Stimulation Through the T Cell Receptor Leads to Interactions Between SHB and Several Signaling Proteins

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

Michael Welsh1, Zhou Songyang5, J Daniel Frantz2,4, Thomas TruÈ b2,4, Kris A Reedquist3,4, TorbjoÈ rn Karlsson1, Masaya Miyazaki2,4, Lewis C Cantley5, Hamid Band3,4 and Steven E Shoelson2,4

1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA; 3Lymphocyte Biology Section, Division of Rheumatology and Immunology, and 4Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA; 5Division of Signal Transduction, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts, USA

Shb is a recently described Src homology 2 (SH2) the TCR associated CD3 and z chains (Weiss and domain-containing adaptor protein. Here we show that Littman, 1994). The ITAMs are repeated sequences of Shb is expressed in lymphoid tissues, and is recruited YXXL separated by 6 ± 8 amino acids and their into signaling complexes upon activation of Jurkat T tyrosine phosphorylation generates binding sites for cells. Grb2 binds proline-rich motifs in Shb via its SH3 Src homology 2 (SH2) domains of signaling molecules, domains. As a result, a number of proteins detected in such as those of ZAP 70 (Chan et al., 1992; Hutchcroft anti-Shb and anti-Grb2 immunoprecipitates are shared, et al., 1992). This initiates a cascade of reactions including phosphoproteins of 22, 36/38, 55/57 and involving the adaptor protein p36/38 (Weber et al., 70 kDa. Shb-association with p22, which represents the 1992; Gilliland et al., 1992; Sieh et al., 1994), Lnk T cell receptor associated z chain, occurs through the (Huang et al., 1995), Grb2 (Ravichandran et al., 1993) Shb SH2 domain. The central region of Shb binds p36/ and the signaling pathways of phospholipase C g, PI-3 38. Since this interaction was inhibited by phosphotyr- kinase and MAP kinase (Ravichandran et al., 1993; osine, this region of Shb is likely to contain a non-SH2 Secrist et al., 1991; Thompson et al., 1992). Eventually, PTB (phosphotyrosine binding) domain. The Shb PTB a signal is propagated which induces altered gene domain was found to preferentially bind the sequence transcription, IL-2 production, dierentiation and a Asp-Asp-X-pTyr when incubated with a phosphopeptide modi®cation of the balance between cell proliferation library. A peptide corresponding to a phosphorylation and apoptosis (Weiss and Littman, 1994; Perlmutter et site in 34 kDa Lnk inhibited association between Shb al., 1993). and p36/38. Overexpression of Shb in Jurkat cells led to Shb is a recently characterized adaptor protein increased basal phosphorylation of Shb-associated p36/ (Welsh et al., 1994), with an SH2 domain in its C- 38 and p70 proteins. Inactivation of the Shb SH2 domain terminus and proline-rich sequences in its N-terminus. by an R522K mutation resulted in a reduced stimulation The cDNA sequence contains two potential translation of tyrosine phosphorylation of several proteins in initiation sites, the ®rst corresponding to a protein response to CD3 crosslinking when expressed in Jurkat product migrating as 66 kDa and containing ®ve cells. Together, our results show three distinct domains proline-rich motifs (Welsh et al., 1994; Karlsson and of Shb all participate in the formulation of multimeric Welsh, 1996) and the second to a product migrating as signaling complexes in activated T cells. These results 55 kDa and containing two proline-rich motifs. The indicate that the Shb protein functions in T cell receptor proline-rich sequences of Shb have been shown to signaling. interact with the Src homology 3 (SH3) domains of the p85 subunit of PI-3 kinase, Src tyrosine kinase and the Keywords: Shb; Jurkat T cells; T cell receptor; p36/38; epidermal growth factor-associated adaptor protein SH2 domain; PTB domain Eps8 (Karlsson et al., 1995). The SH2 domain of Shb binds the autophosphorylated PDGF b-receptor and the FGF receptor-1 (Karlsson et al., 1995). The consensus motif preferentially recognized by the SH2

Introduction domain of Shb was pYTTL (Karlsson et al., 1995), with a strong selection for leucine in position 3 downstream T-cell activation is initiated by the engagement of the from the phosphotyrosine residue. With this knowledge T-cell receptor (TCR) (Weiss and Littman, 1994; in mind, it seemed appropriate to test for Shb- Perlmutter et al., 1993; Chan et al., 1994). This causes interactions in T cells, in which the ITAMs (with a rapid tyrosine phosphorylation of ITAM (immuno- paired YXXL phosphopeptide motifs) play an essential receptor tyrosine-based activation motif) sequences on role in tyrosine kinase dependent signaling. The present data demonstrate the interactions of Shb with ITAM motifs and several other signaling components. We furthermore characterize the central part of Shb as Correspondence: M Welsh, Department of Medical Cell Biology, Box corresponding to a phosphotyrosine binding (PTB) 571, Biomedicum, 75123 Uppsala, Sweden and SE Shoelson, Joslin domain. All three regions of Shb appear signi®cant for Diabetes Center, Harvard Medical School, One Joslin Place, Boston, Shb's interactions in these cells. Together, these results MA, 02215, USA Received 3 July 1997; revised 29 September 1997; accepted 29 suggest that Shb plays an important role for T-cell September 1997 signaling. Shb in T cell signaling MWelshet al 892 the Shb immunoprecipitates were the 55/57 kDa Results phosphoproteins, and next the time-course after CD3 crosslinking for the tyrosine phosphorylation of these Human Shb is expressed in multiple tissues and recruited was studied (Figure 2b). The tyrosine phosphorylation into tyrosine kinase signaling in activated Jurkat cells of the 55/57 kDa products present in the Shb Northern blot analysis of multiple human tissues shows immunoprecipitates rapidly increased (within 30 s) that Shb mRNA is widely expressed as two transcripts and remained elevated for up to 60 min of stimula- of 2.5 and 3.1 kb present in all 16 tissues analysed tion. To assess whether these products corresponded (Figure 1). Mouse tissues contain a predominant 3.1 kb to Shb or were Shb associated, Jurkat cell extracts mRNA (Welsh et al., 1994). Although the levels of from unstimulated or CD3 crosslinked cells were expression were similar in most tissues, prostate boiled in 1% SDS, diluted and subjected to contains slightly more and peripheral blood leukocytes immunoprecipitation with the Shb antibody (Figure somewhat less Shb mRNA (Figure 1). Thus, it is likely 2c). The purpose of this procedure was to disrupt pre- that lymphocytes express the Shb mRNA, since these existing complexes between Shb and other proteins. cells are present in spleen, thymus and blood Western blot analysis of anti-Shb immunoprecipitates leukocytes. under these conditions revealed Shb immunoreactivity To assess a functional role of Shb in T cell corresponding to the molecular weights 55 ± 59 kDa signaling, phosphotyrosine proteins present in anti- (Figure 2c). Probing the same blot with the Shb immunoprecipitates of anti-CD3-stimulated Jur- phosphotyrosine antibody PY-20, revealed the ab- kat T cells were determined (Figure 2a). Phosphotyr- sence of detectable amounts of phosphotyrosine osine proteins of molecular weights 22 kDa, 36/ proteins in these immunoprecipitates, suggesting that 38 kDa, 55/57 kDa, 70 kDa, 100 kDa and 190 kDa the phosphorylated 55/57 products indeed are Shb- were all found to co-precipitate with Shb after TCR associated. One of the supernatants after the anti-Shb stimulation. Although these six phosphotyrosine immunoprecipitation in Figure 2c was transferred to a proteins were commonly seen in the anti-Shb fresh tube and subjected to a second immunoprecipi- immunoprecipitates, their relative abundance varied tation with the Shb antibody (Figure 2c). Shb between dierent experiments (see Figure 3a). The immunoreactivity was absent under these conditions, precipitation of these products was speci®c for the Shb suggesting ecient precipitation of Shb in the ®rst antibody compared with normal rabbit serum (NRS). immunoprecipitation. The results in Figure 2a and b When testing for Shb immunoreactivity, a broad band suggest that Shb is rapidly recruited into tyrosine of 55 ± 59 kDa was observed which was speci®cally kinase signaling upon T cell activation, implying a immunoprecipitated with the Shb antibody (Figure functional role for Shb in this process. 2a). In Jurkat cell extracts, the Shb antibody recognizes in addition to the 55 ± 59 kDa Shb Comparison of Shb and Grb2 co-immunoprecipitation of products present in the immunoprecipitates bands of phosphotyrosine proteins 66, 77 and 80 kDa (see Figure 3d). Previously, Shb isoforms of 55, 66 and 77 kDa have been described, To identify the Shb-associated phosphotyrosine pro- with the 66 kDa product representing Shb initiated at teins, anti-CD3 activated Jurkat cells were lysed and the ®rst methionine codon, the 55 kDa protein Shb proteins co-immunoprecipitated with the Shb or Grb initiated at the second methionine codon and the antibodies were compared. Co-precipitated proteins 77 kDa component probably representing a post- were identi®ed by blotting for phosphotyrosine (Figure translationally modi®ed Shb (Karlsson and Welsh, 3a,b). Tyrosine phosphorylation levels increased 1996). Major phosphotyrosine products observed in following CD3 crosslinking including those of the anti-Shb precipitated proteins of 36/38 kDa, 55 ± 57 kDa, 70 kDa and 190 kDa. The 36/38 kDa, 55/57 and 70 kDa proteins were also immunoprecipitated he br pl lu li sm ki pa sp th pr te ov in co pbl using the Grb2 antibody. The immunoprecipitation of the 36/38 kDa product was more ecient with the

Grb2 antibody. Shorter exposure of the ECL reaction also revealed an approximate doubling of the phosphotyrosine content of p36/38 associated with Grb2 in this experiment (results not shown). When assessing the co-immunoprecipitation of phosphotyr- 3.1 — 2.5 — osine proteins in the 20 ± 30 kDa molecular weight range with the Shb and Grb2 antibodies, again similarities in the co-immunoprecipitation patterns were observed (Figure 3b). Both the Shb and the Grb2 antibodies co-immunoprecipitated two phosphotyrosine proteins of 22 and 25 kDa solely in the CD3 activated cells. The 22 kDa product is likely to be the z Figure 1 Northern blot of human tissues. (a) A multiple tissue chain (Chan et al., 1991). Northern blot was probed for the Shb cDNA. The positions of Similarities in the co-immunoprecipitation patterns the 3.1 and 2.5 kb mRNAs have been indicated. The tissues are between Shb and Grb2 suggested that the two might heart (he), brain (br), placenta (pl), lung (lu), liver (li), skeletal associate with one another. This was further investi- muscle (sm), kidney (ki), pancreas (pa), spleen (sp), thymus (th), prostate (pr), testis (te), ovary (ov), intestine (in), colon (co) and gated by testing Shb-immunoprecipitates for the peripheral blood leukocytes (pbl) presence of Grb2 (Figure 3c). Grb2 immunoreactivity Shb in T cell signaling MWelshet al 893 was demonstrated in Shb immunoprecipitates from introduction of Pro to Leu point mutations at both unstimulated and CD3 activated Jurkat cells positions 49 and 206 (Figure 3d). (Figure 3c). The speci®city of the Shb immunoprecipita- Finally, the association between Shb and the Grb2- tion of Grb2 was demonstrated in another experiment related protein GRAP (TruÈ b et al., 1997; Feng et al., (Figure 3c). No Grb2 immunoreactivity was detected 1996) was tested by transfecting a Myc-tagged GRAP after precipitation with NRS whereas Grb2 immunor- cDNA into the Jurkat cells and immunoprecipitation eactivity was again observed after immunoprecipitation with the Myc antibody (Figure 3e). The results show with the anti-Shb antibody. The data suggest an no detectable Shb-like reactivity in the Myc antibody association between Shb and Grb2 in Jurkat cells. immunoprecipitates of mock transfected cells, whereas The domain-interactions responsible for the associa- the GRAP-tagged cDNA transfected cells showed Shb- tion between Shb and Grb2 were investigated by reactivity corresponding to the p66 Shb isoform when performing GstGrb2 fusion protein binding experi- analysed by Western blotting. The co-immunoprecipi- ments (Figure 3d). A large amount of the p66 Shb tation of this was independent of CD3 cross-linking. component, and some of the p55 Shb isoform were Thus the data suggest that both Grb2 and GRAP are found in association with the GstGrb2 fusion protein. associated with Shb isoforms in the Jurkat cells, and These interactions were independent of activation of that this association shows little or no dependence on the Jurkat cells. Since the Shb antibody used here was activation of the T cell receptor. generated by immunization with a Shb fusion protein, reactivity with the GstGrb fusion protein itself was Association of phosphotyrosine proteins with various Shb tested (Figure 3d, bl=fusion protein not mixed with fusion proteins and identi®cation of p22 as the CD3 the Jurkat cell extract). No detectable reactivity with associated z chain the 66 and 55 kDa components was seen. The association between GstGrb2 and p66 and p55 Shb To obtain an understanding for the mechanisms behind was lost by inactivation of the Grb SH3 domains by the association of Jurkat cell phosphotyrosine proteins

b a Blot: PY20 Blot: PY-20 – + – + — 190 time: 0 0.5 1 2 5 15 60

97 — — 100

69 — — 70 IgGHC — — 55/57

46 — c — 36/38 – + – – + –

21 — — 22

NRS i.p. SHB i.p.

Blot: Shb 55/59 – + – + IgGHC —

— 55-59 Shb IgGHC — i.p.: shb shb shb+ shb shb shb+ shb shb NRS i.p. SHB i.p. Blot: Shb PY20

Figure 2 Shb immunoprecipitation of phosphotyrosine proteins. (a) Jurkat cells (107) were unstimulated (7) or stimulated by CD3 crosslinking for 2 min (+) before lysis and immunopreicipitation using normal rabbit serum (NRS) or the Shb antibody (Shb i.p.). The immunoprecipitates were electrophoresed on 9% acrylamide/0.24% Bis gels before Western transfer and incubation with the phosphotyrosine antibody PY-20. The blot has been divided into subsections to visualize the dierent phosphotyrosine proteins since these required dierent ECL exposure times. Shb immunoreactivity in the NRS and Shb immunoprecipitates is also shown. The molecular weight standards and IgG heavy chain (IgGHC) are indicated to the left and the phosphotyrosine products to the right. (b) Jurkat cells (107) were stimulated for the time points indicated by CD3 cross-linking, lysed and immunoprecipitated using the Shb antibody. The immunoprecipitates were subjected to Western blotting using the phosphotyrosine antibody PY-20. The reactivity with p55/57 kDa doublet is shown. (c) Jurkat cells (107) without treatment (7) or stimulated for 2 min with the CD3 crosslinking antibody (+) were lysed, nuclei pelleted, and the supernatants boiled for 2 min in 1% SDS. The samples were then diluted tenfold in lysis buer and Shb was then immunoprecipitated. The supernatant after immunoprecipitation from the untreated lysate (7) was transferred to another tube and subjected to a second Shb immunoprecipitation. The samples were subjected to Western blotting for Shb and phosphotyrosine. The positions of 55/59 Shb and IgG heavy chain (IgGHC) are indicated Shb in T cell signaling MWelshet al 894 to Shb, fusion proteins corresponding to the dierent p55 Shb product produced in the pET system bound parts of Shb were synthesized and used for binding the 22, 36/38, and 190 kDa proteins in a CD3 receptor experiments (Figure 4a). GST itself did not associate stimulation- and phosphorylation-dependent manner. with any of the Jurkat cell proteins. The GSTSH2 In addition to these products, small amounts of a fusion protein corresponding to the SH2 domain of 70 kDa phosphotyrosine protein was also found to Shb bound two phosphotyrosine proteins of 22 and 36/ associate with the p55 Shb protein. Although weak in 38 kDa in cell extracts from CD3-crosslinked Jurkat this experiment, the association of the 70 kDa cells. No detectable association of these was observed component to p55 Shb was strong in other experi- in the unstimulated cell extracts. Simultaneous addition ments (see Figure 5b). The production of p66 Shb in of 20 mM phosphotyrosine (PY) to the extract from the pET system was inecient, and thus the CD3-stimulated cells greatly reduced the binding of the corresponding experiments using this Shb isoform 22 and 36/38 kDa proteins. Using a Shb fusion protein could not be carried out. The PY-20 phosphotyrosine corresponding to the proline-rich motifs of p55 Shb antibody showed considerable background reactivity and the central region of Shb but lacking the SH2 with the GSTSH2 fusion protein (seen as a broad band domain (GSTp55DSH2), one major product from of 36 kDa), the GSTp55DSH2 fusion protein and its stimulated cells was observed; the p36/38 doublet. proteolytic degradation products (72 kDa and smaller) The association of this phosphotyrosine product was and the p55 Shb (as 62 kDa protein due to the completely blocked by 20 mM phosphotyrosine. The presence of pET-derived sequences).

a c Blot: PY20 Blot: Grb2 220 — – + – – + + – + – + + +

— Grb2

cell extr. Shb i.p. NRS Shb i.p. 97 —

d 66 — Blot: shb + – + bl – + bl 77 — 66 — 55 — 46 — p36/38 extr. wt Grb2 mut-49/206 Grb2

shb grb2 shb grb2 e cell extr. immunoprecipitation Blot: Shb b – + – + Blot: PY20 – + – – + +

IgGLC — — p66 Shb

21 — — p55 Shb shb grb2 shb grb2 Transfection: mock Myc-GRAP cell extr. immunoprecipitation

Figure 3 Association of Grb2, GRAP and other phosphotyrosine proteins with Shb. (a) Phosphotyrosine proteins associated with Shb and Grb2. Jurkat cells (56107 cells) that were either unstimulated (7) or stimulated by CD3 crosslinking (+) were lysed and immunoprecipitated using the Shb or Grb2 antibodies. The immunoprecipitates were electrophoresed on 9% polyacrylamide/0.24% Bis SDS-gels prior to transfer onto Immobilon ®lters. The ®lters were incubated with the PY-20 antibody and a secondary goat anti mouse-HRP antibody before ECL. In parallel, cell extracts (cell extr, 106 cells) were analysed. Molecular weight markers are indicated to the left and p36/38 to the right. (b) Phosphotyrosine proteins associated with Shb and Grb2 in the 20 ± 30 kDa molecular weight range. Cells were treated and analysed as in a. The position for the 21 kDa molecular weight marker and IgG light chain are indicated. (c) Shb co-immunoprecipitation of Grb2. Cell extracts (extr, 106 cells) or Shb immunoprecipitates (shb i.p., 26107 cells) from untreated (7) CD3 crosslinked (+) cells were subjected to SDS-gel electrophoresis on 12% polyacrylamide/ 0.32% Bis gels prior to Western transfer. The blot was incubated with the Grb2 antibody followed by an incubation with protein A/G HRP prior to ECL. In another experiment (two right lanes), the anti-Shb immunoprecipitation was compared with precipitation using normal rabbit serum (NRS). The position of Grb2 is indicated. (d) Binding of p66 and p66 Shb to wild-type or SH3 domain inactivated (mut 49/206) GstGrb2. Cell extracts (107 cells) from untreated (7) or CD3 crosslinked (+) cells were incubated with the fusion proteins and subjected to Western blotting using the Shb-antibody preblocked with 1 mg/ml GstGrb2. Blotting of fusion protein not mixed with extract is shown (bl) as well as an aliquot (extr, 106 cells) of the initial cell extract. The positions of p55, 66 and 77 Shb are indicated. (e) Association between Shb and Myc-tagged GRAP. Cells were mock transfected or transfected with the myc-tagged GRAP construct before lysis and immunoprecipitation using the Myc antibody. The samples were analysed by Western blotting using the Shb antibody. Untreated cells (7) or CD3 crosslinked cells (+) were immunoprecipitated from 56107 cells each. The positions of p66 and p55 Shb are indicated Shb in T cell signaling MWelshet al 895 To identify the nature of p22, blots were probed to the wild-type Shb protein was much more ecient with a CD3 z antibody (Figure 4b). The cell extracts than that to p55 Shb containing the R522K mutation contained a strong band of 16 kDa present in both (Figure 4b) which inactivates the Shb SH2 domain unstimulated and CD3 crosslinked cells and a weaker (Waksman et al., 1992). The binding of other 22 kDa band only present in the cell extracts from CD3 crosslinked cells. The 22 kDa product represents the tyrosine phosphorylated z chain, whereas the 16 kDa protein the unphosphorylated form of the z a chain (Chan et al., 1991). Immunoprecipitation with Blot: PY20 the Shb antibody revealed the coprecipitation of p22 only in extracts from CD3 crosslinked cells (Figure 4b). When testing the association of the p22 z chain to — 22 immobilized, bacterially produced p55 Shb, the binding

concentration zeta-1 peptide: 0 10 100 a Blot: PY20 b – + py – + py – + py – + py – + Blot: PY20 220 — — 190 gst55shb 55shb 97 — ∆sh2 66 —

46 — — 36/38 70 —

31 — — 22 21 — 36/38

peptide: 0 lnk 0 lnk lnk ∆lnk gst gstsh2 gst55shb 55shb cell ex. concentration: 0 10 0 10 100 100 ∆sh2

c b Blot: PY-20 Blot: CD3 ζ Blot: PY-20 peptide: 0 0 lnk 0 lnk + + – + – + + + — 190 p36/38 p36/38 — — 70 GstGrb2SH2 22 — extr p55Shb GstGrb2SH2 — 36/38 16 — R522KWT R522K W T d Blot: Shb cell extr. Shb i.p. p55 Shb p55 Shb Peptide conc: 0 5 50 200 Figure 4 Association of phosphotyrosine proteins and the CD3 associated z chain with Shb fusion proteins. (a) Cells from untreated (7) or CD3 cross-linked Jurkat cells (+) were lysed — gst55shb∆sh2 and mixed with the immobilized fusion proteins (107 cells) for 30 min on ice. In some cases, 20 mM phosphotyrosine (PY, Figure 5 Peptide competition experiments. (a) Eects of the zeta- pH=7.5) was added to the cell extract prior to mixing with the 1 peptide on the binding of p22 to Shb. Bacterially produced p55 fusion proteins. The samples were electrophoresed before protein Shb was incubated with Jurkat cell extracts from CD3 crosslinked transfer and incubation with the phosphotyrosine antibody PY- cells in the presence of dierent concentrations of phosphopeptide 20, goat anti-mouse HRP as a secondary antibody and ECL. (in mM). Binding of tyrosine phosphorylated p22 was analysed by Aliquots of the cell extracts (106 cells) were also electrophoresed. Western blotting using the phosphotyrosine antibody PY-20. (b) The positions of molecular weight markers are shown to the left, Cell extracts from CD3 cross-linked cells were incubated with the and the positions of p22, p36/38 and p190 to the right. The fusion p55 Shb fusion protein (55Shb) or the GST p55 Shb fusion proteins used were GST (gst), the GST-Shb SH2 domain (gstsh2), protein lacking the SH2 domain (gst55shbDsh2) in the presence of the GST-p55Shb lacking the SH2 domain (gst55shbDsh2) and the dierent micromolar concentrations of lnk or Dlnk peptides. The p55 Shb in pET (55shb). The PY-20 antibody showed background samples were then subjected to Western blotting using the reactivity with the gstsh2 fusion protein (36 kDa), gst55shbDsh2 phosphotyrosine antibody PY-20. The positions of p36/38 and (72 kDa and smaller degradation products) and 55shb (62 kDa). p70 are indicated. (c) Binding of p36/38 to p55 Shb or the The background reactivity with the fusion proteins was equal in GSTGrb2 SH2 domain fusion protein was determined with or all three lanes for each fusion protein and dominates the reactivity without the addition of 100 mM lnk peptide. The bound proteins in the unstimulated lanes. (b) Cells from untreated (7) or CD3 were electrophoresed and subjected to Western blotting as in b. crosslinked (+) Jurkat cells were lysed and immunoprecipitated The positions of p36/38 and background reactivity of the PY-20 (26107 cells) with the Shb antibody. Alternatively, aliquots of the antibody with the GSTGrb2 SH2 domain fusion protein are cell extracts (107 cells) were mixed with immobilized, bacterially indicated. (d) Binding of the gst55shbDsh2 fusion protein to produced p55 Shb or p55 Shb carrying the R522K mutation. phosphotyrosine-Sepharose beads in the presence of dierent After washing, the samples were electrophoresed on 12% micromolar concentrations of the lnk peptide. Binding was acrylamide/0.32% Bis gels before electrical transfer and incuba- performed for 30 min at 208C in 150 mM NaCl, 50 mM Tris, tion of the blot with the z chain and PY-20 antibodies. Aliquots pH=7.5. The samples were washed three times in the binding of cell extracts (106 cells) were also electrophoresed. The positions buer and subjected to Western blotting using the Shb antibody. of z chain isoforms 16 and 22 kDa are indicated, as well as 36/38, The ®gure shows only binding of the full-sized (72 kDa) fusion 70 and 190 kDa phosphotyrosine proteins protein and a 68 kDa proteolytic fragment Shb in T cell signaling MWelshet al 896 phosphotyrosine proteins to wild-type or R522K p55 relative anity of 1.4 or higher at the dierent Shb was similar (Figure 4b). The small amount of p22 positions are listed in Table 1. There was a relatively remaining bound to the R522K Shb protein could be strong selection for aspartic acid in position 3 and a indirect via other phosphotyrosine proteins. The results weaker selection for aspartic acid in position 2 in Figure 4 suggest distinct domain interactions upstream of the phosphotyrosine. No preferential between Shb and phosphotyrosine proteins p22, p36/ binding (41.5) of individual amino acids in the other 38, p70 and p190. p22 is the CD3 associated z chain positions could be observed. Thus the consensus and binds primarily to the Shb SH2 domain. sequence for the Shb PTB domain recognition site is Asp-Asp-X-pTyr in these positions. High anity binding between the Shb PTB domain and its target Peptide inhibition experiments sequence may require sequence speci®city in positions A phosphotyrosine peptide corresponding to an ITAM 74to77 relative the phosphotyrosine residue as well. motif of the z chain inhibited almost completely the It should be noted that the lnk peptide which contains association of the putative z chain 22 kDa phosphotyr- an aspartic acid in position 3 upstream of the osine protein to the p55 bacterially produced Shb phosphotyrosine inhibits the association between the protein at 100 mM (Figure 5a). Assessment by Shb PTB domain and p36/38. densitometric analysis of the association of p36/38 to the p55 Shb protein after addition of this peptide at Transfection experiments 100 mM in the same experiment revealed a 71% inhibition of binding (results not shown). A phospho- To study the eects of Shb-overexpression on T cell tyrosine peptide corresponding to a proposed phos- receptor signaling in Jurkat cells, JMC-T Jurkat cells phorylation site of the Lnk protein (Y297) and were transfected with the Shb cDNA by electropora- extending 8 amino acid residues towards the N- tion. The results demonstrated strong expression of the terminus (lnk peptide) caused a signi®cant, dose- 66 kDa Shb isoform (Figure 6a). This band migrated dependent inhibition of the association of the p36/38 as a broad band, possibly as a doublet in these phosphoproteins to bacterially expressed GSTp55DSH2 experiments. Elevated expression of the 55 kDa Shb and p55 Shb (Figure 5b). However, some binding isoform was also observed, although to a much lesser remained even at a peptide concentration of 100 mM, degree. Besides these two Shb isoforms, several possibly representing indirect binding via other prominent components of lower molecular weights adaptors, such as Grb2 and GRAP (TruÈ b et al., were seen (Figure 6a), probably representing proteoly- 1997; Fukazawa et al., 1995). A corresponding peptide tic degradation products. (Dlnk) comprising the same six residues C-terminally, The phosphotyrosine protein content of control or but only extending three amino acids towards the N- Shb-transfected cells was also studied (Figure 6b). terminus relative to the phosphotyrosine, failed to Comparison of the phosphotyrosine protein pattern signi®cantly in¯uence the binding of p36/38 to p55 Shb assessed by Western blotting using the phosphotyrosine (Figure 5b). This latter peptide is expected to only antibody PY-20 revealed increased phosphotyrosine in¯uence SH2 domain recognition, contrary to the lnk contents of proteins of 36/38 and 70 kDa in the peptide which is predicted to inhibit both the SH2 and unstimulated Jurkat cells after Shb transfection. These PTB domain interactions due to its N-terminal phosphotyrosine proteins appear identical to the extension (Wolf et al., 1995; TruÈ b et al., 1995). The corresponding components that interacted with the binding of the z chain to the p55 Shb protein was not p55 Shb protein (Figure 4a). After stimulation by CD3 in¯uenced by the lnk peptide (results not shown). In crosslinking the content of phosphotyrosine proteins Figure 5c, the eects of the lnk peptide on the was similar in the control and the Shb transfected cells association of p36/38 with the Grb2 SH2 domain is (Figure 6b). shown. The lnk peptide inhibited eciently the binding To assess the eects of the Shb SH2 domain of p36/38 to p55 Shb, whereas that to the Grb2 SH2 inactivation mutation R522K on TCR dependent domain was not aected. Background reactivity of the phosphotyrosine signaling, regular Jurkat cells were phosphotyrosine antibody with the GSTGrb2 SH2 mock transfected or transfected with the mutated domain fusion protein has been indicated. The lnk Shb cDNA by electroporation (Figure 6c). A peptide inhibited at 5 mM the binding of the moderate increase in the contents of the p66 and GSTp55DSH2 fusion protein to phosphotyrosine the p55 Shb proteins was observed. When the blot beads in in vitro experiments (Figure 5d). The was probed for phosphotyrosine content, the mock combined data with the lnk peptide in Figure 6b and transfected cells displayed increased phosphotyrosine 6d suggest that the GSTp55DSH2 fusion protein contents of several proteins after TCR stimulation contains a PTB domain which interacts with this peptide. Table 1 Recognition speci®city of the Shb PTB domain PTB domain binding sequence pY±3 pY±2 pY±1 pY+1 pY+2 pY+3 To assess the preferred binding sequence of the Shb D (2.5) D (1.9) S (1.4) D (1.4) D (1.5) D (1.5) PTB domain, immobilized GSTp55DSH2 fusion D (1.4) protein was mixed with a phosphopeptide library The GST55ShbDSH2 fusion protein was mixed with the phosphopep- containing combinations of all amino acids except tide library GAXXXpYXXXAKKK (with X any amino acid except C and W) and preferential binding at the dierent positions was cysteine and tryptophane in positions 1-3 N-terminally determined as previously reported (Songyang et al., 1994). The values and C-terminally relative the phosphotyrosine residue. are relative enrichments for the amino acids indicated. Values below Amino acids binding to the Shb PTB domain with a 1.4 are not included. Values above 1.5 are indicated with bold letters Shb in T cell signaling MWelshet al 897 by CD3 crosslinking (Figure 6d). These were of Shb cDNA, Jurkat cells transfected with the Shb molecular weights 36/38, 66, 70 and 100 kDa. In the cDNA construct lacking the entire SH2 domain Jurkat cells transfected with the R522K mutated Shb (ShbDSH2) displayed decreased tyrosine phosphor- cDNA the increase in tyrosine phosphorylation of ylation of the 36/38, 66 and 100 kDa phosphopro- several of these in response to CD3 crosslinking was teins in response to TCR stimulation. The combined diminished, especially for those of molecular weights data in Figure 6 suggest that Shb plays a role for of 36/38, 66 and 100 kDa (Figure 6d). To further TCR dependent signaling and complex formation of address a role of Shb for TCR signaling, another phosphotyrosine proteins. Shb construct which was entirely deleted of Transfection of Jurkat cells with the R522K mutated sequences coding for the SH2 domain was trans- Shb cDNA and subsequent clonal selection for fected into Jurkat cells, and the tyrosine phosphor- neomycin resistance yielded one clone named Jur- ylation in response to CD3 crosslinking determined katR522K-1 which over-expressed the 66 and 55 ± (Figure 6e). As in the case of the R522K mutated 59 kDa Shb isoforms (Figure 7). No signs of elevated

a c Blot: Shb Blot: Shb – + – +

— 66

— 55

Transfection: Mock R522K Shb — 66

— 55 d Blot: PY-20 – + – +

Transfection: mock Shb 97 — — 100 69 — b — 66 Blot: PY-20 – – + + 46 — — 36/38

Transfection: Mock R522K Shb

— 70 e Blot: PY20 – + – +

— 36/38 — 100 kDa

— 66 kDa

Transfection: mock + + — 36/38 kDa shb + + Transfection: mock Shb∆SH2 Figure 6 Transfection of the Shb cDNA. (a) SV40T-expressing Jurkat cells (JMC-T, Fukazawa et al., 1995) were mock-transfected or transfected with the Shb cDNA in pcDNA1 and were either untreated (7) or incubated with the CD3 antibody for 2 min (+). Cell extracts were prepared and analysed by Western blotting as in Figure 1. The blot was incubated with the Shb antibody and followed by ECL. The ®gure shows the Shb-reactivity of untreated extracts. The positions of p66 and p55 Shb are indicated. (b) Phosphotyrosine proteins of mock or Shb-transfected cells. Untreated (7) or CD3 cross-linked cell extracts taken 2 min after antibody addition (+) were blotted as in a and probed for phosphotyrosine proteins using the PY-20 antibody. The positions of the p36/38 and 70 kDa proteins are indicated. (c) Regular Jurkat cells were electroporated with pcDNA1 (mock) or pcDNA1 containing the R522K mutated Shb cDNA and maintained for 2 days before incubated in the absence (7) or presence (+) of the CD3 crosslinking antibody for 2 min. The cells were lysed (26106) and subjected to Western blotting for Shb after electrophoresis on 6% acrylamide/0.16% Bis gels. The positions of p55 and p66 Shb are indicated. Equal amounts of protein were electrophoresed in the dierent lanes. (d) The blot in c was probed for phosphotyrosine. The positions of molecular weight markers (left) and the p36/37, p66 and p100 proteins (right) are indicated. (e) A Shb cDNA lacking the SH2 domain (ShbDSh2) was constructed by inserting the 1.6 kb BamHI fragment of the Shb cDNA into the BamHI site of the expression vector pcDNA3.1 Myc-His. Jurkat cells were transfected by electroporation in the presence of empty vector (mock) or vector containing the partial Shb cDNA (ShbDSh2). The transfected cells were incubated in the absence (7) or presence (+) of the CD3 crosslinking antibody, and cell extracts were prepared for electrophoresis and Western blotting. The blot shows phosphotyrosine proteins of the molecular weights indicated Shb in T cell signaling MWelshet al 898 contents of the 77 and 80 kDa Shb immunoreactivity 190 kDa proteins after CD3 crosslinking (Figure 7), was observed in these cells (Figure 7). The Jur- although the dierence in tyrosine phosphorylation of katR522K-1 cells displayed a pattern of tyrosine the 66 kDa protein could be more easily visualized by phosphorylation in response to CD3 activation which a shorter ECL exposure (results not shown). The was very similar to that of the Jurkat cells transiently decreased tyrosine phosphorylation of 36/38, 66 and expressing the mutated Shb cDNA (Figure 7). 100 kDa proteins was also observed in the Jurkat cells Compared with a clone of neomycin resistant Jurkat transiently expressing the R522K mutated Shb (see cells (Jurkat-neo), the JurkatR522K-1 cells showed less Figure 6c). The JurkatR522K-1 cells also displayed a tyrosine phosphorylation of the 36/38, 66, 70, 100 and decreased MAP kinase activation after CD3 stimulation compared with the Jurkat-neo cells, as assessed by decreased tyrosine phosphorylation of the MAP kinase TEY motif (Figure 7). MAP kinase p42 was found to Blot: PY-20 be the main MAP kinase product tyrosine phosphory- – + – + lated in response to CD3 activation. Probing the same blot for the presence of total p44 MAP kinase revealed — 190 equal amounts of this protein in the Jurkat-neo and — 100 JurkatR522K-1 cells (Figure 7). Thus, the altered 70 pattern of tyrosine phosphorylation seen in Jurkat cells expressing Shb with an inactivated SH2 domain is 66 paralleled by decreased MAP kinase activation.

— 36/38 Discussion Jurkat- JurkatR522K- neo 1 In the present investigation we have attempted to assess the role of the adaptor protein Shb in signaling downstreams of the T cell receptor. Based on bacterial expression and transfection experiments, three Shb- isoforms of 55, 66 and 77 kDa have been described Blot: Shb – + – + previously, of which the ®rst two result from translation initiation at the second and ®rst methio- — 80 nine codons, respectively, whereas the 77 kDa isoform — 66 is likely to result from some post-translational — 55-59 modi®cation (Karlsson and Welsh, 1996). Subsequent studies of other cell types have revealed the existence of Jurkat- JurkatR522K- additional Shb isoforms. In PC12 cells, for instance, neo 1 three major Shb isoforms of 55, 57 and 59 kDa can be detected, all of which show elevated contents after transfection of these cells with the 2.3 kb human Shb cDNA (Karlsson and Welsh, unpublished data). Thus Blot: Phosphorylated MAPK the 2.3 kb Shb cDNA can generate many dierent Shb – + – + isoforms dierentially in various cells, presumably due — p42 MAPK to several translational and post-translational pro- cesses. All the observed Jurkat Shb isoforms, with the Jurkat- JurkatR522K- exception of the 80 kDa product, thus seem to be neo 1 related to the published Shb coding sequence. The present data show that transfection of Jurkat cells with the Shb cDNA dramatically alters the relative abundance of dierent Shb isoforms compared with Blot: MAPK the untransfected cells. A possible explanation for this – + – + is Shb overexpression in itself, which could shift the processing of the Shb mRNA or proteins. — p44 MAPK Jurkat- JurkatR522K- Immunoprecipitation experiments reveal the rapid neo 1 phosphorylation of two bands of 55 and 57 kDa which are likely to be Shb-associated proteins, since these Figure 7 Phosphorylation of Jurkat cells stably expressing R522K Shb. Jurkat cells were transfected by electroporation were not detected as phosphotyrosine proteins in the with pSV2-neo alone (Jurkat-neo) or the R522K mutated Shb Shb-immunoprecipitates performed after SDS-dena- cDNA in pcDNA1 together with pSV2-neo (JurkatR522K-1). turation of the cell extracts, despite ecient immuno- Transfected cells were plated in multi-well dishes and selected for precipitation of the Shb isoforms under these neomycin resistance in the presence of 1 mg/ml G418. Cells from one clone of each were stimulated for 2 min by CD3 crosslinking conditions. No evidence for tyrosine phosphorylation (+) or left unstimulated (7), after which cell extracts were of p66 Shb was obtained, although this isoform was electrophoresed and subjected to Western blotting as in Figure 6c. not eciently immunoprecipitated in the present The blot was tested for phosphotyrosine (PY-20), Shb (Shb), experiments. In a previous study, p66 Shb (given the activated (tyrosine phosphorylated) MAP kinases and total p44 apparent molecular weight of 69 kDa, Karlsson et al., MAP kinase. The positions of tyrosine phosphorylated proteins 190, 100, 70, 66 and 36/38 kDa, as well as 80, 66 and 55 ± 59 kDa 1995) was immunoprecipitated, suggesting that the Shb, p42 MAP kinase and p44 MAP kinase are indicated antibody has the ability to interact with this Shb Shb in T cell signaling MWelshet al 899 isoform. However, the inecient immunoprecipitation or lnk peptides (TruÈ b et al., 1997), and thus is retained with the Shb-antiserum is likely to have additional in the conditions of Figure 5b (see Figure 5c). The explanations besides a relatively low anity for native combined peptide inhibition data suggest the existence Shb isoforms. One possibility is proteolysis. This of at least two tyrosine phosphorylation sites on p36/ option seems probable, both considering the pattern 38, one interacting with the Shb PTB domain and the of reactivity of the antibody with components of lower other with the Grb2 SH2 domain. molecular weight on blots after immunoprecipitation, The Grb2 and GRAP proteins associate mainly with but also the pattern of blotting reactivity after p66 Shb. The data suggest that this interaction occurs transfection of the Shb cDNA. Another explanation between the SH3 domains and the proline-rich that should be kept in mind is masking of antigenic sequences of Shb. In previous work we tested the epitopes on Shb by binding to other cellular interaction between the N-terminal SH3 domain of components. This is particularly relevant for the Grb2 and the 4th and 5th proline-rich motifs of Shb interaction between Shb and p36/38, since the PTB (Karlsson et al., 1995) and could only detect a weak domain which is a major part of the association association. The most ecient association was pre- between the two was also used for immunization to sently observed between p66 Shb (which contains all generate the antibody. ®ve proline-rich motifs) and full-sized Grb2 GST The combined data suggest that Shb is an important fusion protein containing both SH3 domains, whereas adaptor linking the T cell receptor with other signaling inactivation of these by site-directed mutagenesis components in Jurkat cells. The SH2 domain seems to abolished the interaction. have one major interactions; the z chain. Previous Transfection and expression of the Shb cDNA assessment of the preferred binding sequence of this enhanced the basal tyrosine phosphorylation state of SH2 domain revealed strong selection for leucine in several proteins, mainly the 36/38 and 70 kDa proteins. position 3 downstream of the phosphotyrosine, making The 70 kDa protein was also observed to complex with binding to ITAM motifs on the T cell receptor Shb in the binding experiments. The results can be associated CD3 and z chains a possibility. The ITAMs interpreted to suggest that an increased Shb content are regularly spaced motifs containing two phosphotyr- facilitates complex formation in the absence of T cell osine residues, each with a leucine in position three C- receptor stimulation, and that such complex formation terminal to phosphotyrosine. The present data indeed enhances the tyrosine phosphorylation state of some of suggest that Shb is likely to bind via its SH2 domain to its components. Based on the these observations, it can the ITAM motif, although it is not clear whether only be speculated that Shb-mediated complex formation one or several of the ITAM phosphotyrosine residues is may play a role in the function of p36/38, previously recognized. Besides the z chain, we have obtained data implicated in the possible regulation of the Ras suggesting that p36/38 is also a binding substrate for the pathway (Sieh et al., 1994; Buday et al., 1994). Shb SH2 domain based on GST-fusion protein Although we failed to detect an increased activation experiments. However, the interaction of p36/38 with of MAP kinases in Jurkat cells over-expressing the p55 Shb is not likely to involve the SH2 domain to any wild-type Shb (results not shown), clonally selected large extent, since the Dlnk peptide which contains the Jurkat cells overexpressing R522K mutated Shb pYTPL sequence failed to aect the interaction between displayed decreased MAP kinase activation in re- p55 Shb and p36/38 and since the SH2 domain sponse to CD3 stimulation, suggesting a role for Shb inactivation mutation (R522K Shb) bound p36/38 in this process. Furthermore, the data suggest that the almost as well as the wild-type p55 Shb. Shb-Grb2 complex may be targeted in parts to the T The data suggest that the central portion of Shb is a cell receptor by binding of the Shb SH2 domain to the PTB domain, and that the main phosphotyrosine z chain. This notion is supported by the failure of the protein interacting with this PTB domain is p36/38. R522K mutated Shb, which has an inactivated SH2 The fusion protein containing this part of Shb, but domain, to eciently bind the z chain. As a lacking the SH2 domain, associated in a phosphotyr- consequence of this, the pattern of Jurkat cell tyrosine osine dependent manner with p36/38. The association phosphorylation in response to TCR activation is of p36/38 to the PTB-domain fusion protein or p55 dramatically altered. There has been a signi®cant Shb was inhibited in a dose-dependent manner by a lnk recent controversy about the potential role of Grb2 peptide extending 8 amino acid residues towards the N- versus Shc in linking the z chain to Sos and the Ras terminus, but not by a similar peptide extending only pathway (Ravichandran et al., 1993; Osman et al., three residues towards the N-terminus. A long N- 1995). It is possible that potential dierences in these terminal peptide extension relative the phosphotyrosine studies partly derive from the participation of Shb. is a characteristic feature of PTB-domain recognition In summary, our data suggest that Shb forms (Wolf et al., 1995; TruÈ b et al., 1995). Finally, the multimeric complexes in Jurkat cells, consisting of the binding of the PTB-domain fusion protein to z chain, Shb, p36/38 and Grb2 (or GRAP). This phosphotyrosine beads was signi®cantly inhibited at complex in the activated T cell has the potential to 5 mM of lnk peptide. Although the lnk peptide dock other signaling components via SH2, SH3 and produced a substantial inhibition of the association proline-rich domains and phosphotyrosine residues of p36/38 to the Shb PTB domain, some remaining present on p36/38. Such other signaling components binding was observed (Figure 5b). This could re¯ect include the 55/57, 70, 100 and 190 kDa phosphotyr- indirect association through other adaptors which bind osine proteins found to interact with Shb, as well as both p36/38 and Shb. Two such candidates are Grb2 phospholipase C g, Sos and p85 PI-3 kinase, of which and GRAP which interact with p36/38 via their SH2 the three latter have been detected in association with domains (TruÈ b et al., 1997; Fukazawa et al., 1995). The Grb2 and p36/38. In conclusion, our results suggest binding of these to p36/38 is not in¯uenced by the Dlnk that Shb, which encompasses three distinct structural Shb in T cell signaling MWelshet al 900 modules plays a signi®cant role in assembling of 106 cells/ml. The cells were collected by centrifugation proximal signaling complexes downstream of the T and suspended in RPMI 1640 medium lacking serum (108 cell receptor. cells/ml) before stimulation with the SBV-3Tb CD3 antibody (Reedquist et al., 1996) at 378C for the time periods given. The cells were lysed in 50 mM Tris, pH=7.5, 150 mM NaCl, 0.5% Triton X-100, 10 mM NaF, 1 mM NaVanadate, Materials and methods 1mM PMSF and 100 U/ml aprotinin (108 cells/ml) on ice. After 30 min, nuclei were pelleted by centrifugation at Peptides and reagents 12 000 g for 15 min at +48C, the supernatants were cleared The anity puri®ed Shb antiserum has been previously with 0.5 ml protein A Sepharose/ml extract for 15 min described (Karlsson and Welsh, 1996). The Grb2 antibody before immunoprecipitated with 15 mg/ml anity puri®ed (recognizes the C-terminal portion of the protein), the Shb antibody (Karlsson and Welsh, 1996), or 1.5 mg/ml MAP kinase p44 (ERK1) and the CD3 z chain antibody Grb2 antibody for 90 min on ice. Immunoprecipitates were were from Santa Cruz Biotechnology, CA. The phospho- pelleted by protein A Sepharose (50 ml). Alternatively, tyrosine antibody PY-20 was obtained from Transduction extracts were incubated with the immobilized fusion Laboratories and the antibody recognizing tyrosine proteins (1 ± 5 mg) for 30 min on ice. The samples were phosphorylated MAP kinases was from New England electrophoresed on SDS-polyacrylamide gels (Laemmli, Biolabs. An enhanced chemiluminescence system and 1970), transferred electrophoretically onto Immobilon protein A/G-peroxidase were from Pierce, Rockford, IL. ®lters (Millipore) and subsequently incubated with block- The goat anti-mouse and rabbit peroxidase-conjugated ing solution, primary antibody, secondary antibody (goat antibodies were from Sigma, St Louis, MO. The peptides anti-mouse HRP, goat anti-rabbit HRP or protein A/G used were: zeta1=NQLpYNELNLGRRGEpYDVLDKR, HRP) before washing and enhanced chemiluminescence lnk=HLRAIDNQpYTPLSQL and Dlnk=DNQpYTP- (ECL). In some experiments, SV40 T-expressing Jurkat cells LSQL. These were synthesized by FMOC chemistry and (JMC-T) were transfected with the Shb cDNA inserted into HPLC puri®ed before use (Piccione et al., 1993). pcDNA1 (Invitrogen, LaJolla, CA) or the Myc-tagged GRAP construct by electroporation 2 days before the binding experiments (Fukazawa et al., 1995). DNA constructs and fusion proteins The GstGrb2 fusion protein was kindly provided by Dr M Northern blot analysis Moran. To inactivate the SH3 domains of Grb2, the SH3 domain proline residues at position 49 and 206 were The 2.3 kb human Shb cDNA was isolated and 25 ng were 32 9 converted to leucine residues by polymerase chain reactions labeled with P-dATP to 2.6610 d.p.m./mgusinga using mutant primers to inactivate the SH3 domains. random hexamer labeling system (Stratagene). The probe Similarly, mutant primers were used to introduce an was hybridized to multiple human tissue mRNAs Northern R552K mutation of the Shb cDNA by polymerase chain blots (Clontech) according to recommendations by the reaction. The Shb cDNA from the ®rst BalIsiteat manufacturer. The membranes were washed at 658Cin nucleotide 574 to the 3' end was introduced into the NotI 0.1% SDS+15 mM NaCl, 1.5 mM Na citrate before site of pET 30a after blunt-end formation by T4 DNA exposure to a storage phosphor screen for 6 h (Molecular polymerase. This vector produces the p55 Shb protein with Dynamics). additional His-tag, protein S-tag, thrombin and enteroki- nase sequences in the N-terminus. The p55 Shb fusion protein was produced and puri®ed on Ni-beads according to the manufacturer (Novagen). The GSTp55DSH2 domain Abbreviations fusion protein construct was obtained by introducing the SH2, Src Homology 2; SH3, Src Homology 3; PTB, 1.1 kb BamHI fragment from the p55 Shb-pET construct Phosphotyrosine binding; ITAM, Immunoreceptor tyro- into the BamHI site of pGEX4T-2. The GSTSH2 domain sine-based activation motif; GST, Glutathione S-transfer- fusion protein construct has previously been described ase; PDGF, Platelet-derived growth factor; FGF, (Welsh et al., 1994). Gst fusion proteins were puri®ed by Fibroblast growth factor; TCR, T cell receptor. binding to glutathione-agarose beads. For the myc-tagged GRAP (TruÈ b et al., 1997) cDNA construct, an XhoIsite was introduced immediately upstream of the cDNA start Acknowledgements codon. In addition, nucleotides coding for a Myc epitope We are grateful to Dr E Ottinger for providing the zeta-1 EQKLISEDL were introduced between the site encoding peptide, Dr S Pluskey for providing the phosphotyrosine- GRAP residue 217 and the stop codon, followed by a Sepharose beads and Dr Lena Claesson-Welsh for sugges- BamHI site. The XhoI±BamHI fragment of the myc-tagged tions and reviewing the manuscript. The work was GRAP sequence was inserted into pSRaneo and used for supported by NIH (grants DK45943 and AR36308), transfection. Diabetes and Endocrinology Research Center (Dk36836), the Swedish Medical Research Council (12X-10822, 12P- 11493), the Swedish Diabetes Association, the Swedish Binding experiments Society for Medicine, the Novo Nordisk Fund, the Juvenile

Jurkat cells were cultured at 378Cin5%CO2 in RPMI 1640 Diabetes Foundation International and the American containing 10% fetal calf serum and antibiotics at a density Cancer Society (IM-770).

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