimore, 1997), and the analysis blasts (suggesting that it is a target of Src in vivo) and of Src-associated proteins such as Sam68 (Fumagalli et al., in normal cells following treatment with several growth 1994; Taylor and Shalloway, 1994) and Efs (Ishino et al., factors. Treatment of cells with cytochalasin D also 1995). Many substrates are cytoskeletal or membrane resulted in rapid tyrosine phosphorylation of Fish, components (Davis et al., 1991; Wu et al., 1991; Rothberg concomitant with activation of Src. These data suggest et al., 1992; Flynn et al., 1993; Turner and Miller, 1994), that Fish is involved in signalling by tyrosine kinases, others are enzymes (Cobb et al., 1994), and others adaptor and imply a specialized role in the actin cytoskeleton. or docking proteins (McGlade et al., 1992; Sakai et al., Keywords: cytochalasin D/Fish/PX domain/Src/tyrosine 1994; Ishino et al., 1995; Carpino et al., 1997; Yamanashi kinase substrate and Baltimore, 1997). Yet, despite the seeming plethora of substrates, it seems likely that physiologically relevant substrates remain undiscovered; for example, there are mutant forms of Src which seem able to phosphorylate Introduction all known substrates, yet fail to transform (Erpel et al., 1995). Perhaps some relevant substrates are not stably The viral Src protein (v-Src) was discovered more than associated with Src, or are not abundant in cells, and were 20 years ago. In the intervening years intense study of therefore missed by the available strategies. We therefore both v-Src and its cellular counterpart, c-Src, has focused sought a new way to identify tyrosine kinase substrates. on issues of regulation, transforming activity and physio- logical function. The Src family of protein tyrosine kinases comprises Results eight members in mammals, some broadly expressed, some restricted to certain tissues (Cooper, 1990; Bolen A method to identify tyrosine kinase substrates et al., 1992). All are tightly regulated in vivo; catalytic We wanted to develop a method for identifying substrates activity is repressed by interactions involving the SH3 for tyrosine kinases that could be applied to a variety of domain, the SH2 domain, and the phosphorylated kinases and cell types, and that was based on a cDNA C-terminus (Superti-Furga and Courtneidge, 1995). Recent expression system so that sequence information was imme- crystal structures of Src and Hck have revealed exactly diately available. We therefore chose to adapt a method, how this repression takes place (Sicheri et al., 1997; described by Kavanaugh et al (1995), that was used to Williams et al., 1997; Xu et al., 1997). Morphological identify binding partners for the Shc phosphotyrosine- transformation is frequently the result of the expression binding (PTB) domain. The method in Figure 1A and of catalytically de-repressed forms of Src family kinases, described in detail in Materials and methods, consisted of especially in fibroblasts (Cooper, 1990). There is also a phosphorylating a λgt11 cDNA expression library, derived large body of circumstantial evidence supporting the view from mRNA of mouse Swiss 3T3 fibroblasts, with Src that hyperactive Src family kinases may play a role in kinase, and then detecting phosphorylated proteins with several human epithelial malignancies, particularly of the an anti-phosphotyrosine antibody. Successive rounds of colon and breast (Cartwright et al., 1989, 1990; Ottenhoff- phosphorylation and antibody screening (Figure 1B) were 4346 © Oxford University Press Fish, a new adaptor protein Fig. 1. Identification of potential substrates of Src. (A) Phosphorylation screening protocol. Petri dishes containing a Swiss 3T3 cell cDNA expression library in λgt11 were overlaid with nitrocellulose filters containing IPTG and incubated for 16 h to induce expression of recombinant proteins. Replicate filters were incubated with extracts containing baculovirally expressed human Src kinase and ATP. Individual plaques containing tyrosine-phosphorylated protein were identified with an anti-phosphotyrosine (anti-pTyr) antibody using a standard immunoblotting protocol. Positive cDNA clones were purified by successive rounds of phosphorylation with Src kinase and immunodetection with anti-pTyr antibody. (B) Immunodetection of positive plaques. Immunoblot analysis of filters with an anti-pTyr antibody during (i) primary and (ii and iii) secondary rounds of phosphorylation screening showing various positive clones (arrow heads). used to plaque-purify several cDNAs, and the inserts were ation sites, it contained part of an SH3 domain. We chose then sequenced. to analyse this clone further. Several of these clones, which we have named Tks (tyrosine kinase substrates), are listed in Table I. Concep- Identification of Fish, a novel multi-SH3 tual translation showed that each insert corresponded to a domain-containing protein partial cDNA with an open reading frame containing at Several overlapping cDNA clones encompassing the entire least one, and usually several, motifs which closely coding region of Tks-5 were isolated from a mouse resemble the consensus sequence, EEEIYG/EEFD, for embryonal cDNA library with a probe corresponding to phosphorylation by v-Src kinase (Songyang and Cantley, the original Tks-5 clone. These cDNAs represented two Cas 1995). One clone, Tks-7, was identical to part of p130 , distinct transcripts (data not shown), encoding isoforms a known substrate of both Src and focal adhesion kinase, of 1124 and 1081 residues (Figure 2A). The larger and a Crk binding protein (Sakai et al., 1994; Polte and isoform contained two insertions of 15 and 28 amino Hanks, 1995; Harte et al., 1996). Tks-1, Tks-2 and acids (underlined), which were absent in the smaller Tks-9 correspond to partial cDNAs for recently described isoform. The predicted translational start site, which is proteins with C-terminal SH3 domains. Tks-1 encodes a shared by both transcripts, is preceded by stop codons in portion of a mouse cDNA product (designated s19) which all three reading frames and conforms quite closely to the was identified as a Src SH3-domain binding protein consensus for translation initiation (Kozak, 1991). It is phox (Yamabhai and Kay, 1997). Tks-2 corresponds to part of noteworthy that the initiator methionine of p47 , the SH3P7, a protein identified on the basis of its ability to protein most closely related to that described here, is in interact with SH3-binding peptide ligands (Sparks et al., the analogous position (see Figure 2C). These data strongly 1996). Tks-2 also shares significant sequence similarity suggest that this residue is indeed the translational start with a neuronal actin binding protein, drebrin E (Toda site. Both predicted isoforms have five SH3 domains et al., 1993). Tks-9 corresponds to the mouse homologue (boxed), a phox homology (PX) domain (overlined), and of human CIP4, a proposed target protein of the activated consensus sequences for SH3 domain binding (double form of the Rho-family GTPase Cdc42 (Aspenstrom, underlined) and Src phosphorylation (ringed). The 15 1997) and of a rat protein, STP, that was identified amino acid insertion (underlined) occurs between the PX independently as a possible regulator of cation transport domain and the first SH3 domain, and the 28 amino acid in yeast (Tsuji et al., 1996). A portion of CIP4 was also insertion (underlined) is between the first and second SH3 described as Trip10, a thyroid hormone receptor interacting domains. The original λgt11 clone is encompassed by the protein (Lee et al., 1995). Tks-3 was identical to a arrows. Outside of the described domains, the amino portion of Mem3, a protein encoded by a mouse maternal acid sequence shows no significant homology with other embryonic mRNA, which is related to a yeast vacuolar proteins in any accessible databases (data not shown) and sorting protein (Hwang et al., 1996). The Tks-5 and Tks- is therefore unlikely to contain any catalytic domain. We 14 clones encoded novel proteins. Inspection of Tks-5 have named the protein encoded by these clones Fish (five showed that in addition to three possible Src phosphoryl- SH3 domains). 4347 P.Lock et al. phox The overall topography of Fish is shown schematically other and to the two SH3 domains of p47 (Volpp et al., in Figure 2B, where it is compared with other proteins 1989), as shown in Figure 2D. Pairwise comparison of which also contain PX and/or SH3 domains. The PX the Fish SH3 domains indicate that they share 27.5–48% domain, whose function is as yet unknown, was recently identity with each other, 24–47% identity with the two phox described as a conserved domain in many eukaryotic SH3 domains of p47 , but only 13.7–23.5% identity proteins (Ponting, 1996), including those shown in Figure with the SH3 domains of Src and the p85 subunit of the 2B and C. The PX domain of Fish is most closely related phosphatidylinositol 3-kinase (PI 3-K). phox phox to the PX domains of p47 and p40 (Volpp et al., 1989; Wientjes et al., 1993), Bem1 and Scd2 (Chenevert Expression and tyrosine phosphorylation of Fish et al., 1992; Chang et al., 1994), and Cpk (MacDougall To assess the expression profile of Fish mRNA we analysed et al., 1995; Molz et al., 1996; Virbasius et al., 1996; various adult mouse tissues by Northern blot hybridization G.Plowman, K.Joho and S.A.Courtneidge, unpublished using a probe representing the Tks-5 cDNA (Figure 3). data). All of the SH3 domains are most similar to each We detected a prominent transcript of ~10.5 kb in most tissues examined, with the exception of spleen and testis Table I. A summary of clones obtained from anti-phosphotyrosine which contained relatively low and undetectable levels of screening of a phosphorylated mouse fibroblast expression library Fish mRNA, respectively. We also noted much weaker expression of a message of ~6 kb in those tissues containing Clone Possible Src Size Identity the 10.5 kb mRNA. Whether the smaller transcript corres- phosphorylation (bp) (functional information) sites ponds to a differentially spliced mRNA or perhaps a Fish- related transcript, remains to be established. Both the 10.5 Tks-1 QRLSYGAFT 838 s19 and 6 kb transcripts are significantly larger than the Fish (Src SH3 binding protein) coding region (~3.5 kb) present in the two Fish cDNAs Tks-2 REQRYQEQH 533 SH3P7 that we identified, suggesting that they may also contain PETSYGREH (SH3 ligand binding protein) extensive 5- and/or 3-untranslated sequences. EEGTYEVPP We raised polyclonal antibodies against a GST fusion QDTLYEEP protein containing the original Tks-5 cDNA product, and Tks-3 EGPIYEGLI 1250 Mem3 (relative of yeast used these to examine the expression of Fish in cells vacuolar sorting protein (Figure 4A). Three forms of Fish, of ~130, ~140 and VPS35) ~150 kDa, were detected in immunoprecipitates from NIH Tks-5 LKVKYEEPE 1020 novel (SH3 domain containing 3T3 cells. We think it unlikely that p130 and p140 EEPEYDVPA adaptor protein now named represent proteolytic breakdown products of the p150 EETIYENEG Fish) form of Fish, since they were also detected in lysates of Cas Tks-7 PQDIYDVPP 364 p130 [substrate of Src and cells generated under denaturing conditions (data not LPNQYGQEV focal adhesion kinase (FAK), shown), and perhaps represent products of other RNA LLDVYDVPP Crk binding protein] splice variants. Transient transfection of COS cells with HHSVYDVPP an expression construct encoding the small form of Fish Tks-9 EVQKYEAWL 1000 homologue of human CIP4, (see Figure 2A) gave rise to two bands, the largest of LGAGYGLLS Trip10 and rat STP (Cdc42 which was 150 kDa. The smaller product most likely EVQKYEAWG interacting protein, thyroid DTPIYTEFD hormone receptor interacting corresponds to a truncated or proteolytically degraded protein, cation transport product, since expression of a construct encoding Fish regulator) with a myc epitope tag at its N-terminus also generated Tks-14 EEPVYIEMV 789 novel (contains two YxxM two analogous products that were each recognized by the SEAIYEEMK motifs) myc antibody (data not shown). To test whether Fish was EEMKYPLPE indeed a substrate for Src, we co-transfected 293 cells Fig. 2. Amino acid sequence of murine Fish. (A) Predicted amino acid sequence of two Fish isoforms. The sequence shown was compiled from five overlapping cDNAs that were isolated from a murine day 11.5 embryonal cDNA library using a probe corresponding to Tks-5, a partial cDNA for Fish that was identified in the initial screen for Src substrates using the Swiss 3T3 cDNA library. The boundaries of the Tks-5 cDNA product are marked by arrows and potential Src tyrosine phosphorylation sites therein are indicated with ovals. Insertions of 15 and 28 amino acids that were identified in one clone, and are likely to be derived by alternative mRNA splicing, are underlined. Sequence numbers are shown at the right. The phox homology (PX) domain is overlined and five SH3 domains are boxed. Three polyproline motifs that represent potential SH3 binding sites are doubly underlined. (B) Topology of selected proteins containing a PX domain. Domains are represented by boxes as indicated: PX domains are white; SH3 domains are black; the C2 domain is light grey; the phosphatidylinositol 3-kinase catalytic domain (PI 3-K) is dark grey; the p110 homology domain is dashed; the pleckstrin homology (PH) domain is striped and the Rho-GAP domain is checked. (C) Comparison of the phox homology (PX) domains of Fish and selected proteins. This alignment was created using a similar approach to that reported previously (Ponting, 1996). Briefly, iterative profile searches were conducted using SearchWise (Birney et al., 1996) and multiple alignments using Clustal W (Thompson et al., 1994). Definition of the domain boundaries and introduction of gaps (indicated by dashes) required inspection and some hand editing, and was helped by protein termini and neighbouring domain boundaries. Names of proteins and species are shown at the left of the alignment using the following abbreviations: h is human, m is mouse, dm is Drosophila melanogaster,scis S.cerevisiae and sp is S.pombe. PLD1 refers to phospholipase D1. Sequence numbers are shown at the end of the PX domain. Numbers at the top of the alignment refer to the Fish sequence. Amino acids are shown using the single letter code. Colours are used to indicate conserved features. The basic residues histidine, lysine and arginine (H, K and R, respectively) are shown in green, the hydrophobic residues alanine, phenylalanine, isoleucine, leucine, methionine, proline, valine and tryptophan (A, F, I, L, M, P, V and W, respectively) are shown in purple, the aromatic residues phenylalanine, tryptophan and tyrosine (F, W and Y, respectively) are shown in yellow and the acidic residues aspartate and glutamate (D and E, respectively) are shown in pink. (D) Comparison of the phox phox SH3 domains of Fish with those of p47 . Multiple alignments of the SH3 domains from Fish, p47 , Src and PI 3-K were conducted using MegAlign (DNAStar). The % amino acid identity is shown. 4348 Fish, a new adaptor protein 4349 P.Lock et al. Fig. 3. Tissue distribution of Fish mRNA. Northern blot analysis of poly(A) RNA from the indicated adult mouse tissues using a P-labelled Fish cDNA probe (upper panel). Hybridization analysis of the same Northern blot using a β-actin cDNA probe (lower panel). with cDNAs for Fish, with or without wild-type, activated or kinase-inactive Src. Anti-phosphotyrosine immuno- blotting revealed that Fish was tyrosine-phosphorylated when co-expressed with active forms of Src (Figure 4B). Fig. 4. Expression and tyrosine phosphorylation of Fish by transient We next determined whether Fish was also tyrosine transfection. (A) The Fish cDNA (short form) encodes a 150 kDa phosphorylated in Src-transformed fibroblasts. A com- protein. Fish was immunoprecipitated from extracts of NIH 3T3 cells (lane 1) or COS cells transfected with an expression vector containing parison of NIH 3T3 cells, and counterparts transformed the Fish cDNA in the anti-sense (–)(lane 2) or sense ()(lane 3) with an activated allele of Src in which the regulatory orientation. Samples were analysed by SDS–PAGE and C-terminal tyrosine is replaced with phenylalanine, showed immunoblotting with anti-Fish.1 serum. The Fish cDNA product and a that Fish was tyrosine-phosphorylated in Src-transformed possible premature termination product are indicated by arrow heads. cells (Figure 5A). To determine whether Fish was likely Three forms of endogenous Fish in NIH 3T3 cells, designated p150, p140 and p130, are also indicated. (B) Fish is tyrosine-phosphorylated to be a direct substrate of Src, we used Rat1 fibroblasts in 293 cells which coexpress active Src. Lysates of 293 cells transformed with a temperature-sensitive version of v-Src. transfected with either NmycFish cDNA alone, encoding an At the non-permissive temperature, a low basal level of N-terminal myc epitope-tagged form of Fish, or in combination with tyrosine phosphorylation of Fish was detected. However, cDNAs for SrcK, SrcK– or SrcY527F, as indicated, were analysed by SDS–PAGE and immunoblotting with anti-Fish.2 (upper panel) and within 10 min of placing the cells at the permissive anti-Src (EC10; middle panel). Anti-Fish.2 immunoprecipitates from temperature, the tyrosine phosphorylation of Fish these lysates were analysed by SDS–PAGE and immunoblotting with increased, and was maximal within 1 h of the shift (Figure anti-pTyr (lower panel). 5B). These kinetics are similar to those seen for the re- activation of Src kinase activity (S.A.Courtneidge, unpub- lished observations). proteins. We are currently characterizing these proteins Fish contains several motifs and domains that are known further, although our preliminary analyses have ruled out Ctn to be involved in protein–protein interactions; we therefore rasGAP, Fak, AFAP-110 and p120 . looked for Fish-associated proteins. Fish isolated from NIH Many proteins phosphorylated on tyrosine in Src-trans- 3T3 cells was not associated with tyrosine-phosphorylated formed cells are also transiently phosphorylated in normal proteins (Figure 5C). In contrast, in Src-transformed cells in response to growth factors or other stimuli. We cells Fish immunoprecipitates contained several tyrosine therefore asked whether growth factor stimulation resulted phosphoproteins. Several of these proteins, most notably in tyrosine phosphorylation of Fish. Our initial tests used those of 125, 115 and 65 kDa, were recognized by two NIH 3T3 cells, where we were unable to find convincing different antibodies raised against different regions of evidence for Fish phosphorylation in response to a variety Fish, and are therefore likely to represent Fish-associated of stimuli (data not shown). However treatment of Rat1 4350 Fish, a new adaptor protein Fig. 5. Phosphorylation of Fish in Src-transformed NIH 3T3 cells and temperature-sensitive v-Src-transformed fibroblasts, and association with tyrosine-phosphorylated proteins. (A) Fish is tyrosine-phosphorylated in Src-transformed NIH 3T3 cells. SDS lysates prepared from NIH 3T3 cells (Cl7) and SrcY527F-transformed NIH 3T3 cells (527) were immunoprecipitated either with rabbit Ig or anti-Fish.2 antibody, and analysed by SDS–PAGE and immunoblotting with anti-pTyr (upper panel) or anti-Fish.2 (lower panel). The apparent molecular weight of the tyrosine- phosphorylated species is 150 kDa. (B) Fish is tyrosine-phosphorylated in temperature-sensitive v-Src transformed fibroblasts following a shift to the permissive temperature. Lysates were prepared from ts68.10 cells following a time course of shifting from the non-permissive temperature (34.5°C) to the permissive temperature (39.5°C) and immunoprecipitated with anti-Fish.2 antibody, followed by analysis by SDS–PAGE and immunoblotting with anti-pTyr (upper panel) or anti-Fish.2 (lower panel). The apparent molecular weights of the two forms of Fish detected in Rat1 cells are 140 and 150 kDa; only p150 is tyrosine-phosphorylated. (C) Fish is associated with several tyrosine-phosphorylated proteins. Lysates prepared from NIH 3T3 cells (Cl7) or SrcY527F-transformed NIH 3T3 cells (527) were immunoprecipitated with anti-Fish.1 or anti-Fish.2 antibodies and analysed by SDS–PAGE. Proteins were transferred to nylon and immunoblotted with anti-pTyr. Tyrosine-phosphorylated proteins that appear to co-associate with Fish in immunoprecipitates with both antibodies (bands at ~65, ~115 and ~125kDa) are indicated by arrows. fibroblasts with PDGF (Figure 6), LPA and bradykinin (data not shown) did lead to an increase in tyrosine phosphorylation of Fish. The kinetics of this phosphoryl- ation was quite slow, with increases still being detected 2 h after stimulation. It will be interesting to determine whether tyrosine phosphorylation of Fish has a functional Fig. 6. Fish is tyrosine-phosphorylated in Rat1 cells in response to role in the cell’s response to growth factors. PDGF. Lysates prepared from Rat1 cells following treatment with One response commonly elicited by growth factors, and 50 ng/ml PDGF for the times indicated were immunoprecipitated with anti-Fish.2 antibodies and analysed by SDS–PAGE. Proteins were frequently involving proteins with SH3 domains and PX transferred to nylon and immunoblotted with anti-pTyr (upper panel) domains, is reorganization of the cortical actin cyto- and anti-Fish.2 (lower panel). The apparent molecular weights of the skeleton. Indeed, as shown in Figure 7A, disruption of two forms of Fish detected in Rat1 cells are 140 and 150 kDa; only the actin cytoskeleton by treatment with cytochalasin D p150 is tyrosine-phosphorylated. (cytD) resulted in a dramatic change in the profile of tyrosine-phosphorylated proteins, with both increases and decreases in phosphorylation being detected. One set of on the level of Src in the cell, but it did lead to a proteins which showed increased phosphorylation had modest increase in its intrinsic kinase activity (Figure 8), molecular weights in the range of 150 kDa. Immuno- suggesting that it is activated under the same conditions precipitation and anti-phosphotyrosine immunoblotting that we detected Fish phosphorylation. We have detected confirmed that these were the Fish proteins (Figure 7B, this activation using both a Src-specific monoclonal anti- left panel). Identical results were obtained when latrunculin body and anti-cst.1, an antibody that recognizes Src, Fyn B (a marine toxin that also disrupts the actin cytoskeleton) and Yes. The average activation that we detected in five was used (data not shown). Further analysis showed that separate experiments was 1.8-fold. Fish proteins became phosphorylated within 2 min of cytD treatment (Figure 7B, right panel), and at sub- Discussion micromolar concentrations of the drug (Figure 7C). These results strongly suggest that the tyrosine phosphorylation Despite the large number of substrates for Src already of Fish is connected to the integrity of the actin cyto- described in the literature, several proteins phosphorylated skeleton. Src-transformed cells have a disorganized actin on tyrosine in Src-transformed cells remain uncharacter- cytoskeleton, and many of the known Src substrates are ized, and many important targets may have so far gone cytoskeletal proteins. Yet to our knowledge, there have undetected because of their low abundance. We therefore been no reports that disruption of the actin cytoskeleton sought a method to detect substrates for Src that would not can activate Src. To examine this, we measured Src activity, rely on association with Src or relatively high abundance in both by autophosphorylation and enolase phosphorylation, the cell, and that would allow rapid cloning of the from untreated and cytD-treated cells. cytD had no effect cDNAs encoding them. The method we devised requires 4351 P.Lock et al. Fig. 7. Fish is tyrosine-phosphorylated in response to treatment with cytochalasin D. (A) Treatment of NIH 3T3 cells with cytD results in changes in total cellular phosphotyrosine. Lysates from NIH 3T3 cells treated with either DMSO alone or 10 μM cytD for 10 min were analysed by SDS–PAGE. Proteins were transferred to nylon and immunoblotted with anti-pTyr. Black arrows indicate bands that have increased following cytD treatment and white arrow heads indicate bands that have decreased. The two panels derive from the same immunoblot, with the top panel being a lighter exposure than the bottom. (B) Fish is tyrosine-phosphorylated rapidly following cytochalasin D treatment. Lysates from NIH 3T3 cells treated with either DMSO alone or 10 μM cytD for 10 min were immunoprecipitated with anti-Fish.2 antibodies, analysed by SDS–PAGE and immunoblotted with anti-pTyr (left panel). NIH 3T3 cells were treated with 10 μM cytD for the times indicated, and lysates were prepared and immunoprecipitated with anti-Fish.2 antibodies. Proteins were analysed by SDS–PAGE and immunoblotted with anti-pTyr (right panel). (C) Fish is tyrosine-phosphorylated following treatment with low concentrations of cytochalasin D. Lysates were prepared from NIH 3T3 cells treated with the different concentrations of cytD as indicated for 15 min , and immunoprecipitated with anti-Fish.2 antibodies. Proteins were analysed by SDS–PAGE, and immunoblotted with anti-pTyr (upper panel) and anti-Fish.2 (lower panel). a bacteriophage expression library, an enriched source of cells (C.Abram and S.A.Courtneidge, unpublished data). Src kinase (e.g. baculovirus-infected insect cell extracts) All of the cDNA products that were identified contained and phosphotyrosine antibodies. Using this approach we at least one consensus Src phosphorylation site (Songyang identified cDNAs for several candidate Src substrates. and Cantley, 1995), suggesting that enzyme specificity is One of the cDNA clones, Tks-7, encoded a fragment of retained under the conditions we used to phosphorylate Cas a known Src substrate, p130 (Sakai et al., 1994), the cDNA library. This is perhaps not surprising, since implying that the method is a valid way to detect tyrosine substrate recognition by tyrosine kinases, like SH2 domain kinase substrates. Intriguingly, several of the cDNAs specificity, appears to be dictated by very few residues identified by this method encode proteins with SH3 surrounding the phosphorylation site and is therefore likely Cas domains (p130 , SH3P7, s19, CIP4/STP and Fish), to be independent of substrate conformation (Songyang although their SH3 domains per se were clearly not and Cantley, 1995). We believe that this method will required to facilitate phosphorylation by Src and sub- be generally applicable, using expression libraries from sequent detection since none of the Tks cDNAs encode different tissues and the tyrosine kinase of choice. Indeed, an intact SH3 domain (data not shown). an analogous method to ours for identifying substrates of The Tks-5 clone, encoding Fish, is characterized further serine/threonine as well as tyrosine kinases was reported here. We identified Fish as an in vitro substrate for Src recently (Fukunaga and Hunter, 1997). The MAP kinase both when expressed as a β-galactosidase fusion protein ERK1 was used to phosphorylate a HeLa cell cDNA (Figure 1) and as a GST fusion protein when incubated library in the presence of [γ- P]ATP and positive clones with a Src immunoprecipitate (data not shown). Fish were detected by autoradiography. Using this method a was also tyrosine-phosphorylated in 293 cells when co- number of known substrates of MAP kinases, as well as expressed with an activated form of Src, and endogenous the novel kinase MNK1, were identified. In contrast to Fish was phosphorylated in Src-transformed NIH 3T3 this method, we used enriched rather than purified protein cells. Furthermore, tyrosine phosphorylation of Fish kinase for the phosphorylation step. In addition, we used increased within minutes of transferring cells containing a commercial λgt11 cDNA library while Fukunaga and a temperature-sensitive form of v-Src from non-permissive Hunter (1997) constructed a library using a novel expres- to permissive temperature. These findings suggest that sion vector. Fish is indeed a direct substrate of Src in Src-transformed cells. We are currently analysing several other proteins Fish—a new adaptor protein identified by this method, and have found that at least Fish is a new, broadly expressed adaptor protein, two others are substrates for activated Src in mammalian which appears to associate with several proteins in 4352 Fish, a new adaptor protein bind to the same or related ligands, although the binding specificities of the Fish SH3 domains remain to be determined. Interestingly, the Fish PX domain is also most phox related to that of p47 suggesting that these proteins may be the products of genes which arose from a common ancestor. The region of Fish encoded by the Tks-5 cDNA contained three consensus Src phosphorylation sites, desig- nated Y552, Y557 and Y619 (see Figure 2A). These sites conform to consensus binding sites for the SH2 domains of Abl (Y-E-E-P), Nck or Crk (Y-D-X-P), and Grb2 (Y-X- N-X), respectively (Songyang et al., 1993). Conceivably, tyrosine-phosphorylated forms of Fish, such as those found in Src-transformed fibroblasts, could associate with one or more of these proteins in vivo. As well as SH3 domains, Fish also has three polyproline segments which encompass one or more P-X-X-P motif that could allow it to bind to SH3 domains (Pawson, 1995). Fish also has many potential sites of serine phosphorylation. Perhaps the most unusual feature of Fish is the presence at the N-terminus of a phox homology or PX domain. This domain, which is ~100–110 amino acids in length, has only recently been described (Ponting, 1996), and its function is not yet known. To our knowledge Fish is the Fig. 8. Src is activated by cytochalasin D treatment. (A) Src is first protein containing a PX domain to be implicated in activated by cytochalasin D treatment. Lysates of NIH 3T3 cells a tyrosine-kinase signalling pathway. PX domains have treated with either DMSO alone or 10 μM cytD for 10 min were several well conserved residues, many of them hydro- immunoprecipitated with anti-Src antibodies. The immunoprecipitates phobic, and two almost invariably conserved basic were subjected to an in vitro kinase assay using enolase as a substrate. Proteins were analysed by SDS–PAGE and autoradiography. (B) The residues, which may be important for function. The pattern level of Src is not altered by cytochalasin D treatment. Lysates of NIH of conserved hydrophobic residues strongly suggests that 3T3 cells treated with either DMSO alone, or 10 μM cytD for 10 min, the PX domain includes an N-terminal segment predomin- were immunoprecipitated with anti-Src antibodies. Proteins were antly composed of β-strand-like sequences and both central analysed by SDS–PAGE, transferred to nylon and immunoblotted with anti-cst.1. and C-terminal α-helical structures that are separated by a conserved proline-rich sequence of indeterminate Src-transformed cells. Three forms of Fish were detected secondary structure (Ponting, 1996; data not shown). It in NIH 3T3 cells. These forms are unlikely to be has been proposed that the proline-rich sequence might proteolytic breakdown products because they were constitute a binding site for SH3 domains although this detected even when cells were lysed in hot extraction has not been tested directly (Ponting, 1996). buffer containing SDS. They may occur as a result of Where studied, PX domain-containing proteins are often post-translational modifications, although this cannot be associated with processes involving the actin cytoskeleton, accounted for by tyrosine phosphorylation since Fish is membranes and/or GTP-binding proteins. For example the not detectably phosphorylated in non-transformed NIH Saccharomyces cerevisiae protein Bem1, and Scd2, its 3T3 cells. In addition, preliminary experiments suggest orthologue in Shizosaccharomyces pombe, appear to that neither serine/threonine phosphorylation nor glycos- coordinate rearrangement of the cortical cytoskeleton ylation can account for the three forms. The most likely during cell polarization in response to mating factors explanation is that they are encoded by different RNA (Chenevert et al., 1992; Chang et al., 1994). Bem1 interacts splice products. Indeed we identified two distinct cDNAs directly with Cdc24, a guanine nucleotide exchange factor for Fish in the embryonal cDNA library, the smaller of for the Rho-family GTPase Cdc42, and probably indirectly which encoded a protein of ~150 kDa when expressed with Cdc42 itself (Peterson et al., 1994). Similar inter- in COS cells. The p140 and p130 forms of Fish may actions have been reported for Scd2 in S.pombe (Chang be generated by other splice variations. The complexity et al., 1994). Bem1 has also been shown to interact with of Fish isoforms is also evident in different cell types. Ste20p, a serine/threonine kinase belonging to the p21- For example in human platelets a single band of 150 activated kinase (PAK) family, as well as to actin (Leeuw kDa was detected, whereas all three forms were found et al., 1995). Another bud emergence gene, Bem3, also in human fibroblasts and vascular smooth muscle cells has a PX domain (Zheng et al., 1994). Interestingly, Bem3 (data not shown). Rat1 fibroblasts have p150 and p140 is a GTPase activating protein (GAP) for certain Rho- forms (Figure 6). The fact that in all of these cases family proteins indicating that, like Bem1, it also interacts two different antibodies raised against the original with GTP binding proteins. Two components of the phox murine Fish sequence recognized each of these proteins neutrophilic NADPH oxidase complex, p40 and phox confirms that they are indeed isoforms of Fish. p47 , each have a PX domain very similar to that of Fish contains many potential binding sites for other Fish. Activation of the oxidase, and production of super- proteins; the most notable are the five SH3 domains. oxide and peroxide in response to invading microorgan- Strikingly, all five are most related to each other and to isms, or to agonists such as fMLP, involves recruitment phox phox phox the p47 SH3 domains, suggesting that they may all of the cytoplasmic proteins p40 and p47 (and other 4353 P.Lock et al. phox proteins including p67 and the small GTP-binding posed from the observation that it is most closely related to protein Rac2) to the plasma membrane, where they associ- the cytosolic NADPH oxidase components of neutrophils, phox phox ate with membrane-bound flavocytochrome b (Segal p47 and p40 . Interestingly, a recent report described and Abo, 1993; Dorseuil et al., 1996). Significantly, the identification of Posh, a Rac-interacting adaptor protein oxidase activation is concomitant with phagocytosis, a with four SH3 domains, which has a striking similarity to phox membrane process requiring rearrangement of the cortical p67 (Tapon et al., 1998). The expression of the p47 actin cytoskeleton (Hall, 1992). Recently a new PI 3-K and p67 proteins is restricted to phagocytic cells (Segal was described in mouse and Drosophila (variously known and Abo, 1993), whereas both Posh and Fish are more as Cpk, 68 D or p170) that also contains a PX domain broadly expressed. In neutrophils, the respiratory burst (MacDougall et al., 1995; Molz et al., 1996; Virbasius can be initiated by the chemoattractant and G-protein- et al., 1996). Another enzyme which contains a PX domain coupled receptor agonist fMLP (Segal and Abo, 1993). and is involved in membrane-associated phospholipid However, superoxide formation has also been observed in metabolism is phospholipase D1 (Ponting and Parker, many other cell types, and has indeed been linked to 1996). Finally, other PX domain-containing proteins with carcinogenesis (Burdon, 1995). One exciting possibility known functions include Vam7, which is involved in is that Fish forms part of the superoxide-generating vacuolar morphogenesis (Wada and Anraku, 1992), Mvp1 machinery in non-phagocytic cells, a hypothesis which is and Vps17, which are involved in sorting of proteins to currently being tested. vacuoles (Kohrer and Emr, 1993; Ekena and Stevens, 1995) and Mdm1, which is involved in organelle inheritance Materials and methods (McConnell and Yaffe, 1992). It is hard to predict from the sequence of the PX domain what it might interact with; Phosphorylation screening both phospholipids and proteins are candidate ligands. It The phosphorylation screening method we used to identify Src substrates was adapted from a technique designed to identify binding partners of is now possible to undertake functional studies of PX the Shc phosphotyrosine binding (PTB) domain (Kavanaugh et al., domain-containing proteins bearing targeted mutations, as 1995). In the primary screen for Src substrates, 210 individual clones well as binding studies of the isolated domains in vitro, from a λgt11 library containing mouse Swiss 3T3 fibroblast cDNA in order to resolve this issue. (Clontech; ML1023b) were plated on four 150 mm Petri dishes. Plates were overlaid with nitrocellulose filters impregnated with 10 mM Fish was tyrosine-phosphorylated at cytochalasin D isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated at 37°C for concentrations that disrupt the cortical actin cytoskeleton, 16 h to induce expression of recombinant lacZ fusion proteins. Replica and also in Src-transformed fibroblasts, which have a filters were washed extensively in TBST (10 mM Tris–HCl pH 7.4, dramatically altered actin cytoskeleton. Furthermore, Fish 150 mM NaCl, 0.1% Triton X-100) then equilibrated for1hin kinase was tyrosine-phosphorylated in response to treatment of buffer (TBST containing 10 mM MgCl and 2 mM MnCl ). Filters were 2 2 blocked for 1 h at room temperature in kinase buffer containing 3% Rat1 fibroblasts with either PDGF, LPA or bradykinin. bovine serum albumin (BSA). Recombinant proteins were phosphorylated Each of these mitogens activates the Rho family of small by incubating filters at 30°C for 60 min in kinase buffer supplemented GTP binding proteins and causes changes in the actin with one tenth volume of an Sf9 extract containing baculovirus-derived cytoskeleton such as stress fibre, lamellipodia and filopodia human Src, 250 μM ATP and 100 μM sodium orthovanadate. Filters were washed briefly with kinase buffer alone then incubated in stripping formation (Hall, 1998). These data suggest that Fish may buffer (62.5 mM Tris–HCl pH 7.0, 2% SDS, 100 mM 2-β-mercapto- play a role in cytoskeletal changes. ethanol) at 50°C for 30 min to remove possible associated phospho- Many of the Src substrates identified so far are cyto- proteins, including Src itself or Sf9 cell-derived proteins, which might skeletal proteins, including p110 AFAP, paxillin, vinculin interfere with the screening. Tyrosine-phosphorylated proteins were detected with an anti-phosphotyrosine monoclonal antibody, 4G10, using and cortactin, which has led to the suggestion that in standard immunoblotting methodology (see below). Twelve positive transformed cells Src can regulate cytoskeletal structures clones were plaque-purified by successive rounds of phosphorylation (Brown and Cooper, 1996). In normal cells, Src is activated screening. The cDNA inserts were amplified by polymerase chain by mitogens such as PDGF, and is required for DNA reaction (PCR) using Taq DNA polymerase (Boehringer Mannheim) and synthesis in response to several growth factors (Erpel subcloned into pCRII (Invitrogen Corp.). The cDNA inserts were sequenced with the aid of T7 and SP6 oligonucleotide primers. and Courtneidge, 1995). More recently, it has also been reported that PDGF stimulation of cells results in the Isolation of a full-length Fish cDNA translocation of Src to regions of the cell periphery that To identify the full-length Fish cDNA, a murine day 11.5 embryo cDNA also show dense actin staining. This effect was seen within library (Clontech, ML1027b) (a generous gift of G.Plowman, SUGEN) 30–60 min of PDGF addition, and required an intact actin was screened by hybridization with a 1 kb P-labelled DNA fragment corresponding to the Tks-5 cDNA that was isolated during the initial cytoskeleton and the activity of Rho family proteins screen for Src substrates. Two cDNA clones were identified, λME14 (Fincham et al., 1996), suggesting that the activity of Src and λME5E, which contained ~1.3 kb of additional 5 sequence and may be under the control of the actin cytoskeleton. By 0.5 kb of 3 sequence relative to Tks-5, respectively. Radiolabelled DNA this mechanism, the cytoskeleton could control the access probes corresponding to fragments at the 5-end of λME14 and the 3- of Src to substrates such as Fish, which might explain the end of λME5E were then used to rescreen the embryo cDNA library. A cDNA designated λMEA3C was identified which contains an additional rather slow kinetics of Fish phosphorylation following 0.3 kb of sequence at the 5-end relative to λME14, including a PDGF stimulation. Alternatively, Fish could be on a presumptive translational initiation codon. Two non-identical clones, pathway downstream of Src that controls the activity of λME14(3) and λME5A, were found to contain ~0.6 kb of additional Rho family proteins and their effect on the actin cytoskel- 3-sequence relative to λME5E including two stop codons at the 3-end of the Fish coding sequence. λMEA3C and λME14 were shown to differ eton. In this regard, it is interesting that tyrosine kinases with respect to putative alternative exons of 45 and 84 bp which are are proposed to participate both upstream and downstream present in λME14 but absent from λMEA3C, indicating the existence of Rho proteins (Ridley and Hall, 1994; Nobes et al., of at least two alternatively spliced transcripts. Two Fish cDNA sequences 1995; Kranenburg et al., 1997). were predicted on the basis of the overlapping cDNAs identified. The An alternative potential function for Fish can be pro- amino acid sequence of the corresponding Fish isoforms, which include 4354 Fish, a new adaptor protein both the 15 and the 28 residue insertions, or lack these sequences, permissive temperatures used in experiments with ts68.10 cells were respectively, was deduced on the basis of these cDNAs. 34.5 and 39.5°C, respectively. Cells were treated with 50 ng/ml PDGF (UBI) at 37°C for the times stated. Extracts were prepared from NIH Analysis of RNA expression 3T3, 527, 293, ts68.10, Rat1 or COS cells, by washing the adherent A multiple tissue Northern blot containing 2 μg of poly(A) RNA from cells with ice cold TBS (25 mM Tris–HCl pH 7.5, 150 mM NaCl) various adult mouse tissues (Clontech) was hybridized with a P- containing 100 μMNa VO and 2 mM dithiothreitol (DTT). Cells were 3 4 labelled DNA probe corresponding to the 1 kb Tks-5 cDNA (shown in lysed in 0.5 to 2.0 ml of either NP40 lysis buffer (20 mM HEPES Figure 2A) under high stringency conditions (65°C, 0.1 SSC, 0.1% pH 7.0, 150 mM NaCl, 1% Nonidet P40) or RIPA lysis buffer (20 mM SDS). The blot was stripped according to the suppliers protocol and Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, rehybridized with a P-labelled β-actin probe. 0.1% SDS) containing 100 μMNa VO , 10 mM NaF, 2 mM DTT, 3 4 10 μg/ml aprotinin, 20 μM leupeptin and 100 μM phenylmethylsulfonyl Constructs fluoride (PMSF) for 10–15 min at 4°C. Extracts were clarified by Expression plasmids encoding wild-type and epitope-tagged versions of centrifugation at 10 000 g for 10 min and the protein concentration the smaller Fish isoform (see above) were constructed. For the wild- determined. Extracts of Sf9 cells expressing human Src baculovirus were type construct, the following DNA fragments were subcloned into the prepared essentially as described above except that the cells were NotI and XbaI sites of pBluescript II KS (pBSIIKS, Stratagene): a harvested by centrifugation in 50 ml tubes, prior to lysis. For immunopre- 1423 bp EagI–EcoRI fragment from λMEA3C which contains 54 bp of cipitations, samples containing 150–400 μg of total protein were incub- 5-untranslated region (UTR) and sequences encoding residues 1–412; ated with either 1–2 μl of crude pre-immune serum or antiserum, or a 566 bp EcoRI–SacI fragment from λME14 which encodes residues 1 μg of affinity-purified antibody, and ~7 μl packed volume of protein 413–602; and a 1548 bp SacI partial XbaI fragment originating from A–Sepharose beads (Pharmacia) or protein A–agarose beads (Santa λME14(3) which codes for amino acids 603–1081, contains 80 bp of Cruz) for 1 h at 4°C. Immunocomplexes were washed four times in ice 3-UTR and an additional 36 bp of polylinker sequences from pBSIIKS. cold RIPA buffer containing 100 μMNa VO and 1–2 mM DTT. 3 4 The resulting plasmid, pBS-Fish, was digested with EagI and XbaI, Samples were resuspended in SDS sample buffer (80mM Tris pH 6.8, releasing a 3414 bp fragment encompassing the complete Fish coding 2% SDS, 75 mM DTT, 10% glycerol, 1.25% Bromophenol Blue), heated to 95°C for 5 min and subjected to SDS–PAGE using 7.5 or 9% region. Recessed 3-termini were filled using Klenow enzyme polyacrylamide gels. Kinase assays were carried out as described (Boehringer Mannheim), and BstXI adaptors (Invitrogen) were ligated previously (Kypta et al., 1990). Briefly, 50 μg lysate was immunoprecipit- onto the ends. The fragment was subcloned into the BstXI sites of ated with 1 μg of anti-Src antibody (327) and 20 μl protein A/G plus pEF-BOS (Mizushima and Nagata, 1990) in the forward and reverse agarose (Santa Cruz) followed by washing four times with ice cold orientations to generate the plasmids pEF-BOS Fish() and pEF-BOS RIPA buffer containing 100 μMNa VO and 1 mM DTT, and once with Fish(–), respectively. The plasmid pEF-BOS NmycFish, encoding two 3 4 kinase buffer (20 mM HEPES pH 7.4, 10 mM MgCl , 2 mM MnCl , copies of the myc epitope recognized by the 9E10 antibody fused to the 2 2 1 mM DTT). The reactions were carried out in 20 μl kinase buffer N-terminus of Fish, was generated by ligating the following fragments containing 15 μM unlabelled ATP, 3 μg acid-denatured enolase and into the BamHI and XbaI sites of pEF-BOS Nmyc (a gift from P.Orban, 10μCi [γ- P]ATP for 10 min at 30°C, and stopped with SDS-sample EMBL, Germany): a 294 bp BglII–ClaI fragment derived by PCR using buffer. The samples were analysed by SDS–PAGE and autoradiography Vent polymerase (New England Biolabs), which encodes Fish residues using Fuji RX film. 1–96; a 950 bp ClaI–EcoRI fragment encoding residues 97–412; and a 2119 bp EcoRI–XbaI fragment encoding residues 413–1081 and including Immunoblotting 80 bp of 3-UTR plus 36 bp of polylinker sequences. SDS–PAGE gels were electrophoretically transferred to nitrocellulose filters (Scleicher and Schuell) or PVDF (Millipore) using a Millipore Antibodies and peptides semi-dry blotting aparatus. Filters were incubated in blocking solution The Fish-specific polyclonal antiserum, Fish.1, was generated by (either PBS or TBS containing 0.1% Tween 20 and 2–3% BSA) for 1 h immunising rabbits with a purified glutathione S-transferase (GST) fusion at room temperature. Filters were washed three times in wash solution protein containing residues 457–787 of Fish. A second polyclonal anti- (PBS or TBS containing 0.1% Tween 20) and incubated with blocking Fish antibody, Fish.2, was generated by immunising rabbits with a GST solution plus the primary antibody for1hat room temperature using fusion protein containing residues 807–908. Fish.2 antiserum was affinity the following antibody dilutions: α-pTyr, 1:1000–1500; α-Fish.1, 1:7500– purified by passing over a column containing the GST fusion protein, 10 000; α-Fish.2, 0.5 μg/ml; EC10, 2 μg/ml; α-cst.1, 1:200. Filters were coupled to 3M Emphaze™ Biosupport Medium AB1 (Pierce) according washed three times in wash solution and incubated with the secondary to manufacturer’s instructions. The anti-phosphotyrosine (α-pTyr) mono- detection reagent for1hat room temperature. For blots probed with a clonal antibody 4G10 was from Upstate Biotechnology Incorporated rabbit polyclonal antibody, filters were incubated with blocking solution (UBI). The mouse monoclonal antibodies, EC10 (UBI) and 327 (vSrc-1, containing either protein A-horseradish peroxidase (HRP) conjugate Calbiochem) and the rabbit polyclonal antibody, anti-cst.1 (Courtneidge (Amersham) at a dilution of 1:1000–2500 or donkey anti-rabbit HRP and Smith, 1984) were used to detect Src. (Amersham) at 1:20 000. For blots probed with mouse monoclonal antibodies, filters were incubated with sheep anti-mouse HRP conjugate Cell culture, baculoviral infection and DNA transfection (Amersham) at a dilution of 1:5000. Filters were washed three times in COS cells were grown in RPMI 1640 medium containing 10% fetal calf wash solution and the protein bands detected using enhanced chemi- serum (FCS) and antibiotics at 37°C in 5% CO . 293 cells, Rat1 cells, luminescence (ECL, Amersham or Supersignal, Pierce) in conjunction ts68.10 cells which express a temperature-sensitive mutant of Src with Fuji RX film. (Courtneidge and Bishop, 1982), NIH 3T3 cells and their derivative, 527 cells, which stably express a mutant form of chicken Src with an EMBL Accession Number activating Y527F substitution, were grown in Dulbecco’s modified The accession number for the Fish cDNA sequence is AJ007012. Eagle’s medium (DMEM) containing 10% FCS and antibiotics at 37°C in 10% CO . Sf9 insect cells were maintained in Grace’s medium Acknowledgements containing 7.5% FCS at 27°C. COS cells were transfected by electropor- ation. Briefly, 210 cells were transfected with 10 μg of pEF-BOS We thank A.Verhagen and G.Superti-Furga for many helpful discussions. Fish() or pEF-BOS Fish (–). Transfectants were analysed ~40 h post- Thanks to members of the EMBL DNA sequencing and animal facilities transfection. 293 cells were transfected with Lipofectamine™ (Gibco- for sequence analysis and in raising antisera, respectively. Thanks BRL) according to manufacturer’s instructions. 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(1991) Identification and characterization of a novel cytoskeleton-associated pp60src substrate. Mol. Cell. Biol., 11, 5113–5124. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX domain‐containing protein, and Src substrate

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Copyright © European Molecular Biology Organization 1998
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Abstract

The EMBO Journal Vol.17 No.15 pp.4346–4357, 1998 A new method for isolating tyrosine kinase substrates used to identify Fish, an SH3 and PX domain-containing protein, and Src substrate 1,2 3 1 Kalff et al., 1992). In normal cells, Src family kinases act Peter Lock , Clare L.Abram , Toby Gibson 1,3,4 in signal transduction cascades. Typically they transduce and Sara A.Courtneidge signals from cell surface receptors, of both the kinase European Molecular Biology Laboratory, 69012 Heidelberg, Germany and non-kinase type (Erpel and Courtneidge, 1995), and and SUGEN Inc., 351 Galveston Drive, Redwood City, CA 94063, mediate the activation of a variety of pathways, including USA those involving the mitogen-activated protein (MAP) kin- Present address: The Ludwig Institute for Cancer Research, ases (Dikic et al., 1996; Luttrell et al., 1996; Sadoshima Royal Melbourne Hospital, Parkville 3052, Australia and Izumo, 1996), the phosphatidylinositol 3-kinase (PI Corresponding author 3-K) (Cantley et al., 1991), and Myc (Barone and e-mail: [email protected] Courtneidge, 1995). P.Lock and C.L.Abram contributed equally to this work Over the years, many putative substrates of Src have been identified by a variety of methods, including direct We describe a method for identifying tyrosine kinase analysis of candidate proteins such as tensin (Davis et al., substrates using anti-phosphotyrosine antibodies to 1991) and paxillin (Turner and Miller, 1994), the use of screen tyrosine-phosphorylated cDNA expression lib- phosphotyrosine antibodies for proteins such as actin raries. Several potential Src substrates were identified filament associated protein (AFAP-110; Flynn et al., 1993), ctn including Fish, which has five SH3 domains and a cortactin (Wu et al., 1991), p120 (Reynolds et al., 1992), Cas dok recently discovered phox homology (PX) domain. Fish p130 (Sakai et al., 1994) and p62 (Carpino et al., is tyrosine-phosphorylated in Src-transformed fibro- 1997; Yamanashi and Baltimore, 1997), and the analysis blasts (suggesting that it is a target of Src in vivo) and of Src-associated proteins such as Sam68 (Fumagalli et al., in normal cells following treatment with several growth 1994; Taylor and Shalloway, 1994) and Efs (Ishino et al., factors. Treatment of cells with cytochalasin D also 1995). Many substrates are cytoskeletal or membrane resulted in rapid tyrosine phosphorylation of Fish, components (Davis et al., 1991; Wu et al., 1991; Rothberg concomitant with activation of Src. These data suggest et al., 1992; Flynn et al., 1993; Turner and Miller, 1994), that Fish is involved in signalling by tyrosine kinases, others are enzymes (Cobb et al., 1994), and others adaptor and imply a specialized role in the actin cytoskeleton. or docking proteins (McGlade et al., 1992; Sakai et al., Keywords: cytochalasin D/Fish/PX domain/Src/tyrosine 1994; Ishino et al., 1995; Carpino et al., 1997; Yamanashi kinase substrate and Baltimore, 1997). Yet, despite the seeming plethora of substrates, it seems likely that physiologically relevant substrates remain undiscovered; for example, there are mutant forms of Src which seem able to phosphorylate Introduction all known substrates, yet fail to transform (Erpel et al., 1995). Perhaps some relevant substrates are not stably The viral Src protein (v-Src) was discovered more than associated with Src, or are not abundant in cells, and were 20 years ago. In the intervening years intense study of therefore missed by the available strategies. We therefore both v-Src and its cellular counterpart, c-Src, has focused sought a new way to identify tyrosine kinase substrates. on issues of regulation, transforming activity and physio- logical function. The Src family of protein tyrosine kinases comprises Results eight members in mammals, some broadly expressed, some restricted to certain tissues (Cooper, 1990; Bolen A method to identify tyrosine kinase substrates et al., 1992). All are tightly regulated in vivo; catalytic We wanted to develop a method for identifying substrates activity is repressed by interactions involving the SH3 for tyrosine kinases that could be applied to a variety of domain, the SH2 domain, and the phosphorylated kinases and cell types, and that was based on a cDNA C-terminus (Superti-Furga and Courtneidge, 1995). Recent expression system so that sequence information was imme- crystal structures of Src and Hck have revealed exactly diately available. We therefore chose to adapt a method, how this repression takes place (Sicheri et al., 1997; described by Kavanaugh et al (1995), that was used to Williams et al., 1997; Xu et al., 1997). Morphological identify binding partners for the Shc phosphotyrosine- transformation is frequently the result of the expression binding (PTB) domain. The method in Figure 1A and of catalytically de-repressed forms of Src family kinases, described in detail in Materials and methods, consisted of especially in fibroblasts (Cooper, 1990). There is also a phosphorylating a λgt11 cDNA expression library, derived large body of circumstantial evidence supporting the view from mRNA of mouse Swiss 3T3 fibroblasts, with Src that hyperactive Src family kinases may play a role in kinase, and then detecting phosphorylated proteins with several human epithelial malignancies, particularly of the an anti-phosphotyrosine antibody. Successive rounds of colon and breast (Cartwright et al., 1989, 1990; Ottenhoff- phosphorylation and antibody screening (Figure 1B) were 4346 © Oxford University Press Fish, a new adaptor protein Fig. 1. Identification of potential substrates of Src. (A) Phosphorylation screening protocol. Petri dishes containing a Swiss 3T3 cell cDNA expression library in λgt11 were overlaid with nitrocellulose filters containing IPTG and incubated for 16 h to induce expression of recombinant proteins. Replicate filters were incubated with extracts containing baculovirally expressed human Src kinase and ATP. Individual plaques containing tyrosine-phosphorylated protein were identified with an anti-phosphotyrosine (anti-pTyr) antibody using a standard immunoblotting protocol. Positive cDNA clones were purified by successive rounds of phosphorylation with Src kinase and immunodetection with anti-pTyr antibody. (B) Immunodetection of positive plaques. Immunoblot analysis of filters with an anti-pTyr antibody during (i) primary and (ii and iii) secondary rounds of phosphorylation screening showing various positive clones (arrow heads). used to plaque-purify several cDNAs, and the inserts were ation sites, it contained part of an SH3 domain. We chose then sequenced. to analyse this clone further. Several of these clones, which we have named Tks (tyrosine kinase substrates), are listed in Table I. Concep- Identification of Fish, a novel multi-SH3 tual translation showed that each insert corresponded to a domain-containing protein partial cDNA with an open reading frame containing at Several overlapping cDNA clones encompassing the entire least one, and usually several, motifs which closely coding region of Tks-5 were isolated from a mouse resemble the consensus sequence, EEEIYG/EEFD, for embryonal cDNA library with a probe corresponding to phosphorylation by v-Src kinase (Songyang and Cantley, the original Tks-5 clone. These cDNAs represented two Cas 1995). One clone, Tks-7, was identical to part of p130 , distinct transcripts (data not shown), encoding isoforms a known substrate of both Src and focal adhesion kinase, of 1124 and 1081 residues (Figure 2A). The larger and a Crk binding protein (Sakai et al., 1994; Polte and isoform contained two insertions of 15 and 28 amino Hanks, 1995; Harte et al., 1996). Tks-1, Tks-2 and acids (underlined), which were absent in the smaller Tks-9 correspond to partial cDNAs for recently described isoform. The predicted translational start site, which is proteins with C-terminal SH3 domains. Tks-1 encodes a shared by both transcripts, is preceded by stop codons in portion of a mouse cDNA product (designated s19) which all three reading frames and conforms quite closely to the was identified as a Src SH3-domain binding protein consensus for translation initiation (Kozak, 1991). It is phox (Yamabhai and Kay, 1997). Tks-2 corresponds to part of noteworthy that the initiator methionine of p47 , the SH3P7, a protein identified on the basis of its ability to protein most closely related to that described here, is in interact with SH3-binding peptide ligands (Sparks et al., the analogous position (see Figure 2C). These data strongly 1996). Tks-2 also shares significant sequence similarity suggest that this residue is indeed the translational start with a neuronal actin binding protein, drebrin E (Toda site. Both predicted isoforms have five SH3 domains et al., 1993). Tks-9 corresponds to the mouse homologue (boxed), a phox homology (PX) domain (overlined), and of human CIP4, a proposed target protein of the activated consensus sequences for SH3 domain binding (double form of the Rho-family GTPase Cdc42 (Aspenstrom, underlined) and Src phosphorylation (ringed). The 15 1997) and of a rat protein, STP, that was identified amino acid insertion (underlined) occurs between the PX independently as a possible regulator of cation transport domain and the first SH3 domain, and the 28 amino acid in yeast (Tsuji et al., 1996). A portion of CIP4 was also insertion (underlined) is between the first and second SH3 described as Trip10, a thyroid hormone receptor interacting domains. The original λgt11 clone is encompassed by the protein (Lee et al., 1995). Tks-3 was identical to a arrows. Outside of the described domains, the amino portion of Mem3, a protein encoded by a mouse maternal acid sequence shows no significant homology with other embryonic mRNA, which is related to a yeast vacuolar proteins in any accessible databases (data not shown) and sorting protein (Hwang et al., 1996). The Tks-5 and Tks- is therefore unlikely to contain any catalytic domain. We 14 clones encoded novel proteins. Inspection of Tks-5 have named the protein encoded by these clones Fish (five showed that in addition to three possible Src phosphoryl- SH3 domains). 4347 P.Lock et al. phox The overall topography of Fish is shown schematically other and to the two SH3 domains of p47 (Volpp et al., in Figure 2B, where it is compared with other proteins 1989), as shown in Figure 2D. Pairwise comparison of which also contain PX and/or SH3 domains. The PX the Fish SH3 domains indicate that they share 27.5–48% domain, whose function is as yet unknown, was recently identity with each other, 24–47% identity with the two phox described as a conserved domain in many eukaryotic SH3 domains of p47 , but only 13.7–23.5% identity proteins (Ponting, 1996), including those shown in Figure with the SH3 domains of Src and the p85 subunit of the 2B and C. The PX domain of Fish is most closely related phosphatidylinositol 3-kinase (PI 3-K). phox phox to the PX domains of p47 and p40 (Volpp et al., 1989; Wientjes et al., 1993), Bem1 and Scd2 (Chenevert Expression and tyrosine phosphorylation of Fish et al., 1992; Chang et al., 1994), and Cpk (MacDougall To assess the expression profile of Fish mRNA we analysed et al., 1995; Molz et al., 1996; Virbasius et al., 1996; various adult mouse tissues by Northern blot hybridization G.Plowman, K.Joho and S.A.Courtneidge, unpublished using a probe representing the Tks-5 cDNA (Figure 3). data). All of the SH3 domains are most similar to each We detected a prominent transcript of ~10.5 kb in most tissues examined, with the exception of spleen and testis Table I. A summary of clones obtained from anti-phosphotyrosine which contained relatively low and undetectable levels of screening of a phosphorylated mouse fibroblast expression library Fish mRNA, respectively. We also noted much weaker expression of a message of ~6 kb in those tissues containing Clone Possible Src Size Identity the 10.5 kb mRNA. Whether the smaller transcript corres- phosphorylation (bp) (functional information) sites ponds to a differentially spliced mRNA or perhaps a Fish- related transcript, remains to be established. Both the 10.5 Tks-1 QRLSYGAFT 838 s19 and 6 kb transcripts are significantly larger than the Fish (Src SH3 binding protein) coding region (~3.5 kb) present in the two Fish cDNAs Tks-2 REQRYQEQH 533 SH3P7 that we identified, suggesting that they may also contain PETSYGREH (SH3 ligand binding protein) extensive 5- and/or 3-untranslated sequences. EEGTYEVPP We raised polyclonal antibodies against a GST fusion QDTLYEEP protein containing the original Tks-5 cDNA product, and Tks-3 EGPIYEGLI 1250 Mem3 (relative of yeast used these to examine the expression of Fish in cells vacuolar sorting protein (Figure 4A). Three forms of Fish, of ~130, ~140 and VPS35) ~150 kDa, were detected in immunoprecipitates from NIH Tks-5 LKVKYEEPE 1020 novel (SH3 domain containing 3T3 cells. We think it unlikely that p130 and p140 EEPEYDVPA adaptor protein now named represent proteolytic breakdown products of the p150 EETIYENEG Fish) form of Fish, since they were also detected in lysates of Cas Tks-7 PQDIYDVPP 364 p130 [substrate of Src and cells generated under denaturing conditions (data not LPNQYGQEV focal adhesion kinase (FAK), shown), and perhaps represent products of other RNA LLDVYDVPP Crk binding protein] splice variants. Transient transfection of COS cells with HHSVYDVPP an expression construct encoding the small form of Fish Tks-9 EVQKYEAWL 1000 homologue of human CIP4, (see Figure 2A) gave rise to two bands, the largest of LGAGYGLLS Trip10 and rat STP (Cdc42 which was 150 kDa. The smaller product most likely EVQKYEAWG interacting protein, thyroid DTPIYTEFD hormone receptor interacting corresponds to a truncated or proteolytically degraded protein, cation transport product, since expression of a construct encoding Fish regulator) with a myc epitope tag at its N-terminus also generated Tks-14 EEPVYIEMV 789 novel (contains two YxxM two analogous products that were each recognized by the SEAIYEEMK motifs) myc antibody (data not shown). To test whether Fish was EEMKYPLPE indeed a substrate for Src, we co-transfected 293 cells Fig. 2. Amino acid sequence of murine Fish. (A) Predicted amino acid sequence of two Fish isoforms. The sequence shown was compiled from five overlapping cDNAs that were isolated from a murine day 11.5 embryonal cDNA library using a probe corresponding to Tks-5, a partial cDNA for Fish that was identified in the initial screen for Src substrates using the Swiss 3T3 cDNA library. The boundaries of the Tks-5 cDNA product are marked by arrows and potential Src tyrosine phosphorylation sites therein are indicated with ovals. Insertions of 15 and 28 amino acids that were identified in one clone, and are likely to be derived by alternative mRNA splicing, are underlined. Sequence numbers are shown at the right. The phox homology (PX) domain is overlined and five SH3 domains are boxed. Three polyproline motifs that represent potential SH3 binding sites are doubly underlined. (B) Topology of selected proteins containing a PX domain. Domains are represented by boxes as indicated: PX domains are white; SH3 domains are black; the C2 domain is light grey; the phosphatidylinositol 3-kinase catalytic domain (PI 3-K) is dark grey; the p110 homology domain is dashed; the pleckstrin homology (PH) domain is striped and the Rho-GAP domain is checked. (C) Comparison of the phox homology (PX) domains of Fish and selected proteins. This alignment was created using a similar approach to that reported previously (Ponting, 1996). Briefly, iterative profile searches were conducted using SearchWise (Birney et al., 1996) and multiple alignments using Clustal W (Thompson et al., 1994). Definition of the domain boundaries and introduction of gaps (indicated by dashes) required inspection and some hand editing, and was helped by protein termini and neighbouring domain boundaries. Names of proteins and species are shown at the left of the alignment using the following abbreviations: h is human, m is mouse, dm is Drosophila melanogaster,scis S.cerevisiae and sp is S.pombe. PLD1 refers to phospholipase D1. Sequence numbers are shown at the end of the PX domain. Numbers at the top of the alignment refer to the Fish sequence. Amino acids are shown using the single letter code. Colours are used to indicate conserved features. The basic residues histidine, lysine and arginine (H, K and R, respectively) are shown in green, the hydrophobic residues alanine, phenylalanine, isoleucine, leucine, methionine, proline, valine and tryptophan (A, F, I, L, M, P, V and W, respectively) are shown in purple, the aromatic residues phenylalanine, tryptophan and tyrosine (F, W and Y, respectively) are shown in yellow and the acidic residues aspartate and glutamate (D and E, respectively) are shown in pink. (D) Comparison of the phox phox SH3 domains of Fish with those of p47 . Multiple alignments of the SH3 domains from Fish, p47 , Src and PI 3-K were conducted using MegAlign (DNAStar). The % amino acid identity is shown. 4348 Fish, a new adaptor protein 4349 P.Lock et al. Fig. 3. Tissue distribution of Fish mRNA. Northern blot analysis of poly(A) RNA from the indicated adult mouse tissues using a P-labelled Fish cDNA probe (upper panel). Hybridization analysis of the same Northern blot using a β-actin cDNA probe (lower panel). with cDNAs for Fish, with or without wild-type, activated or kinase-inactive Src. Anti-phosphotyrosine immuno- blotting revealed that Fish was tyrosine-phosphorylated when co-expressed with active forms of Src (Figure 4B). Fig. 4. Expression and tyrosine phosphorylation of Fish by transient We next determined whether Fish was also tyrosine transfection. (A) The Fish cDNA (short form) encodes a 150 kDa phosphorylated in Src-transformed fibroblasts. A com- protein. Fish was immunoprecipitated from extracts of NIH 3T3 cells (lane 1) or COS cells transfected with an expression vector containing parison of NIH 3T3 cells, and counterparts transformed the Fish cDNA in the anti-sense (–)(lane 2) or sense ()(lane 3) with an activated allele of Src in which the regulatory orientation. Samples were analysed by SDS–PAGE and C-terminal tyrosine is replaced with phenylalanine, showed immunoblotting with anti-Fish.1 serum. The Fish cDNA product and a that Fish was tyrosine-phosphorylated in Src-transformed possible premature termination product are indicated by arrow heads. cells (Figure 5A). To determine whether Fish was likely Three forms of endogenous Fish in NIH 3T3 cells, designated p150, p140 and p130, are also indicated. (B) Fish is tyrosine-phosphorylated to be a direct substrate of Src, we used Rat1 fibroblasts in 293 cells which coexpress active Src. Lysates of 293 cells transformed with a temperature-sensitive version of v-Src. transfected with either NmycFish cDNA alone, encoding an At the non-permissive temperature, a low basal level of N-terminal myc epitope-tagged form of Fish, or in combination with tyrosine phosphorylation of Fish was detected. However, cDNAs for SrcK, SrcK– or SrcY527F, as indicated, were analysed by SDS–PAGE and immunoblotting with anti-Fish.2 (upper panel) and within 10 min of placing the cells at the permissive anti-Src (EC10; middle panel). Anti-Fish.2 immunoprecipitates from temperature, the tyrosine phosphorylation of Fish these lysates were analysed by SDS–PAGE and immunoblotting with increased, and was maximal within 1 h of the shift (Figure anti-pTyr (lower panel). 5B). These kinetics are similar to those seen for the re- activation of Src kinase activity (S.A.Courtneidge, unpub- lished observations). proteins. We are currently characterizing these proteins Fish contains several motifs and domains that are known further, although our preliminary analyses have ruled out Ctn to be involved in protein–protein interactions; we therefore rasGAP, Fak, AFAP-110 and p120 . looked for Fish-associated proteins. Fish isolated from NIH Many proteins phosphorylated on tyrosine in Src-trans- 3T3 cells was not associated with tyrosine-phosphorylated formed cells are also transiently phosphorylated in normal proteins (Figure 5C). In contrast, in Src-transformed cells in response to growth factors or other stimuli. We cells Fish immunoprecipitates contained several tyrosine therefore asked whether growth factor stimulation resulted phosphoproteins. Several of these proteins, most notably in tyrosine phosphorylation of Fish. Our initial tests used those of 125, 115 and 65 kDa, were recognized by two NIH 3T3 cells, where we were unable to find convincing different antibodies raised against different regions of evidence for Fish phosphorylation in response to a variety Fish, and are therefore likely to represent Fish-associated of stimuli (data not shown). However treatment of Rat1 4350 Fish, a new adaptor protein Fig. 5. Phosphorylation of Fish in Src-transformed NIH 3T3 cells and temperature-sensitive v-Src-transformed fibroblasts, and association with tyrosine-phosphorylated proteins. (A) Fish is tyrosine-phosphorylated in Src-transformed NIH 3T3 cells. SDS lysates prepared from NIH 3T3 cells (Cl7) and SrcY527F-transformed NIH 3T3 cells (527) were immunoprecipitated either with rabbit Ig or anti-Fish.2 antibody, and analysed by SDS–PAGE and immunoblotting with anti-pTyr (upper panel) or anti-Fish.2 (lower panel). The apparent molecular weight of the tyrosine- phosphorylated species is 150 kDa. (B) Fish is tyrosine-phosphorylated in temperature-sensitive v-Src transformed fibroblasts following a shift to the permissive temperature. Lysates were prepared from ts68.10 cells following a time course of shifting from the non-permissive temperature (34.5°C) to the permissive temperature (39.5°C) and immunoprecipitated with anti-Fish.2 antibody, followed by analysis by SDS–PAGE and immunoblotting with anti-pTyr (upper panel) or anti-Fish.2 (lower panel). The apparent molecular weights of the two forms of Fish detected in Rat1 cells are 140 and 150 kDa; only p150 is tyrosine-phosphorylated. (C) Fish is associated with several tyrosine-phosphorylated proteins. Lysates prepared from NIH 3T3 cells (Cl7) or SrcY527F-transformed NIH 3T3 cells (527) were immunoprecipitated with anti-Fish.1 or anti-Fish.2 antibodies and analysed by SDS–PAGE. Proteins were transferred to nylon and immunoblotted with anti-pTyr. Tyrosine-phosphorylated proteins that appear to co-associate with Fish in immunoprecipitates with both antibodies (bands at ~65, ~115 and ~125kDa) are indicated by arrows. fibroblasts with PDGF (Figure 6), LPA and bradykinin (data not shown) did lead to an increase in tyrosine phosphorylation of Fish. The kinetics of this phosphoryl- ation was quite slow, with increases still being detected 2 h after stimulation. It will be interesting to determine whether tyrosine phosphorylation of Fish has a functional Fig. 6. Fish is tyrosine-phosphorylated in Rat1 cells in response to role in the cell’s response to growth factors. PDGF. Lysates prepared from Rat1 cells following treatment with One response commonly elicited by growth factors, and 50 ng/ml PDGF for the times indicated were immunoprecipitated with anti-Fish.2 antibodies and analysed by SDS–PAGE. Proteins were frequently involving proteins with SH3 domains and PX transferred to nylon and immunoblotted with anti-pTyr (upper panel) domains, is reorganization of the cortical actin cyto- and anti-Fish.2 (lower panel). The apparent molecular weights of the skeleton. Indeed, as shown in Figure 7A, disruption of two forms of Fish detected in Rat1 cells are 140 and 150 kDa; only the actin cytoskeleton by treatment with cytochalasin D p150 is tyrosine-phosphorylated. (cytD) resulted in a dramatic change in the profile of tyrosine-phosphorylated proteins, with both increases and decreases in phosphorylation being detected. One set of on the level of Src in the cell, but it did lead to a proteins which showed increased phosphorylation had modest increase in its intrinsic kinase activity (Figure 8), molecular weights in the range of 150 kDa. Immuno- suggesting that it is activated under the same conditions precipitation and anti-phosphotyrosine immunoblotting that we detected Fish phosphorylation. We have detected confirmed that these were the Fish proteins (Figure 7B, this activation using both a Src-specific monoclonal anti- left panel). Identical results were obtained when latrunculin body and anti-cst.1, an antibody that recognizes Src, Fyn B (a marine toxin that also disrupts the actin cytoskeleton) and Yes. The average activation that we detected in five was used (data not shown). Further analysis showed that separate experiments was 1.8-fold. Fish proteins became phosphorylated within 2 min of cytD treatment (Figure 7B, right panel), and at sub- Discussion micromolar concentrations of the drug (Figure 7C). These results strongly suggest that the tyrosine phosphorylation Despite the large number of substrates for Src already of Fish is connected to the integrity of the actin cyto- described in the literature, several proteins phosphorylated skeleton. Src-transformed cells have a disorganized actin on tyrosine in Src-transformed cells remain uncharacter- cytoskeleton, and many of the known Src substrates are ized, and many important targets may have so far gone cytoskeletal proteins. Yet to our knowledge, there have undetected because of their low abundance. We therefore been no reports that disruption of the actin cytoskeleton sought a method to detect substrates for Src that would not can activate Src. To examine this, we measured Src activity, rely on association with Src or relatively high abundance in both by autophosphorylation and enolase phosphorylation, the cell, and that would allow rapid cloning of the from untreated and cytD-treated cells. cytD had no effect cDNAs encoding them. The method we devised requires 4351 P.Lock et al. Fig. 7. Fish is tyrosine-phosphorylated in response to treatment with cytochalasin D. (A) Treatment of NIH 3T3 cells with cytD results in changes in total cellular phosphotyrosine. Lysates from NIH 3T3 cells treated with either DMSO alone or 10 μM cytD for 10 min were analysed by SDS–PAGE. Proteins were transferred to nylon and immunoblotted with anti-pTyr. Black arrows indicate bands that have increased following cytD treatment and white arrow heads indicate bands that have decreased. The two panels derive from the same immunoblot, with the top panel being a lighter exposure than the bottom. (B) Fish is tyrosine-phosphorylated rapidly following cytochalasin D treatment. Lysates from NIH 3T3 cells treated with either DMSO alone or 10 μM cytD for 10 min were immunoprecipitated with anti-Fish.2 antibodies, analysed by SDS–PAGE and immunoblotted with anti-pTyr (left panel). NIH 3T3 cells were treated with 10 μM cytD for the times indicated, and lysates were prepared and immunoprecipitated with anti-Fish.2 antibodies. Proteins were analysed by SDS–PAGE and immunoblotted with anti-pTyr (right panel). (C) Fish is tyrosine-phosphorylated following treatment with low concentrations of cytochalasin D. Lysates were prepared from NIH 3T3 cells treated with the different concentrations of cytD as indicated for 15 min , and immunoprecipitated with anti-Fish.2 antibodies. Proteins were analysed by SDS–PAGE, and immunoblotted with anti-pTyr (upper panel) and anti-Fish.2 (lower panel). a bacteriophage expression library, an enriched source of cells (C.Abram and S.A.Courtneidge, unpublished data). Src kinase (e.g. baculovirus-infected insect cell extracts) All of the cDNA products that were identified contained and phosphotyrosine antibodies. Using this approach we at least one consensus Src phosphorylation site (Songyang identified cDNAs for several candidate Src substrates. and Cantley, 1995), suggesting that enzyme specificity is One of the cDNA clones, Tks-7, encoded a fragment of retained under the conditions we used to phosphorylate Cas a known Src substrate, p130 (Sakai et al., 1994), the cDNA library. This is perhaps not surprising, since implying that the method is a valid way to detect tyrosine substrate recognition by tyrosine kinases, like SH2 domain kinase substrates. Intriguingly, several of the cDNAs specificity, appears to be dictated by very few residues identified by this method encode proteins with SH3 surrounding the phosphorylation site and is therefore likely Cas domains (p130 , SH3P7, s19, CIP4/STP and Fish), to be independent of substrate conformation (Songyang although their SH3 domains per se were clearly not and Cantley, 1995). We believe that this method will required to facilitate phosphorylation by Src and sub- be generally applicable, using expression libraries from sequent detection since none of the Tks cDNAs encode different tissues and the tyrosine kinase of choice. Indeed, an intact SH3 domain (data not shown). an analogous method to ours for identifying substrates of The Tks-5 clone, encoding Fish, is characterized further serine/threonine as well as tyrosine kinases was reported here. We identified Fish as an in vitro substrate for Src recently (Fukunaga and Hunter, 1997). The MAP kinase both when expressed as a β-galactosidase fusion protein ERK1 was used to phosphorylate a HeLa cell cDNA (Figure 1) and as a GST fusion protein when incubated library in the presence of [γ- P]ATP and positive clones with a Src immunoprecipitate (data not shown). Fish were detected by autoradiography. Using this method a was also tyrosine-phosphorylated in 293 cells when co- number of known substrates of MAP kinases, as well as expressed with an activated form of Src, and endogenous the novel kinase MNK1, were identified. In contrast to Fish was phosphorylated in Src-transformed NIH 3T3 this method, we used enriched rather than purified protein cells. Furthermore, tyrosine phosphorylation of Fish kinase for the phosphorylation step. In addition, we used increased within minutes of transferring cells containing a commercial λgt11 cDNA library while Fukunaga and a temperature-sensitive form of v-Src from non-permissive Hunter (1997) constructed a library using a novel expres- to permissive temperature. These findings suggest that sion vector. Fish is indeed a direct substrate of Src in Src-transformed cells. We are currently analysing several other proteins Fish—a new adaptor protein identified by this method, and have found that at least Fish is a new, broadly expressed adaptor protein, two others are substrates for activated Src in mammalian which appears to associate with several proteins in 4352 Fish, a new adaptor protein bind to the same or related ligands, although the binding specificities of the Fish SH3 domains remain to be determined. Interestingly, the Fish PX domain is also most phox related to that of p47 suggesting that these proteins may be the products of genes which arose from a common ancestor. The region of Fish encoded by the Tks-5 cDNA contained three consensus Src phosphorylation sites, desig- nated Y552, Y557 and Y619 (see Figure 2A). These sites conform to consensus binding sites for the SH2 domains of Abl (Y-E-E-P), Nck or Crk (Y-D-X-P), and Grb2 (Y-X- N-X), respectively (Songyang et al., 1993). Conceivably, tyrosine-phosphorylated forms of Fish, such as those found in Src-transformed fibroblasts, could associate with one or more of these proteins in vivo. As well as SH3 domains, Fish also has three polyproline segments which encompass one or more P-X-X-P motif that could allow it to bind to SH3 domains (Pawson, 1995). Fish also has many potential sites of serine phosphorylation. Perhaps the most unusual feature of Fish is the presence at the N-terminus of a phox homology or PX domain. This domain, which is ~100–110 amino acids in length, has only recently been described (Ponting, 1996), and its function is not yet known. To our knowledge Fish is the Fig. 8. Src is activated by cytochalasin D treatment. (A) Src is first protein containing a PX domain to be implicated in activated by cytochalasin D treatment. Lysates of NIH 3T3 cells a tyrosine-kinase signalling pathway. PX domains have treated with either DMSO alone or 10 μM cytD for 10 min were several well conserved residues, many of them hydro- immunoprecipitated with anti-Src antibodies. The immunoprecipitates phobic, and two almost invariably conserved basic were subjected to an in vitro kinase assay using enolase as a substrate. Proteins were analysed by SDS–PAGE and autoradiography. (B) The residues, which may be important for function. The pattern level of Src is not altered by cytochalasin D treatment. Lysates of NIH of conserved hydrophobic residues strongly suggests that 3T3 cells treated with either DMSO alone, or 10 μM cytD for 10 min, the PX domain includes an N-terminal segment predomin- were immunoprecipitated with anti-Src antibodies. Proteins were antly composed of β-strand-like sequences and both central analysed by SDS–PAGE, transferred to nylon and immunoblotted with anti-cst.1. and C-terminal α-helical structures that are separated by a conserved proline-rich sequence of indeterminate Src-transformed cells. Three forms of Fish were detected secondary structure (Ponting, 1996; data not shown). It in NIH 3T3 cells. These forms are unlikely to be has been proposed that the proline-rich sequence might proteolytic breakdown products because they were constitute a binding site for SH3 domains although this detected even when cells were lysed in hot extraction has not been tested directly (Ponting, 1996). buffer containing SDS. They may occur as a result of Where studied, PX domain-containing proteins are often post-translational modifications, although this cannot be associated with processes involving the actin cytoskeleton, accounted for by tyrosine phosphorylation since Fish is membranes and/or GTP-binding proteins. For example the not detectably phosphorylated in non-transformed NIH Saccharomyces cerevisiae protein Bem1, and Scd2, its 3T3 cells. In addition, preliminary experiments suggest orthologue in Shizosaccharomyces pombe, appear to that neither serine/threonine phosphorylation nor glycos- coordinate rearrangement of the cortical cytoskeleton ylation can account for the three forms. The most likely during cell polarization in response to mating factors explanation is that they are encoded by different RNA (Chenevert et al., 1992; Chang et al., 1994). Bem1 interacts splice products. Indeed we identified two distinct cDNAs directly with Cdc24, a guanine nucleotide exchange factor for Fish in the embryonal cDNA library, the smaller of for the Rho-family GTPase Cdc42, and probably indirectly which encoded a protein of ~150 kDa when expressed with Cdc42 itself (Peterson et al., 1994). Similar inter- in COS cells. The p140 and p130 forms of Fish may actions have been reported for Scd2 in S.pombe (Chang be generated by other splice variations. The complexity et al., 1994). Bem1 has also been shown to interact with of Fish isoforms is also evident in different cell types. Ste20p, a serine/threonine kinase belonging to the p21- For example in human platelets a single band of 150 activated kinase (PAK) family, as well as to actin (Leeuw kDa was detected, whereas all three forms were found et al., 1995). Another bud emergence gene, Bem3, also in human fibroblasts and vascular smooth muscle cells has a PX domain (Zheng et al., 1994). Interestingly, Bem3 (data not shown). Rat1 fibroblasts have p150 and p140 is a GTPase activating protein (GAP) for certain Rho- forms (Figure 6). The fact that in all of these cases family proteins indicating that, like Bem1, it also interacts two different antibodies raised against the original with GTP binding proteins. Two components of the phox murine Fish sequence recognized each of these proteins neutrophilic NADPH oxidase complex, p40 and phox confirms that they are indeed isoforms of Fish. p47 , each have a PX domain very similar to that of Fish contains many potential binding sites for other Fish. Activation of the oxidase, and production of super- proteins; the most notable are the five SH3 domains. oxide and peroxide in response to invading microorgan- Strikingly, all five are most related to each other and to isms, or to agonists such as fMLP, involves recruitment phox phox phox the p47 SH3 domains, suggesting that they may all of the cytoplasmic proteins p40 and p47 (and other 4353 P.Lock et al. phox proteins including p67 and the small GTP-binding posed from the observation that it is most closely related to protein Rac2) to the plasma membrane, where they associ- the cytosolic NADPH oxidase components of neutrophils, phox phox ate with membrane-bound flavocytochrome b (Segal p47 and p40 . Interestingly, a recent report described and Abo, 1993; Dorseuil et al., 1996). Significantly, the identification of Posh, a Rac-interacting adaptor protein oxidase activation is concomitant with phagocytosis, a with four SH3 domains, which has a striking similarity to phox membrane process requiring rearrangement of the cortical p67 (Tapon et al., 1998). The expression of the p47 actin cytoskeleton (Hall, 1992). Recently a new PI 3-K and p67 proteins is restricted to phagocytic cells (Segal was described in mouse and Drosophila (variously known and Abo, 1993), whereas both Posh and Fish are more as Cpk, 68 D or p170) that also contains a PX domain broadly expressed. In neutrophils, the respiratory burst (MacDougall et al., 1995; Molz et al., 1996; Virbasius can be initiated by the chemoattractant and G-protein- et al., 1996). Another enzyme which contains a PX domain coupled receptor agonist fMLP (Segal and Abo, 1993). and is involved in membrane-associated phospholipid However, superoxide formation has also been observed in metabolism is phospholipase D1 (Ponting and Parker, many other cell types, and has indeed been linked to 1996). Finally, other PX domain-containing proteins with carcinogenesis (Burdon, 1995). One exciting possibility known functions include Vam7, which is involved in is that Fish forms part of the superoxide-generating vacuolar morphogenesis (Wada and Anraku, 1992), Mvp1 machinery in non-phagocytic cells, a hypothesis which is and Vps17, which are involved in sorting of proteins to currently being tested. vacuoles (Kohrer and Emr, 1993; Ekena and Stevens, 1995) and Mdm1, which is involved in organelle inheritance Materials and methods (McConnell and Yaffe, 1992). It is hard to predict from the sequence of the PX domain what it might interact with; Phosphorylation screening both phospholipids and proteins are candidate ligands. It The phosphorylation screening method we used to identify Src substrates was adapted from a technique designed to identify binding partners of is now possible to undertake functional studies of PX the Shc phosphotyrosine binding (PTB) domain (Kavanaugh et al., domain-containing proteins bearing targeted mutations, as 1995). In the primary screen for Src substrates, 210 individual clones well as binding studies of the isolated domains in vitro, from a λgt11 library containing mouse Swiss 3T3 fibroblast cDNA in order to resolve this issue. (Clontech; ML1023b) were plated on four 150 mm Petri dishes. Plates were overlaid with nitrocellulose filters impregnated with 10 mM Fish was tyrosine-phosphorylated at cytochalasin D isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated at 37°C for concentrations that disrupt the cortical actin cytoskeleton, 16 h to induce expression of recombinant lacZ fusion proteins. Replica and also in Src-transformed fibroblasts, which have a filters were washed extensively in TBST (10 mM Tris–HCl pH 7.4, dramatically altered actin cytoskeleton. Furthermore, Fish 150 mM NaCl, 0.1% Triton X-100) then equilibrated for1hin kinase was tyrosine-phosphorylated in response to treatment of buffer (TBST containing 10 mM MgCl and 2 mM MnCl ). Filters were 2 2 blocked for 1 h at room temperature in kinase buffer containing 3% Rat1 fibroblasts with either PDGF, LPA or bradykinin. bovine serum albumin (BSA). Recombinant proteins were phosphorylated Each of these mitogens activates the Rho family of small by incubating filters at 30°C for 60 min in kinase buffer supplemented GTP binding proteins and causes changes in the actin with one tenth volume of an Sf9 extract containing baculovirus-derived cytoskeleton such as stress fibre, lamellipodia and filopodia human Src, 250 μM ATP and 100 μM sodium orthovanadate. Filters were washed briefly with kinase buffer alone then incubated in stripping formation (Hall, 1998). These data suggest that Fish may buffer (62.5 mM Tris–HCl pH 7.0, 2% SDS, 100 mM 2-β-mercapto- play a role in cytoskeletal changes. ethanol) at 50°C for 30 min to remove possible associated phospho- Many of the Src substrates identified so far are cyto- proteins, including Src itself or Sf9 cell-derived proteins, which might skeletal proteins, including p110 AFAP, paxillin, vinculin interfere with the screening. Tyrosine-phosphorylated proteins were detected with an anti-phosphotyrosine monoclonal antibody, 4G10, using and cortactin, which has led to the suggestion that in standard immunoblotting methodology (see below). Twelve positive transformed cells Src can regulate cytoskeletal structures clones were plaque-purified by successive rounds of phosphorylation (Brown and Cooper, 1996). In normal cells, Src is activated screening. The cDNA inserts were amplified by polymerase chain by mitogens such as PDGF, and is required for DNA reaction (PCR) using Taq DNA polymerase (Boehringer Mannheim) and synthesis in response to several growth factors (Erpel subcloned into pCRII (Invitrogen Corp.). The cDNA inserts were sequenced with the aid of T7 and SP6 oligonucleotide primers. and Courtneidge, 1995). More recently, it has also been reported that PDGF stimulation of cells results in the Isolation of a full-length Fish cDNA translocation of Src to regions of the cell periphery that To identify the full-length Fish cDNA, a murine day 11.5 embryo cDNA also show dense actin staining. This effect was seen within library (Clontech, ML1027b) (a generous gift of G.Plowman, SUGEN) 30–60 min of PDGF addition, and required an intact actin was screened by hybridization with a 1 kb P-labelled DNA fragment corresponding to the Tks-5 cDNA that was isolated during the initial cytoskeleton and the activity of Rho family proteins screen for Src substrates. Two cDNA clones were identified, λME14 (Fincham et al., 1996), suggesting that the activity of Src and λME5E, which contained ~1.3 kb of additional 5 sequence and may be under the control of the actin cytoskeleton. By 0.5 kb of 3 sequence relative to Tks-5, respectively. Radiolabelled DNA this mechanism, the cytoskeleton could control the access probes corresponding to fragments at the 5-end of λME14 and the 3- of Src to substrates such as Fish, which might explain the end of λME5E were then used to rescreen the embryo cDNA library. A cDNA designated λMEA3C was identified which contains an additional rather slow kinetics of Fish phosphorylation following 0.3 kb of sequence at the 5-end relative to λME14, including a PDGF stimulation. Alternatively, Fish could be on a presumptive translational initiation codon. Two non-identical clones, pathway downstream of Src that controls the activity of λME14(3) and λME5A, were found to contain ~0.6 kb of additional Rho family proteins and their effect on the actin cytoskel- 3-sequence relative to λME5E including two stop codons at the 3-end of the Fish coding sequence. λMEA3C and λME14 were shown to differ eton. In this regard, it is interesting that tyrosine kinases with respect to putative alternative exons of 45 and 84 bp which are are proposed to participate both upstream and downstream present in λME14 but absent from λMEA3C, indicating the existence of Rho proteins (Ridley and Hall, 1994; Nobes et al., of at least two alternatively spliced transcripts. Two Fish cDNA sequences 1995; Kranenburg et al., 1997). were predicted on the basis of the overlapping cDNAs identified. The An alternative potential function for Fish can be pro- amino acid sequence of the corresponding Fish isoforms, which include 4354 Fish, a new adaptor protein both the 15 and the 28 residue insertions, or lack these sequences, permissive temperatures used in experiments with ts68.10 cells were respectively, was deduced on the basis of these cDNAs. 34.5 and 39.5°C, respectively. Cells were treated with 50 ng/ml PDGF (UBI) at 37°C for the times stated. Extracts were prepared from NIH Analysis of RNA expression 3T3, 527, 293, ts68.10, Rat1 or COS cells, by washing the adherent A multiple tissue Northern blot containing 2 μg of poly(A) RNA from cells with ice cold TBS (25 mM Tris–HCl pH 7.5, 150 mM NaCl) various adult mouse tissues (Clontech) was hybridized with a P- containing 100 μMNa VO and 2 mM dithiothreitol (DTT). Cells were 3 4 labelled DNA probe corresponding to the 1 kb Tks-5 cDNA (shown in lysed in 0.5 to 2.0 ml of either NP40 lysis buffer (20 mM HEPES Figure 2A) under high stringency conditions (65°C, 0.1 SSC, 0.1% pH 7.0, 150 mM NaCl, 1% Nonidet P40) or RIPA lysis buffer (20 mM SDS). The blot was stripped according to the suppliers protocol and Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, rehybridized with a P-labelled β-actin probe. 0.1% SDS) containing 100 μMNa VO , 10 mM NaF, 2 mM DTT, 3 4 10 μg/ml aprotinin, 20 μM leupeptin and 100 μM phenylmethylsulfonyl Constructs fluoride (PMSF) for 10–15 min at 4°C. Extracts were clarified by Expression plasmids encoding wild-type and epitope-tagged versions of centrifugation at 10 000 g for 10 min and the protein concentration the smaller Fish isoform (see above) were constructed. For the wild- determined. Extracts of Sf9 cells expressing human Src baculovirus were type construct, the following DNA fragments were subcloned into the prepared essentially as described above except that the cells were NotI and XbaI sites of pBluescript II KS (pBSIIKS, Stratagene): a harvested by centrifugation in 50 ml tubes, prior to lysis. For immunopre- 1423 bp EagI–EcoRI fragment from λMEA3C which contains 54 bp of cipitations, samples containing 150–400 μg of total protein were incub- 5-untranslated region (UTR) and sequences encoding residues 1–412; ated with either 1–2 μl of crude pre-immune serum or antiserum, or a 566 bp EcoRI–SacI fragment from λME14 which encodes residues 1 μg of affinity-purified antibody, and ~7 μl packed volume of protein 413–602; and a 1548 bp SacI partial XbaI fragment originating from A–Sepharose beads (Pharmacia) or protein A–agarose beads (Santa λME14(3) which codes for amino acids 603–1081, contains 80 bp of Cruz) for 1 h at 4°C. Immunocomplexes were washed four times in ice 3-UTR and an additional 36 bp of polylinker sequences from pBSIIKS. cold RIPA buffer containing 100 μMNa VO and 1–2 mM DTT. 3 4 The resulting plasmid, pBS-Fish, was digested with EagI and XbaI, Samples were resuspended in SDS sample buffer (80mM Tris pH 6.8, releasing a 3414 bp fragment encompassing the complete Fish coding 2% SDS, 75 mM DTT, 10% glycerol, 1.25% Bromophenol Blue), heated to 95°C for 5 min and subjected to SDS–PAGE using 7.5 or 9% region. Recessed 3-termini were filled using Klenow enzyme polyacrylamide gels. Kinase assays were carried out as described (Boehringer Mannheim), and BstXI adaptors (Invitrogen) were ligated previously (Kypta et al., 1990). Briefly, 50 μg lysate was immunoprecipit- onto the ends. The fragment was subcloned into the BstXI sites of ated with 1 μg of anti-Src antibody (327) and 20 μl protein A/G plus pEF-BOS (Mizushima and Nagata, 1990) in the forward and reverse agarose (Santa Cruz) followed by washing four times with ice cold orientations to generate the plasmids pEF-BOS Fish() and pEF-BOS RIPA buffer containing 100 μMNa VO and 1 mM DTT, and once with Fish(–), respectively. The plasmid pEF-BOS NmycFish, encoding two 3 4 kinase buffer (20 mM HEPES pH 7.4, 10 mM MgCl , 2 mM MnCl , copies of the myc epitope recognized by the 9E10 antibody fused to the 2 2 1 mM DTT). The reactions were carried out in 20 μl kinase buffer N-terminus of Fish, was generated by ligating the following fragments containing 15 μM unlabelled ATP, 3 μg acid-denatured enolase and into the BamHI and XbaI sites of pEF-BOS Nmyc (a gift from P.Orban, 10μCi [γ- P]ATP for 10 min at 30°C, and stopped with SDS-sample EMBL, Germany): a 294 bp BglII–ClaI fragment derived by PCR using buffer. The samples were analysed by SDS–PAGE and autoradiography Vent polymerase (New England Biolabs), which encodes Fish residues using Fuji RX film. 1–96; a 950 bp ClaI–EcoRI fragment encoding residues 97–412; and a 2119 bp EcoRI–XbaI fragment encoding residues 413–1081 and including Immunoblotting 80 bp of 3-UTR plus 36 bp of polylinker sequences. SDS–PAGE gels were electrophoretically transferred to nitrocellulose filters (Scleicher and Schuell) or PVDF (Millipore) using a Millipore Antibodies and peptides semi-dry blotting aparatus. Filters were incubated in blocking solution The Fish-specific polyclonal antiserum, Fish.1, was generated by (either PBS or TBS containing 0.1% Tween 20 and 2–3% BSA) for 1 h immunising rabbits with a purified glutathione S-transferase (GST) fusion at room temperature. Filters were washed three times in wash solution protein containing residues 457–787 of Fish. A second polyclonal anti- (PBS or TBS containing 0.1% Tween 20) and incubated with blocking Fish antibody, Fish.2, was generated by immunising rabbits with a GST solution plus the primary antibody for1hat room temperature using fusion protein containing residues 807–908. Fish.2 antiserum was affinity the following antibody dilutions: α-pTyr, 1:1000–1500; α-Fish.1, 1:7500– purified by passing over a column containing the GST fusion protein, 10 000; α-Fish.2, 0.5 μg/ml; EC10, 2 μg/ml; α-cst.1, 1:200. Filters were coupled to 3M Emphaze™ Biosupport Medium AB1 (Pierce) according washed three times in wash solution and incubated with the secondary to manufacturer’s instructions. The anti-phosphotyrosine (α-pTyr) mono- detection reagent for1hat room temperature. For blots probed with a clonal antibody 4G10 was from Upstate Biotechnology Incorporated rabbit polyclonal antibody, filters were incubated with blocking solution (UBI). The mouse monoclonal antibodies, EC10 (UBI) and 327 (vSrc-1, containing either protein A-horseradish peroxidase (HRP) conjugate Calbiochem) and the rabbit polyclonal antibody, anti-cst.1 (Courtneidge (Amersham) at a dilution of 1:1000–2500 or donkey anti-rabbit HRP and Smith, 1984) were used to detect Src. (Amersham) at 1:20 000. For blots probed with mouse monoclonal antibodies, filters were incubated with sheep anti-mouse HRP conjugate Cell culture, baculoviral infection and DNA transfection (Amersham) at a dilution of 1:5000. Filters were washed three times in COS cells were grown in RPMI 1640 medium containing 10% fetal calf wash solution and the protein bands detected using enhanced chemi- serum (FCS) and antibiotics at 37°C in 5% CO . 293 cells, Rat1 cells, luminescence (ECL, Amersham or Supersignal, Pierce) in conjunction ts68.10 cells which express a temperature-sensitive mutant of Src with Fuji RX film. (Courtneidge and Bishop, 1982), NIH 3T3 cells and their derivative, 527 cells, which stably express a mutant form of chicken Src with an EMBL Accession Number activating Y527F substitution, were grown in Dulbecco’s modified The accession number for the Fish cDNA sequence is AJ007012. Eagle’s medium (DMEM) containing 10% FCS and antibiotics at 37°C in 10% CO . Sf9 insect cells were maintained in Grace’s medium Acknowledgements containing 7.5% FCS at 27°C. COS cells were transfected by electropor- ation. Briefly, 210 cells were transfected with 10 μg of pEF-BOS We thank A.Verhagen and G.Superti-Furga for many helpful discussions. Fish() or pEF-BOS Fish (–). Transfectants were analysed ~40 h post- Thanks to members of the EMBL DNA sequencing and animal facilities transfection. 293 cells were transfected with Lipofectamine™ (Gibco- for sequence analysis and in raising antisera, respectively. Thanks BRL) according to manufacturer’s instructions. Ten centimetre dishes of P.Orban, G.Plowman and T.Pawson for providing reagents. Thanks to cells at ~70% confluence were transfected with a total of 6 μgofDNA M.Velarde for help with figures. P.L. is very grateful for laboratory space at EMBL courtesy of A.Nebreda and T.Graf. S.A.C. and C.L.A. (pEF-BOS NmycFish alone, or in combination with pSG5-SrcK, acknowledge the support of the Human Frontiers Science Program. P.L. pSG5-SrcK- or pSG5-SrcY527F) in serum free medium OptiMEM was supported by research fellowships from the Alexander von Humboldt (Gibco) for 12–24 h. Cells were lysed 24 h later. For baculoviral Foundation and the Human Frontiers Science Program. infections, adherent cells were infected with recombinant baculovirus containing the human Src cDNA at a multiplicity of infection (MOI) of 1 and cell lysates prepared after ~72 h. References Cell treatment, lysis, immunoprecipitation and kinase assay Aspenstrom,P. 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Journal

The EMBO Journal – Springer Journals

Published: Aug 3, 1998

Keywords: cytochalasin D; Fish; PX domain; Src; tyrosine kinase substrate

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