Báo cáo Y học: Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells pptx

Henriksson2, Bengt Hallberg2and Michael Welsh1 1 Department of Medical Cell Biology, Biomedicum, Uppsala University, Sweden; 2 Department of Medical Biosciences/Pathology, Umea˚ Universi

Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells

Cecilia K Lindholm1, Maria L Henriksson2, Bengt Hallberg2and Michael Welsh1

1 Department of Medical Cell Biology, Biomedicum, Uppsala University, Sweden; 2 Department of Medical

Biosciences/Pathology, Umea˚ University, Sweden

This study addresses the interactions between the adaptor

protein Shb and components involved in T cell signalling,

including SLP-76, Gads, Vav and ZAP70 We show that

both SLP-76 and ZAP70 co-immunoprecipitate with Shb in

Jurkat T cells and that Shb and Vav co-immunoprecipitate

when cotransfected in COS cells We also demonstrate,

utilizing fusion protein constructs, that SLP-76, Gads and

Vav associate independently of each other to different

domains or regions, of Shb Overexpression of an SH2

domain-defective Shb causes diminished phosphorylation of

SLP-76 and Vav and consequently decreased activation of c-Jun kinase upon T cell receptor (TCR) stimulation Shb was also found to localize to glycolipid-enriched membrane microdomains (GEMs), also called lipid rafts, after TCR stimulation Our results indicate that upon TCR stimula-tion, Shb is targeted to these lipid rafts where Shb aids in recruiting the SLP-76–Gads–Vav complex to the T cell receptor f-chain and ZAP70

Keywords: Shb; V av; SLP-76; T cell receptor; lipid rafts

The early events in T cell signalling involve the activation of

Lck, which subsequently phosphorylates the TCR f-chain

and the e-chain of the CD3 complex on ITAM motifs

(immunoreceptor tyrosine-based activation motifs) This

recruits the kinases ZAP70 or Syk that consequently induces

tyrosine phosphorylation of several intracellular substrates,

for example, phospholipase C-c1 (PLC-c1), LAT [1], Vav

and SLP-76 [2] Functional interactions between these

effector molecules are believed to be necessary for T cell

maturation, proliferation and differentiation

One aspect of T cell receptor (TCR) activation relates to

specific areas (microdomains) of the T cell plasma

mem-brane, rich in glycosphingolipids, cholesterol and

GPI-anchored proteins, but poor in phospholipids (reviewed in

[3]) These glycolipid-enriched membrane microdomains

(GEMs), or lipid rafts, are rich in PTKs (protein tyrosine

kinases), monomeric and trimeric G proteins and several

other signaling proteins In resting T cells LAT, Lck, Fyn,

Syk, Cbl and Ras are located in rafts, whereas some other

proteins are recruited to the rafts upon T cell receptor

stimulation The latter group includes SLP-76, Gads, Vav,

ZAP70, TCR f-chain, PLC-c1, Shc and PKC [4,5]

The adapter proteins SLP-76 and Gads and the guanine

nucleotide exchange factor Vav are critical for appropriate

T cell activation SLP-76 and Vav are both phosphorylated upon CD3 stimulation in Jurkat T cells SLP-76 is phosphorylated by ZAP70 [6] at multiple residues (Tyr113, Tyr128 and Tyr145) of which Tyr113 and Tyr128 are responsible for allowing the binding of the Vav SH2 domain [7] SLP-76 has also been shown to associate with Lck, LAT, Grb2 and SLAP-130 in Jurkat T cells Vav is reported to be phosphorylated by Lck on Tyr174 and this process activates the exchange activity of Vav [8,9] However, other studies have suggested

tyrosine-174 on Vav as a putative candidate for phosphorylation by Syk and Zap70 [10] Vav is a guanine nucleotide exchange factor for the GTPases Rac1 and Cdc42, which cause, among other things, activation of the c-Jun N-terminal kinases (JNK) Gads (Grb2-related adaptor downstream of Shc) is not phosphorylated in response to TCR stimulation, but it has been shown to associate with several proteins upon T cell activation, including Shc, SLP-76 and LAT [11,12]

The Shb adapter protein was cloned from a b-cell library

in 1994 [13], but it has since been found to be ubiquitously expressed [14] Shb is involved in FGFR-1 and PDGFR signaling, and also associates with several signaling proteins, including CrkII, Eps8, Grap and Src [14,15] Shb is also of importance for the TCR-dependent immune response in Jurkat cells TCR-mediated activation of NFAT was totally abolished in Jurkat cells expressing a mutant form of Shb with a defective SH2 domain, and endogenous IL-2 production was also decreased in these cells [16] Overex-pression of wild-type Shb, however, had no effects on the CD3 mediated responses in Jurkat cells [14,16] In Jurkat cells, Shb has previously been found to exhibit domain-dependent interactions with Grb2, LAT, PLC-c1 and the f-chain of the T cell receptor [14–16] The SH2 domain of Shb bound the f-chain, the PTB domain bound LAT and the proline-rich regions associate with PLC-c1 and Grb2 There are also four putative tyrosine phosphorylation sites

in Shb that show extensive homology with similar sequences

in other adapter proteins [17], and conform with the consensus sequence Y-X-D/E/T/Q-P-F/Y/W-D/E

Correspondence to C Lindholm, Department of Medical Cell Biology,

Box 571, Biomedicum, 75123, Uppsala, Sweden.

Fax: + 46 18 556401, Tel.: + 46 18 4714033,

E-mail: Cecilia.Lindholm@medcellbiol.uu.se

Abbreviations: TCR, T cell receptor; SH2, Src homology 2; PTB,

phosphotyrosine binding; ITAM, immunoreceptor tyrosine-based

activation motif; PTK, protein tyrosine kinase; ECL, enhanced

chemiluminescence system; JNK, c-Jun N-terminal kinase; GST,

glutathione-S-transferase; PDGF, platelet-derived growth factor;

FGF, fibroblast growth factor; P-Y, phosphotyrosine; MAPK,

mitogen-activated protein kinase; GEMs, glycolipid-enriched

membrane microdomains; LAT, Linker for Activation of T cells.

(Received 13 February 2002, revised 16 May 2002,

accepted 21 May 2002)

In this study, we describe interactions between Shb and

SLP-76, Gads, Vav and ZAP70 and how a functional Shb

SH2 domain is important for phosphorylation of SLP76

and Vav, possibly by ZAP70 The Shb SH2 domain is also

of importance for the activation of the JNK, downstream of

Vav in the T cell signalling cascade We have also observed

the recruitment of Shb to lipid rafts upon TCR stimulation

in Jurkat T cells

M A T E R I A L S A N D M E T H O D S

Antibodies

Anti-phosphotyrosine Ig (4G10) and anti-Vav Ig were

purchased from Upstate Biotechnology (Lake Placid, NY,

USA) CD3 antibody (CD3 pure) was from

Becton-Dickinson (San Jose, CA) Anti-JNK Ig and

anti-(phos-pho-JNK) Ig were from New England BioLabs (Beverly,

MA) HA antibody was from Santa Cruz

Anti-ZAP70 Ig, anti-SLP-76 Ig and anti-Rac1 Ig were from

Transduction Laboratories (Lexington, KY) Rabbit

poly-clonal anti-Gads Ig was a gift from Jane McGlade, Hospital

for Sick Children Research Institute, Toronto, Canada

Affinity purified anti-Shb Ig has been described previously

[18]

DNA constructs

The Shb–SH2–GST fusion protein plasmid has been

described previously [13] The Shb–PTB-Pro–GST plasmid

was described previously under the name p55 ShbDSH2 [14]

and produces a fusion protein corresponding to the PTB

domain and two proline-rich sequences The p55 Shb–pET

plasmid has been described previously [14] and produces the

full-length p55 Shb with an additional His-tag for

purifica-tion on Ni-beads according to the manufacturer (Novagen)

The Shb–PTB–GST plasmid was constructed using the

primers 5¢-GGGATCCTTCCAGGACCCCTAC-3¢ and

5¢-AGAATTCAGGGCTCCCATGTTT-3¢ corresponding

to the Shb cDNA nucleotides 840–1740 The amplified

fragment was digested using EcoRI, and ligated into EcoRI

digested pGEX-2T vector The SLP-76-Pro–GST plasmid

was constructed using the primers 5¢-CGAGGGATCCCT

GCAGAACTCCATCCTGCCTG-3¢ and 5¢-CATTTAAT

GAATTCTCTTCCTCCGC-3¢ corresponding to SLP-76

nucleotides 466–1245 The amplified fragment was cut using

BamH1 and EcoR1 and ligated into pGEX-2TK The

SLP-76-SH2-GST plasmid was constructed using the

primers 5¢-GGAAGGATCCAATTCATTAAATGAAGA

GTG-3¢ and 5¢-GGCTATAACGAATTCTGGGTACCC

TGCAGCATG-3¢ This fragment was also cut with BamH1

and EcoR1 and ligated into pGEX-2TK The

PAK-CD-GST (PAK Crib domain) plasmid has previously been

described previously [19]

The Shb wild-type plasmid contains the Shb cDNA

inserted in pcDNA1 vector, and has been described

previously The Shb R522K plasmid has also been described

previously [14] Briefly, the arginine at position 522 was

converted to a lysine, in full-length Shb cDNA, and inserted

in pcDNA1 expression vector The Shb DPTB-Tyr plasmid

was constructed by the deletion of the nucleotides 880–1603

in the Shb cDNA (containing the four tyrosine

phosphory-lation sites and the PTB domain), using the restriction

enzymes FspI and BstEII The hemeagglutinin-tagged SLP-76 vectors were constructed as follows The SLP-76 fragment (1–534), the SLP-76-Tyr fragment (1–155) and the SLP-76-SH2 fragment (415–534) were cut out from the above mentioned pGEX-2TK vectors using BamH1 and EcoR1 and ligated into pSG5A vector, containing an HA tag The Vav expression plasmid was a kind gift from A Weiss, San Francisco, CA, USA

Transient transfections COS cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FcII and antibiotics The cells were washed three times in serum-free DMEM and transfected with 2 lg of each plasmid or emply vector, as indicated in the figures, using Lipofectamine as recommended by the manufac-turer Jurkat T cells were washed twice in NaCl/Pi and transfected by electroporation (380 V, 950 lF in a 1-cm cuvette) with 15 lg of each plasmid as indicated in figure 3

Binding experiments and immunoprecipitations Jurkat cells or Jurkat cells overexpressing Shb R522K [16], were maintained in RPMI medium supplemented with 10% FcII (Hyclone) and antibiotics COS cells were maintained

in DMEM medium supplemented with 10% FcII Jurkat cells were collected by centrifugation and suspended in RPMI 1640 medium lacking serum before stimulation with the CD3 antibody at 37C for 2 min COS cells were either unstimulated or stimulated with pervanadate for 20 min at

37C Jurkat cells were pelleted by centrifugation and COS cells were scraped together using a rubber policeman The cells were lysed in Triton lysis buffer (0.15MNaCl, 0.05M Tris pH 7.5, 0.5% Triton X-100, 1 mM NaF, 0.1 mM orthovanadate, 100 UÆmL)1trasylol, 2 mM phenylmethane-sulfonyl fluoride) for 10 min Nuclei were pelleted by centrifugation and cell extracts were incubated with the immobilized fusion proteins on glutatione Sepharose beads (Amersham-Pharmacia Biotech, Uppsala, Sweden) or Ni-beads (Novagen) for 30 min on ice and then washed three times with NaCl/Pi/1% Triton Alternatively, the cell extracts were preincubated with protein A Sepharose for

15 min (Jurkat cell extracts only) and then immunoprecipi-tated for 1 h with antibodies against Shb, SLP-76, Vav, ZAP70, HA or preimmune sera (IgG) Immunoprecipitates were pelleted using either protein A or protein G Sepharose (50 lL) and then washed three times with NaCl/Pi/1% Triton The fusion protein complexes, cell extracts or the immuno-complexes were then resolved on SDS/PAGE and subjected to Western blotting onto Immobilon filters (Millipore) in 20% methanol, 190 mMglycine, 23 mMTris and 0.02% SDS The blots were subsequently incubated with blocking solution (5% BSA in NaCl/Pi/0.5% Tween) and primary antibody as indicated Immuno-reactivity was detected using horseradish peroxidase-conjugated secondary antibodies and ECL (Amersham–Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions For detection of Rac1 activity, the amount of Rac1 bound to the PAK-CD fusion protein was determined using Rac1 antisera, and normalized to total Rac1, in cell lysate

Peptide synthesis and peptide inhibition experiments

Peptides corresponding to tyrosine-phosphorylation sites in

SLP-76 and Vav were synthesized The peptides used in this

paper include: Y113: WSSFEEDDYESPND,

SLP-Y128: QDGEDDGDYESPNE, SLP-Y145: APVEDDA

DYEPPPS, Vav-Y174: NEEAEGDEIYEDLMRL and

Vav-Y692: ILANRSDGTYLVRQRV, where Y indicates

phosphotyrosine For peptide inhibition experiments, cell

lysates prepared as above were incubated with

Shb–PTB-Pro–GST, Shb–PTB–GST or p55 Shb–pET fusion protein in

the presence of 100 lMphosphorylated peptide or

phospho-tyrosine (10 mM) at 4C for 30 min After three washes with

NaCl/Pi/1% Triton, the samples were separated by SDS/

PAGE and transferred onto Immobilon filter as described

above Anti-SLP76 Ig and anti-Vav Ig were used to detect the

amount of these proteins bound to the Shb fusion protein

Isolation of GEM fractions

Jurkat cells (5· 107) where either left unstimulated or

stimulated with CD3 antibody at 37C for 3 min, followed

by lysis at 4C for 30 min in 1 mL of Mes-buffered saline

(25 mM Mes, pH 6.5, 150 mM NaCl, 5 mM EDTA, 1%

Triton X-100, 0.05 mMorthovanadate, 100 U mL)1

trasy-lol, 10 mMNaF, 1 mMPefablock) The lysates where then

mixed with 1 mL 80% sucrose in Mes-buffered saline and

transferred to ultracentrifuge tubes The samples were

overlaid with 2 mL of 30% sucrose in Mes-buffered saline,

followed by 5% sucrose in Mes-buffered saline The

Triton-insoluble fractions were separated from the cell lysates by

ultracentrifugation for 22 h at 250 000 g in a Beckman

SW50.1 rotor at 4C Fractions (400 lL) were removed

sequentially starting from the top of the gradient The

proteins in each fraction were precipitated using 20%

trichloroacetic acid The precipitates were washed with

acetone, dried and resuspended in 40 lL of SDS-sample

buffer The fractions (fraction 1–6 contain the lipid rafts)

were then resolved on SDS/PAGE and subjected to Western

blotting as described above The blots were subsequently

incubated with blocking solution (5% BSA in NaCl/Pi/

0.5% Tween) and primary antibody as indicated

Immuno-reactivity was detected as described above

R E S U L T S

Expression of Shb with an R522K mutation in the SH2

domain affects the co-immunoprecipitation of several

tyrosine phosphorylated proteins

To further investigate the protein-interactions of the Shb

adapter protein, we attempted to identify additional binding

partners for Shb in Jurkat T cells Shb immunoprecipitation

in Jurkat T cells revealed several coimmunoprecipitated

proteins (Fig 1) We detected phosphoproteins

correspond-ing to the previously identified proteins PLC-c1 at 160 kDa,

Linker for Activation of T cells (LAT) at 35 kDa and the

TCR f-chain at 22 kDa, but also proteins of 100, 75 and

70 kDa The association between Shb and PLC-c1 was

previously shown to be independent of TCR activation,

whereas the interactions between LAT or the f-chain and

Shb were found to be dependent on TCR activation [14,16]

We have also described the R522K mutation in the SH2

domain of Shb [14,16], and how expression of this Shb mutant decreases the tyrosine phosphorylation of PLC-c1, LAT and the TCR f-chain Jurkat R522K-2 cell lysates displayed reduced tyrosine phosphorylation of proteins migrating as 160, 100, 75 and 35 kDa (Fig 1) upon CD3 stimulation Shb immunoprecipitation of the R522K cell lysates revealed decreased phosphotyrosine content of the bands corresponding to PLC-c1 (160 kDa), LAT (36 kDa) and the f-chain (22 kDa) In addition, the Shb immuno-precipitations exhibited reduced tyrosine phosphorylation

of the 75 and 100 kDa proteins, in the Jurkat R522K-2 cells, unlike the 70-kDa product, which was not, or only slightly, affected Probing the blot with an antibody reactive with SLP-76 revealed a weak band at 75 kDa that was detected

in the CD3-stimulated control cells The R522K-2 cells exhibited the presence of SLP-76 regardless of whether the cells were stimulated with CD3 or not The reduced phosphorylation of the 160-, 100-, 75-, 35- and 22-kDa proteins in Jurkat cell lysates has previously been verified in another clone overexpressing R522K Shb and after tran-sient transfection of the R522K Shb cDNA [14,16]

Fig 1 Shb coimmunoprecipitates with several tyrosine phosphorylated proteins Jurkat-neo or Jurkat R522K-2 cells (107) were left unstimu-lated (–) or were stimuunstimu-lated with anti-CD3 Ig for 2 min in 37 C (+) The cells were then lysed in triton lysis buffer and cell extracts were precleared with protein A Sepharose and then subjected to a-Shb immunoprecipitation The precipitates and cell extracts were resolved

on SDS/PAGE and blotted for SLP-76, Shb and phosphotyrosine using the 4G10 antibody The positions of SLP-76, Shb, LAT and molecular mass markers are indicated.

Shb associates with SLP-76, ZAP70 and Vav

in CD3 stimulated Jurkat T cells

To further elucidate the identity of the 75-kDa protein seen

after a-Shb immunoprecipitation, another blot was probed

with anti-(SLP-76) Ig SLP-76 was present in a-Shb

immunoprecipitates after CD3 stimulation, but not after

immunoprecipitation with preimmune serum (Fig 2A)

Likewise, the 70-kDa protein that coimmunoprecipitated

with Shb was identified as ZAP70 (Fig 2A)

To assess the mode of interactions between Shb and

SLP-76 or ZAP70, GST fusion proteins comprising p55

ShbDSH2 (Shb–PTB-Pro–GST) [14], Shb–PTB–GST,

Shb–SH2–GST [13] or GST (as control) were incubated

with cell extracts from CD3 stimulated or unstimulated

Jurkat cells (Fig 2B) We also tested if phosphotyrosine

could inhibit the binding between fusion protein and cell

protein ZAP70 was found to associate with both the Shb– PTB-Pro-GST fusion protein and the Shb–SH2–GST fusion protein, and these associations were slightly increased after CD3 stimulation The interaction between the SH2 domain and ZAP70 was completely blocked with phospho-tyrosine, whereas binding of ZAP70 to the Shb–PTB-Pro– GST fusion protein was only partially inhibited under these conditions The blot was also stained with amidoblack to verify that equal amounts of Shb–PTB-Pro–GST fusion protein were used

SLP-76 was found to associate with the Shb–PTB-Pro– GST fusion protein, containing both the PTB domain and two of the N-terminal proline-rich domains This interac-tion was only slightly inhibited by phosphotyrosine To test

if the Shb PTB domain could be involved in this interaction

we searched the protein-sequence of SLP-76 for D-D-X-Y, which is the consensus sequence for the Shb PTB domain

Fig 2 Association of Shb with SLP-76 and ZAP70 in CD3 stimulated T cells and pervanadate stimulated COS cells Jurkat cells (107) were unstimulated (–) or stimulated by CD3 cross-linking for 2 min in 37 C (+) before lysis in triton lysis buffer (A) Cell extracts were subjected to immunoprecipitation using normal rabbit serum (IgG) or anti-Shb antibody (Shb) (B) Cell extracts were incubated for 30 min on ice with the indicated immobilized fusion proteins in the presence (+) or absence (–) of 10 m M phosphotyrosine (P-Y) (C) Cell extracts were incubated for

30 min on ice with immobilized p55 Shb–pET fusion protein in the absence or presence of tyrosine-phosphorylated peptides or phosphotyrosine (P-Y) The peptides correspond to tyrosines 113, 128 and 145 in SLP-76 and tyrosines 174 and 692 in Vav SLP-3Y contains all three SLP-peptides (D) Cell extracts were incubated for 30 min on ice with immobilized SLP-76-Pro-GST or SLP-76-SH2-GST fusion proteins (E) COS cells, transfected with Shb and different HA-tagged SLP-76 constructs or vector, as indicated, were stimulated, or not, with pervanadate, for 15 min in

37 C, before lysis The lysates were subjected to immunoprecipitations using anti-HA Ig (F) Jurkat cell extracts were incubated with immobilized p55 Shb–pET fusion protein in the absence (–) or presence (+) of phosphotyrosine (P-Y) The samples in (A–F) were resolved on SDS/PAGE The blots were probed with the antibodies indicated and the blot in (B) was stained using amido-black The positions of SLP-76, ZAP70, Gads and Shb are indicated with arrows.

binding site [14] Three such sequences were found, all

previously reported to be involved in cell signalling [7,20]

Three synthetic phosphopeptides were made corresponding

to these three sites (SLP-Y113, SLP-Y128 and SLP-Y145)

However, no inhibition of SLP-76 binding to the Shb fusion

protein could be seen using these peptides (Fig 2C) In

addition, the Shb PTB domain fusion protein does not

allow binding of SLP-76 (results not shown) The two

LAT-peptides previously reported to inhibit the LAT–Shb

association [16] were also tested, and had no inhibitory

effect on SLP-76, ZAP70 or Vav association to Shb (results

not shown)

To characterize the Shb–SLP-76 interaction, we utilized

fusion proteins corresponding to the SH2 domain of

SLP76 and the proline-rich regions of SLP-76 We found

that Shb associates with the SH2 domain of SLP-76, but

not to the proline-rich regions of SLP-76 (Fig 2D), and

that this association was increased by CD3-stimulation To

test this further, we overexpressed HA-tagged SLP-76 or

different domains of SLP-76, together with wild type Shb

in COS cells, and performed HA-immunoprecipitations

Figure 2E shows that Shb is associated with wild-type

SLP-76 and the SH2 domain of SLP-76 upon pervanadate

stimulation (to increase the degree of protein tyrosine

phosphorylation) in COS cells We then hypothesized that

the interaction between Shb and SLP-76 might be of a

trimeric nature, where the SH2 domain of SLP-76

asso-ciates with a phosphotyrosine motif in Shb, and the proline

rich sequences of Shb interact with some other adaptor,

which also has the ability to bind SLP-76 (Fig 8) After

investigating possible SLP-76-interacting adaptor proteins,

we considered the Grb2-related adaptor Gads a candidate

Gads was found to associate with the p55 Shb fusion

protein, in a phosphotyrosine-independent manner

(Fig 2F), which was very similar to our findings

concern-ing the association of SLP-76 to the Shb–PTB-Pro–GST

fusion protein The same blot was also probed with

anti-(SLP-76) Ig, and the pattern of association was found to be

quite similar to that of Gads The significance of the

SLP-76–Shb interaction was further verified by transient

transfections of Jurkat cells with different Shb mutants, to

study the pattern of tyrosine phosphorylation Cells

cotransfected with SLP-76 and wild-type Shb, exhibited a

normal patern of tyrosine phosphorylation in response to

CD3 stimulation (Fig 3) However, when SLP-76 was

cotransfected with Shb R522K (with a nonfunctional SH2

domain) or Shb DPTB-Tyr (with the four tyrosine

phosphorylation sites and the PTB domain deleted), the

cells exhibited a decreased response to CD3 stimulation

(Fig 3), which was verified using densitometric scannings

Particularly, decreased tyrosine phosphorylation of

pro-teins corresponding to 160 (PLC-c1), 75 and 36 kDa

(LAT) was observed and described in Fig 3, with the

relative phosphorylation of these proteins compared to the

total amount of SLP-76 in the CD3 treated lanes The

75-kDa protein comigrated exactly with SLP-76 These

results indicate that both the SH2 domain and the

PTB-Tyr region of Shb, are vital for the phosphorylation of

several proteins upon TCR engagement, including SLP-76,

PLC-c1 and LAT

The guanine nucleotide exchange factor Vav was

also found to associate with Shb, utilizing fusion

pro-teins (Fig 4A) The PTB domain of Shb mediated this

interaction and the binding could be blocked out with phosphotyrosine (Fig 4A,B) To identify a putative binding site for Shb in the Vav protein, we searched the Vav sequence for the PTB domain consensus binding-site D-D-X-Y Two such motifs were found in Vav and synthetic peptides corresponding to them were made These peptides were used in binding experiments with a GST fusion protein containing only the Shb PTB domain The PTB domain of Shb was found to associate with Vav and this binding is blocked by both phosphotyrosine and the more specific synthetic peptide corresponding to tyrosine 174 in Vav (Vav-Y174) (Fig 4B), which is more known as the autoregulatory site of Vav [21] We also tried to block this binding with the SLP-76 peptides, as Y113 and Y128 in SLP76 have been reported to be responsible for Vav binding

to SLP76 [20], but observed no effect

Attempts to coimmunoprecipitate Vav with Shb in Jurkat cells were unsuccessful, due to an IgG-related band of the same size as Vav, which made detection of Vav impossible

An alternative approach was to express Vav and Shb in COS cells, and then perform a-Shb immunoprecipitations Figure 4C shows how Vav coimmunoprecipitates with Shb upon pervanadate treatment of COS cells transfected with Shb and Vav

Our results suggest that although Vav and SLP-76 are known to interact with each other, they both bind directly and independently to Shb

Phosphorylation of SLP-76 and Vav is dependent

on Shb with a functional SH2 domain

To further study the effects of an R522K mutation in the SH2 domain of Shb on SLP-76 and Vav phosphorylation,

we performed immunoprecipitations of SLP-76 and Vav in

Fig 3 Shb is vital for the phosphorylation of several proteins in Jurkat

T cells Jurkat cells, transfected with SLP-76 and different Shb con-structs, as indicated, were stimulated (+) or not (–) with CD3 antibody for 2 min in 37 C, and lysed The cell extracts were resolved on SDS/ PAGE and the blots were probed with (SLP-76) Ig and 4G10 anti-phosphotyrosine Ig Relative phosphorylation, as assessed by densio-metric scanning of the proteins indicated, is given in the table below the blot The values are given relative the total amount of SLP-76.

Jurkat-R522K-2 and Jurkat-neo cells (Fig 5A,B) Western

blot analyses of these immunoprecipitates revealed in the

Shb SH2 defective clone, Jurkat-R522K-2, a failure of CD3

stimulation to increase the phosphorylation of either

SLP-76 or Vav The basal phosphorylation of Vav was

also lower in the clone expressing the R522K mutant The

same experiment was performed on ZAP70 in these cells,

with a minor difference in stimulation between mutant and

control (Fig 5C) The degree of phosphorylation, relative

unstimulated Jurkat cells, are given below each blot These

findings are consistent with the view that ZAP70 operates

upstream of Shb, whereas SLP-76 and Vav are effectors

downstream of Shb in the signalling pathway following

TCR engagement

Effects of expression of R522K Shb in Jurkat T cells

on JNK and Rac1 activation

The guanine nucleotide exchange factor Vav is known to

activate Rac1, RhoA and Cdc42 Both Rac1 and Cdc42 are

among other things known to cause activation of JNK As

Vav phosphorylation is decreased in the cells expressing Shb

with a defective SH2 domain (Jurkat R522K-2), we decided

to also examine Rac1 and JNK activation upon CD3 stimulation in these cells As displayed in Fig 6A, Rac1 activity (GTP-Rac1), as assessed by association with the PAK-CD fusion protein, is decreased in the Jurkat

R522K-2 cells upon CD3 stimulation, compared to normal Jurkat cells Consequently, JNK activation is also abolished upon CD3 stimulation in the Jurkat R522K-2 cells compared to the Jurkat-neo cells (Fig 6B)

Shb is recruited to GEMs after TCR ligation

It has recently been shown that several proteins involved in TCR signal transduction, including SLP-76, Vav, ZAP70 and Gads, localize to GEMs upon TCR stimulation [22–26]

To assess if this was also the case for Shb, Jurkat T cells were left unstimulated or stimulated with anti-CD3 Ig and then lysed in a Triton X-100-based buffer Lysates were subjected

to sucrose density gradient ultracentrifugation to separate the detergent resistant GEMs from the Triton-soluble

Fig 5 Phosphorylation of SLP-76 and Vav is dependent on the Shb SH2 domain Jurkat-neo or Jurkat R522K-2 cells (107) were left unstimulated (–) or were stimulated with anti-CD3 Ig for 2 min in

37 C (+) The cells were then lysed in triton lysis buffer and cell extracts were precleared with protein A sepharose and then subjected

to immunoprecipitation with (A) anti-SLP-76 Ig (B) anti-Vav Ig or (C) anti-ZAP70 Ig The precipitated proteins were resolved on SDS/PAGE and blotted for phosphotyrosine (4G10), SLP-76, Vav and ZAP70 The positions of SLP-76, Vav and ZAP-70 are indicated The relative phosphorylation, after normalization for the total amount of protein, compared to unstimulated Jurkat cells, is also indicated under each blot.

Fig 4 Association of Shb and Vav in CD3 stimulated T cells and

pervanadate stimulated COS cells (A,B) Jurkat cells (107) were

unstimulated (–) or stimulated by CD3 cross-linking for 2 min in 37 C

(+) before lysis in triton lysis buffer (A) Cell extracts were incubated

for 30 min on ice with the indicated immobilized fusion proteins in the

presence (+) or absence (–) of 10 m M phosphotyrosine (P-Y) (B)

Jurkat cell extracts were incubated for 30 min on ice with immobilized

Shb–PTB–GST fusion protein in the absence or presence of the

indi-cated tyrosine-phosphorylated peptides or phosphotyrosine (P-Y) (C)

COS cells, transfected with Shb and Vav, as indicated, were stimulated,

or not, with pervanadate, for 15 min in 37 C, before lysis The lysates

were subjected to immunoprecipitations using Shb antibody The

samples in (A–C) were washed and resolved on SDS/PAGE and

immunoblotted with the antibodies indicated The positions of Vav

and Shb are indicated with arrows.

fractions Both Shb isoforms (55 and 66 kDa) were found to

localize to the GEM fraction (mainly fractions 3 and 4) after

CD3 stimulation (Fig 7A) The GEM fraction was defined

by the presence of LAT, which is constitutively located to

lipid rafts LAT might therefore be responsible for the

recruitment of Shb to the lipid rafts upon TCR engagment

(Fig 7B,C) SLP-76, Vav and Gads were also seen to

localize to fraction 3, 4 and 5 upon CD3 stimulation

(Fig 7D–F), which is in agreement with previous studies

[23–26]

D I S C U S S I O N

We have previously established a role for Shb in T cell

signalling by describing the domain-specific binding of Shb

to the TCR f-chain, LAT, PLC-c1 and Grb2 [14] [16] We

have also demonstrated that a mutation in the SH2 domain

of Shb affects TCR signalling through MAPK and Ca2+

causing abolished activation of the NFAT element in the

IL-2 promoter [16] In this report, we show interactions

between Shb and SLP-76, Gads, Vav and ZAP70 and that

Shb is recruited to GEMs upon TCR ligation We also

examine how some of these interactions are affected by a mutation in the SH2 domain of Shb, and consequently how the activation of JNK is affected

To further study the effects of the mutation in the Shb SH2 domain, we examined the proteins found to coprecipi-tate with Shb in Jurkat cells Except for the previously studied LAT, PLC-c1 and the TCR f-chain, we also noted the presence of other tyrosine phosphorylated proteins possibly corresponding to SLP-76, Vav and ZAP70 In the cells expressing a nonfunctional SH2 domain, the phos-phorylation of SLP-76 and Vav is decreased This indicates that Shb forms a signalling complex with LAT, SLP-76, Vav and PLC-c1, and that this complex is linked to the TCR via the f-chain, through association of the Shb SH2 domain Phosphorylation of ZAP70 is not affected, or affected very little, by the R522K mutation in the Shb SH2 domain This is consistent with our model (Fig 8), where ZAP70 is bound to the ITAMs of the f-chain, to which Shb with its associated proteins also are attached, and thus brought in proximity to the tyrosine kinase ZAP70 Introduction of the Shb SH2 point mutation decreases the ability of the Shb–LAT–SLP76–Vav complex to interact with ZAP70 In addition, expression of Shb with a deletion

of the PTB domain and the tyrosine phosphorylation sites, also reduces the phosphorylation of phospho-proteins corresponding to PLC-c1, SLP-76 and LAT, implicating the involvement of these regions of Shb in TCR signaling

We have previously investigated the effects of overex-pression of wild-type Shb in Jurkat cells, and seen no differences in the CD3-mediated tyrosine phosphorylation, when Shb is overexpressed compared to mock transfected Jurkat cells [14] This is further confirmed in Fig 3, where cotransfection of Shb and SLP-76 produces Jurkat cells with

a normal CD3-induced tyrosine phosphorylation pattern

To elucidate how SLP-76, Vav and ZAP70 associate with Shb, we utilized fusion proteins comprising different parts of Shb An association of the adapter protein SLP-76 to the p55 Shb–pET and the Shb PTB-Proline-rich fusion protein could be seen both in unstimulated cells and after CD3 crosslinking As the phosphopeptides did not efficiently displace this association and as SLP-76 did not bind directly

to the Shb PTB-domain fusion protein only, we considered

it plausible that the Shb proline-rich motifs associated with SLP-76 via some adapter protein, as SLP-76 lacks an SH3 domain However, this interaction does not explain the CD3-dependency of the SLP-76–Shb interaction; we there-fore performed binding experiments using fusion proteins corresponding to different domains of SLP-76 We also cotransfected Shb together with different SLP-76 mutants in COS cells Both these approaches showed that the SLP-76 SH2 domain associated with Shb after CD3 stimulation in Jurkat cells, or pervanadate treatment in COS cells We have previously reported tyrosine phosphorylation of Shb after CD3 stimulation [16], and therefore suggest that phosphorylated tyrosines in Shb are binding sites for the SLP-76 SH2 domain Gads is a potential adapter that links the Shb proline-rich motifs with SLP-76 as the latter associates with Gads via an SH3-domain/proline–rich motif interaction The strong association of Gads to the Shb fusion protein, and the previously reported association between Shb and other proteins of the Gads family, Grb2 and Grap, all suggest that an association between Shb and Gads exists Accordingly, Gads bridges SLP-76 and Shb,

Fig 6 Activation of Rac1 and JNK is dependent on a functional Shb

SH2 domain Jurkat-neo or Jurkat R522K-2 cells (107) were

unstimulated (–) or stimulated by CD3 crosslinking for 2 min (+)

before lysis in triton lysis buffer (A) Jurkat cell extracts were incubated

for 30 min on ice with immobilized PAK–CD–GST fusion protein.

Fusion-protein complexes and cell extracts were resolved on SDS/

PAGE, and the blot was probed with Rac1 antisera (B) Cell extracts

were resolved on SDS/PAGE and the blot was probed with

anti-(phospho-JNK) Ig, and after stripping, reprobed with anti-JNK Ig.

The positions of Rac1, phospho-JNK and total JNK are indicated in

the figures The relative amount of active Rac1, after normalization for

the total amount of Rac1, is also indicated under the blot in (A).

and the SLP-76 SH2 domain binds to tyrosine

phosphor-ylated Shb

The guanine nucleotide exchange factor Vav associates

directly with the PTB domain of Shb through tyrosine-174

Tyrosine 174 has previously been shown to be of

import-ance for Vav GEF function [9] and is a possible site for

negative regulation of Vav The binding of Vav to the Shb

PTB domain might stabilize its association to SLP-76, and

thus aids in the activation process of Vav

Our data also show abolished CD3-mediated Rac1

activation and JNK phosphorylation, as a consequence of

the mutation in the Shb SH2 domain (R522K) This is

probably due to the abolished activation of Vav in these

cells, as several reports have shown that activated Vav is

required for Rac1-mediated JNK activation in lymphoid

cells [27,28]

Recent attention has focused on the concentration of

effector molecules into subdomains, so-called GEMs or

lipid rafts, upon receptor stimulation These membrane

subdomains are characterized by their detergent insolubility Some proteins involved in T cell signalling are always present in GEMs, for example LAT, which is anchored to the membrane via its palmitoylated tail We presently show that Shb is recruited to the GEMs upon TCR stimulation, where SLP-76, Vav, ZAP70, Gads and Grb2 have all previously been shown to target after CD3 ligation [23,24,26,29]

In conclusion, our results suggest that Shb is important for the early events of T cell signalling Shb is recruited to GEMs, possibly by LAT, after antigen stimulation, and can also associate with several important signalling proteins such as PLC-c1, Vav, Gads, Grb2, GRAP and SLP-76 This generates a signalling complex that is brought in proximity with the ZAP70 tyrosine kinase, the main phosphate-donor and thus activator, of these signaling proteins These interactions are then of importance for the activation of both the MAP kinase and the c-Jun kinase pathways

Fig 7 Recruitment of Shb to GEMs after TCR ligation Jurkat T cells were not treated (NT) or stimulated with CD3 antibody (CD3) for 3 min, followed by lysis in Mes lysis buffer Lysates were subjected to sucrose gradient ultracentrifugation Sequential fractions were removed from the top

of the gradient and indicated as fraction number The fractions were precipitated using TCA, and after washing with acetone, dissolved in 40 lL of SDS sample buffer The samples were resolved on SDS/PAGE, followed by immunoblot analysis using anti-Shb Ig (A), anti-phosphotyrosine Ig (4G10) (B), anti-LAT Ig (C), anti-Gads Ig (D), anti-Vav Ig (E) and anti-(SLP-76) Ig (F).

A C K N O W L E D G E M E N T S

We gratefully acknowledge the skillful technical assistance of Ing-Britt

Hallgren and Ing-Marie Mo¨rsare and peptide synthesis by Dr A˚ke

Engstro¨m We thank Dr Jane McGlade for providing the anti-Gads Ig

and Dr Arthur Weiss for providing the Vav expression plasmid The

work has been supported by grants from the Juvenile Diabetes

Foundation International, Swedish Medical Research Council

(31X-10822), the Swedish Diabetes association, The Novo-Nordisk

Foun-dation and the Family Ernfors Fund.

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