multiple invivo phosphorylated tyrosine phosphatase...

Vol. 7, 1589-1597, December 1996 CeO Growth & Differentiation 1589

Multiple in Vivo Phosphorylated Tyrosine Phosphatase SHP-2Engages Binding to Grb2 via Tyrosine 5841

Wolfgang Vogel2 and Axel UlIrich

Department of Molecular Biology, Max-Planck-lnstitut f#{252}rBiochemie,

Am Klopferspitz 18A, 82152 Martinsried, Germany

Abstract5HP-2 (also named PTPID, syp, or 5H-PTP2) has beenidentified as a phosphotyrosine phosphatasecomprising two src-homology-2 (SH2) domains. Upongrowth factor stimulation, SHP-2 becomes tyrosinephosphorylated, thereby increasing its catalytic activity.Here, we identified SHP-2 to be phosphorylated onmultiple tyrosine residues in response to differentstimuli and unmasked the carboxyl-terminal tyrosine584 as a major phosphorylation site in human cell lines.Tyrosine 584 shares, together with tyrosine 546, theconsensus sequence pY-X-N-X, a characteristic ofpotential binding sites for the SH2 domain of growthfactor receptor-bound protein 2 (Grb2). We show herethat mutation of tyrosine 584, but not tyrosine 546, tophenylalanine totally abolished the binding of Grb2 to5HP-2. By using a systematic mutagenesis approach,phosphorylation of additional tyrosines in each of theSH2 domains of 5HP-2 was detected aftercoexpression of epidermal growth factor receptor, but

not after coexpression of platelet-derived growth factorreceptor, whereas tyrosine 263 located in theinterspace between SH2 and catalytic domain appearsto be exclusively recognized by platelet-derived growthfactor receptor. Immunoprecipitation of SHP-2 from apanel of mammary carcinoma cell lines copurifiesseveral tyrosine phosphorylated proteins; the mostprominent band has an apparent molecular weight ofMr 115,000.

Introduction

The transfer of phosphate groups to tyrosine residues of

various proteins is a crucial event in cellular signaling, leading

to growth, differentiation, and metabolism (1). Protein tyro-

sine phosphorylation is balanced by the action of tyrosine

kinases and PTPases3. Deregulation of these processes by

Received 8/1/96; revised 9/16/96; accepted 9/30/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 1 8 U.S.C. Section 1 734 solely to mdi-cate this fact.1 This investigation was supported by a grant from Sugen, Inc. W. V. iscurrently supported by a fellowship of the Deutsche Forschungsgemein-schaft.2 To whom requests for reprints should be addressed, at Program inMolecular Biology and Cancer, Samuel Lunenfeld Research Institute,Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5. Phone: (416)586-4524; Fax: (416) 586-8857; E-mail: [email protected] The abbreviations used are: PTPase, phosphotyrosine phosphatase;

overexpression or hyperactivation of a tyrosine kinase or by

gene deletion of a PTPase can result in cell transformation

and cancer.

The enzyme families of tyrosine kinases and PTPases are

both comprised of transmembrane and cytosolic forms. In

total, more than 40 mammalian PTPases and even twice as

many tyrosine kinases are known. Although the extracellular

ligand binding of growth factors to RTKs and their activation

by transphosphorylation are well-established mechanisms in

cellular signaling, the targets of both transmembrane and

cytosolic PTPases remain, in most cases, unknown. Upon

growth factor activation, for example by EGF or PDGF, the

corresponding receptors recruit a specific constellation of

cytosolic substrates to the membrane. These become tyro-sine phosphorylated and transmit the signal, evoking RTK-

specific responses. Grb2 functions as an adaptor between

RTK and the GDP/GTP exchanger mSosl and, upon RTK

activation, induces contacts between mSosl and the proto-

oncogene product Ras, which is a central switchpoint in

downstream signaling (2, 3). From activated Ras, a cascade

of serine/threonine kinases branches off, ultimately resulting

in transcription factor phosphorylation and translation of theRTK signal into regulation of gene expression.

Nontransmembrane PTPases are composed of a single

conserved catalytic domain that is flanked by variable se-

quence stretches, presumably defining their intracellular lo-

calization and individual function. A prominent subgroup of

the PTPase family consists of SHP-2 (also named PTP1 D,

SHPTP2, SHPTP3, PTP2C, or syp) and SHP-1 , both char-

acterized by two adjacent SH2 domains at their amino ter-

mini (4-9). SH2 domains are approximately 100 amino acidslong and serve as structural modules directing SHP-1 and

SHP-2, together with many other SH2-containing proteins, to

selected tyrosine phosphorylation sites (1 0). The recognition

site is specified predominantly by the surface requirements

of the SH2 domain binding pocket and the three to fivecarboxyl-terminal amino acids flanking the autophosphoryl-

ation site of the receptor (1 1-13). After ligand activation of

the PDGF receptor, SHP-2 associates with the receptor and

subsequently becomes tyrosine phosphorylated, inducing

an increase in its catalytic activity (4).To understand the significance of SHP-2 phosphorylation

in greater detail, we have mapped five tyrosine phosphoryl-

ation sites within SHP-2, of which tyrosine 584 is a major site

and serves as docking site for Grb2. Furthermore, we

RTK, receptor tyrosine kinase; EGF, epidermal growth factor; PDGF,platelet-derived growth factor; Grb2, growth factor receptor-bound pro-tein 2; EP, chimeric RTK composed of the extracellular ligand-bindingdomain of the EGF receptor and the cytoplasmic, catalytically activedomain of the PDGF receptor; mSosl , mammalian son-of-sevenless;SH2, src-homology-2; P,, inorganic phosphate; Csw, corkscrew; Dos,daughter-of-sevenless.

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elucidateevents.

15� Phosphorylation and Association of SHP-2

Fig. 1. Phosphoamino acidanalysis of SHP-2 from restingcells (left panel) or from PDGF-stimulated cells (right panel).293 cells were transfected withSHP-2 alone or cotransfectedwith PDGF receptor and Ia-beled with [32P]P,. Immunopre-cipitated SHP-2 was isolatedafter SDS-PAGE, hydrolyzed,and separated by two-dimen-sional electrophoresis at pH 1.9and pH 3.5, respectively. Themigration of the standards forphosphoserine (S), phospho-threonine (T), and phosphoty-rosine (Y) and the origin (0) atthe lower right is indicated.

for downstream interaction partners of SHP-2 to

the function of this enzyme in cellular signaling

ResultsSHP-2 Is Phosphorylated on Serine, Threonine, and Ty-rosine Residues. It has been shown previously that SHP-2is strongly phosphorylated on tyrosine residues in response

to PDGF or EGF treatment of various cell lines (4, 7, 14, 15).The tyrosine phosphorylation of SHP-2 results in a remark-

able shift ofthe protein to a higher apparent molecular weightin SDS-PAGE.

To analyze the actual phosphorylation state in more detail,

human embryonic kidney fibroblast 293 cells were trans-fected with cytomegalovirus-based expression plasmids

coding for SHP-2 or cotransfected with plasmids coding for

SHP-2 and PDGF receptor and metabolically labeled with

[32P]P. Immunoprecipitated SHP-2 was purified by SDS-

PAGE and subjected to phosphoamino acid analysis. Fig. 1

shows that SHP-2 phosphorylation in resting cells can bedetected only on serine and, to a lesser extent, on threonine

residues, whereas in cells, which express ligand-activated

PDGF receptor, phosphorylation of SHP-2 occurs mainly on

tyrosine residues. The phosphotyrosine content was esti-mated to account for approximately 95% of the overall phos-phorylated amino acids.

SHP-2 Exhibits Phosphorylation on Multiple TyrosineResidues. To investigate whether tyrosine phosphorylationof SHP-2 is restricted to a single site within the molecule orwhether several tyrosines are modified, we transfectedSHP-2, together with the PDGF receptor, in 293 cells, and

after ligand stimulation we isolated [32P]-labeled SHP-2 pro-tein for digestion with trypsin and two-dimensional phos-phopeptide analysis. As shown in Fig. 2A, a complex patternof at least 10 distinguishable spots indicated phosphoryla-tion of SHP-2 on multiple sites. These were shown by phos-phoamino acid analysis to be predominantly tyrosine resi-dues (Fig. 1). We further cotransfected SHP-2 in 293 cellswith EGF receptor or c-src and analyzed the resulting SHP-2phosphorylation pattern. As observed with the PDGF recep-tor, both the EGF receptor and c-src phosphorylated SHP-2in vivo on several tyrosine residues, although the number and

position of the phosphopeptides were different when corn-pared with the pattern after PDGF receptor phosphorylation(Fig. 2, A-C). Superimposition ofthe peptide maps generatedby coexpression of SHP-2 with either PDGF receptor, EGF

receptor, or c-src suggested that certain sites were phos-phorylated by all three kinases, whereas others appeared tobe specifically modified by each kinase.

To rule out the possibility that the intensity of SHP-2 phos-

phorylation resulted from overexpression in the 293 cell sys-tern, we analyzed the phosphorylation pattern of SHP-2 in

nontransfected cells. The mouse mammary carcinoma cellline 6590 was chosen, because it had previously revealed aconstitutively high expression level of SHP-2 as judged byNorthern blot analysis. Incubation of these cells with 1 m�orthovanadate resulted in an increase of tyrosine phospho-rylation of total cellular proteins, including SHP-2 (data notshown). [32P]-labeled phosphorylated SHP-2 was isolatedfrom 6590 cells and analyzed by phosphopeptide mapping.

As shown in Fig. 2D, SHP-2 was phosphorylated at multiple

sites, indicating that phosphorylation of SHP-2 is not entirelydependent on the overexpression in 293 cells. The phos-phopeptide pattern of SHP-2 from 6590 cells was differentfrom that obtained by PDGF receptor, EGF receptor, or c-srcphosphorylation, presumably because of differences in theSHP-2 sequence between mouse and human.

Analysis of SHP-2 Tyrosine Phosphorylation by Sys-tematic Mutagenesis. To study the relative importance of

specific tyrosine phosphorylation sites in the molecule, we

changed each tyrosine present in SHP-2 to a phenylalanine

residue (Y/F) by site-directed mutagenesis. In total, we con-structed 21 Y/F mutants that were separately coexpressedwith the EP receptor (1 6). By using the EP receptor chimera,we could compare the phosphorylation by the kinase do-mains of PDGF receptor and EGF receptor, while using onlyone type of ligand. The phosphopeptide maps of each of theSHP-2 mutants were compared with the map of the wild-

type protein. Fig. 3, A and B, shows representative phos-phopeptide maps of the mutants Y263F and Y584F. Whenthe Y263F and wild-type maps were superimposed, one

prominent phosphopeptide was absent from the mutant map(letter F in Fig. 3C). Hence, we concluded that tyrosine 263

was a target of the PDGF receptor kinase. This tyrosine was

Cell Growth & Differentiation 1591

Fig. 2. Phosphopeptide mapsof SHP-2 overexpressed in 293cells and cotransfected withPDGF receptor (A), EGF recep-tor (B), c-src (C), or the 6590mouse mammary carcinomacell line (D). Cells were labeledwith [32P]P�. PDGF receptor andEGF receptor were stimulatedwith the corresponding ligand;6590 cells were treated with 1mM orthovanadate. After immu-noprecipitation and SDS-PAGE,SHP-2 was digested withtrypsine and analyzed by thin-layer chromatography in the firstdimension by electrophoresisand in the second dimension bychromatography.

A

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located within the rather short, hydrophilic tryptic peptide

L-L-pY-S-K, which may explain the comparatively far migra-

tion to the upper right corner of the phosphopeptide map. It

is surprising that phosphorylation of Y263 could not be de-

tected after phosphorylation of SHP-2 by EGF receptor or

c-src (Fig. 2, B and C). Using the same strategy, we also

identified tyrosines 546 and 584 as phosphorylation sites.

The phosphopeptide map of the Y584F mutant lacks at least

three spots (letters B, C, and G in Fig. 3C), which might be

explained by the production of several distinct peptides con-

taming the Y584 phosphorylation site. We estimated that

phosphorylation on tyrosine 584 made up more than one-third of all phosphotyrosine content. Furthermore, the phos-

phorylation-induced shift of the wild-type SHP-2 protein tohigher molecular weight by SDS-PAGE was significantly de-

creased for the mutant proteins Y546F and Y584F (Fig. 3D).SHP-2 Is Tyrosine Phosphorylated at Both SH2 Do-

mains by Activated EGF Receptor. To examine the partic-

ular relevance of the 5H2 domains in the phosphorylation ofSHP-2, we generated a premature C-terminal termination of

the polypeptide by mutation of asparagine 21 7 to an amber

stop codon. Expression of this construct, termed SHP-2�, in

293 cells gave rise to a truncated form of SHP-2, which

lacked the PTPase domain and the C-terminal sequence but

retained the original start codon followed by both 5H2 do-

mains.

The SHP-2z� construct was coexpressed in 293 cells either

with the EGF receptor or with the EP receptor chimerae. Fig.

4 shows an antiphosphotyrosine immunoblot of crude ly-

sates from EGF-stimulated cells. Only the EGF receptor, and

not the EP receptor, was able to phosphorylate the SHP-2�

protein on tyrosine residues significantly (Fig. 4, Lanes 3 and

8). Nevertheless, both RTKs were comparably active, asdemonstrated by phosphorylation of wild-type SHP-2 (Fig. 4,

Lanes 2 and 7). By introducing V/F mutations, the phospho-

rylation sites in the SHP-2� protein were localized. Coex-

pression of SHP-2z�-Y1OOF with the EGF receptor displayed

reduced tyrosine phosphorylation when compared with the

original construct (Fig. 4, Lanes 3 and 4). Addition of a

second V/F mutation at position 197 (SHP-2�-Y1OO/197F)

resulted in complete abolition of tyrosine phosphorylation

(Fig. 4, Lane 5). Taken together, these results suggest that

the EGF receptor is capable of phosphorylating both the N-

and C-terminal SH2 domains of SHP-2 at their extremeC-terminal tyrosines 1 00 and 197 (Fig. 3E). In contrast to the

sites Y546 and Y584 phosphorylated by EGF receptor and

PDGF receptor, the 5H2 domain sites were recognized only

by the EGF receptor and not by the PDGF receptor.

Tyrosine Phosphorylation of SHP-2 Leads to Assem-bling of a Multiprotein Complex. A panel of different

mouse mammary carcinoma cell lines was treated with or-

thovanadate to induce tyrosine phosphorylation of SHP-2.

Immunoprecipitates of SHP-2 were analyzed by Westernblotting with antiphosphotyrosine antibodies. Intense phos-

phorylation of SHP-2 was detected in three of six cell lines(Fig. 5). It is surprising that a series of equally strong phos-

phorylated proteins was coprecipitated with SHP-2. Themost abundant protein had an apparent molecular weight of

A

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546 584

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ftEGF-R and PDGF.R

1592 Phosphorylation and Association of SHP-2

100 197 263

-1fl,�-�PP

f ftEGF-R EGF-R PDGF.R

Fig. 3. Tryptic phosphopeptide maps of SHP-2 mutants Y263F (A) and Y584F (B), which are phosphorylated by PDGF receptor, and schematicrepresentation of wild-type SHP-2 phosphopeptide pattern (C). lmmunodetection of SHP-2 in immunoprecipitates from wild-type or Y/F mutant transfected293 cells (D). Localization of tyrosine phosphorylation sites within the SHP-2 sequence (E).

Mr 1 1 5,000; less intense phosphorylated bands were M�

95,000, 175,000, and 1 90,000 in size. As with SHP-2, the Mr

1 1 5,000 species generated a broad signal, presumably mdi-

cating phosphorylatmon at several sites. A small amount of

phosphorylated Mr 1 1 5,000 protein coprecipitated from6380 and 6374 cells not treated with orthovanadate, sug-

gesting that SHP-2 was able to aggregate some Mr 115,000

protein without being phosphorylated (Fig. 5).

Grb2 Interaction with Tyrosine-Phosphorylated SHP-2.The multiprotein complex coprecipitating with SHP-2 from

various cell lines was scanned with a panel of antibodies

raised against different proteins known to be involved in

RTK-mediated signal transduction. In lysates from vanadate-

treated human mammary carcinoma SK-BR-3 cells, the 5H2

domain-containing adaptor protein Grb2 was found to co-

precipitate with SHP-2. Previous results showed a tight af-

finity of SHP-2 to the RTK p1 85HER2Jneu, which is overex-

pressed in human breast cancer cell lines, such as SK-BR-3,

due to gene amplification (4, 1 7). We considered that either

SHP-2 may be directly bound to Grb2 or that both proteins

interact indirectly while individually associated with distinct

autophosphorylation sites of the HER2 receptor. To evaluate

these possibilities, portions of SK-BR-3 lysates were prein-

cubated with the HER2-specific monoclonal antibodies 2C4

and 4D5. After complex formation, the receptor and associ-

ated proteins were removed by binding to protein A-Sepha-

rose. The supernatant was subjected to immunoprecipitation

with anti-SHP-2-specific antibodies. As shown in Fig. 6A,

Western blotting with anti-Grb2 antibodies dotected nearly

identical amounts of coprerip�tatea protein from the initial orHER2-depleted lysates. In the reverse experiment, the co-

precipitation of SHP-2 was demonstrated by analyzing the

Grb2 immunocomplex (Fig. 6B). These data suggest that the

SHP-2-Grb2 complex was formed independently of the as-

sociation of both molecules to the HER2 receptor.

Phosphotyrosine 584 of SHP-2 Is Required for Grb2Binding. To further investigate the molecular basis of the

interaction between SHP-2 and Grb2, we used a panel of

SHP-2 V/F mutants in an in vitro association experiment.

Grb2 was overexpressed in 293 cells and isolated from the

cell lysates by immunocomplexing to protein A-Sepharose.In parallel, SHP-2 and eight V/F mutants were tyrosine phos-

phorylated in vivo by coexpression with the c-src kinase in293 cells. Cell lysates of these transfections were incubated

with aliquots of the Grb2 immunocomplex, and coprecipitat-ing SHP-2 was detected by Western blotting with anti-SHP-2

antibodies. As shown in Fig. 7A, the very distant C-terminalmutation V584F totally abolished binding of SHP-2 to Grb2.

A remarkable reduction in the amount of associated SHP-2

was observed with the mutant V546F. Fig. 7B is a reprobe of

the Western blot with anti-Grb2 antibodies. Equal expression

of SHP-2 and the V/F mutants in the c-src cotransfected

cells was confirmed in separate Western blots (data not

shown). The use of a bacterial-expressed Grb2-glutathione-S-transferase fusion protein instead of the Grb2 immuno-

complex in a similar in vitro association experiment gavesimilar results, indicating that phosphotyrosine 584 is thepredominant binding site for Grb2 (data not shown).

DiscussionOur previous results showed that SHP-2 is activated upon

tyrosine phosphorylation (4). In the study presented here, we

have focused on this phosphorylation event on a molecular

level and have identified Grb2 and a Mr 11 5,000 protein asassociation partners of SHP-2. We could not detect any

A

SHP-2

SHP-2 z�

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Ii 2 3 4 5 6 7 8blot: a PY

IP: a SHP-2, blot: a PY

-20 SHP-2

- 116

- 97

#{163}�pz � �A�III -‘66

blot: aSHP-2

Fig. 5. Immunoprecipitates of SHP-2 copurify a M, 1 15,000 tyrosine-phosphorylated protein. Six mouse mammary carcinoma cell lines weretreated with orthovanadate for 90 mm. SHP-2 was immunoprecipitatedfrom equal amounts of cellular lysates (1 mg). After SDS-PAGE separation,the Westem blot was probed with antiphosphotyrosine antibodies (A),stripped, and reprobed with antibodies against SHP-2 (B).

blot: aSHP-2

Fig. 4. Distinct tyrosines in the SH2 domains of SHP-2 are phosphoryl-ated by the EGF receptor and not by the EP receptor chimera. Wild-typeand truncated (SHP-2�) phosphatase were coexpressed with EGF recep-tor or EP receptor and stimulated with EGF for 5 mm. Proteins fromportions of cell Iysates were separated by SDS-PAGE, transferred tonitrocellulose, and probed with antibodies to phosphotyrosine (A). Theblot was stripped and reprobed with anti-SHP-2 antibodies (B).


4’,

A ‘�

tyrosine phosphorylation of SHP-2 in resting cells, even

when strongly overexpressed in the 293 cell system. Activa-

tion by cotransfected PDGF receptor gave rise to a remark-

able increase in tyrosine phosphorylation of SHP-2, which is

mediated by the receptor. In contrast, a minor part of phos-

phorylation on serine and threonine residues remains un-changed. Hence, the PTPase function seems not to be ad-

ditionally modulated by serine- or threonine-specific kinases.

Similar observations have been made when analyzing theSHP-2 phosphorylation state in resting or growth factor-

activated mouse 3T3 fibroblasts (1 5). On the basis of datafrom Peraldi et a!. (1 8) showing that stimulation of rat pheo-chromocytoma PC12 cells with EGF led to a 2-fold increasein serine phosphorylation and to a slight increase in threoninephosphorylation, but no phosphorylation of tyrosine resi-

dues, cell-type-dependent differences seem to exist. Tryptic

phosphopeptide mapping of SHP-2 revealed that multiple

sites of the protein are tyrosine phosphorylated in human and

mouse cells. Furthermore, the patterns obtained by PDGF

receptor, EGF receptor, or c-src phosphorylation were

clearly different, indicating that each kinase recognized spe-

cific sites and phosphorylated them to a certain degree. Thisobservation could be interpreted as one of the initial stepsdefining the specificity of each tyrosine kinase due to differ-ential activation of a common target and thereby creating a

unique signal.

The intense tyrosine kinase-dependent phosphorylation of

SHP-2 is directed against at least five distinct sites of the

protein. By a systematic mutagenesis approach, we found all

subdomains of the protein, except the catalytic domain, to

be affected. It is conceivable that these different phospho-

rylation sites may reflect distinct functional requirements.The panel of tyrosine to phenylalanine mutants enabled us to

identify the strongly phosphorylated tyrosine 584 as the mainbinding site of Grb2, whereas tyrosine 546 seems to be a

minor site. It is interesting that the overexpression of SHP-2

in hamster (baby hamster kidney) cells transfected with EGFreceptor led to just a minor phosphorylation of tyrosmne 584,

thereby setting off tyrosine 546 as predominant site at the tail

(1 9). Bennett et a!. (20) also reported that SHP-2 is phospho-

rylated in vivo only at tyrosine 542 (equal to 546 in our study),

again using a heterologous expression system with human

PDGF receptor in canine cells. Whereas, if an in vitro phos-

phorylation of glutathione-S-transferase fusion proteins with

purified PDGF receptor was performed, they could readily

detect phosphorylation of tyrosine 584. It is likely that phos-

phorglation of tyrosine 546 and 584 is regulated by yet anadditional mechanism (i.e., alternative splicing of the C-ter-

BAprelnc.

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IP: aSHP-2, IP: aGrb2,blot: aGrb2 blot: aSHP-2

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Fig. 7. In vitro association of Grb2 to SHP-2 wild-type and V/F mutants.Grb2 was overexpressed in 293 cells and immune-complexed to proteinA-Sepharose. Aliquots were incubated with lysates from cells transfectedwith c-src and SHP-2 wild-type or V/F mutants. Immunoprecipitates wereanalyzed by SDS-PAGE, transferred to nitrocellulose, and blotted withantiserum against SHP-2 (A). Subsequently, the blot was stripped andreprobed with anti-Grb2 antibodies (B).


pr.lnca HER2

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Fig. 6. Human mammary carcinoma SK-BR-3 cells were treated with orthovanadate and lysed. From one-half of the lysates, the HER2 receptor complexwas removed by incubation with protein A-Sepharose-coupled anti-HER2 monoclonal antibodies 2C4 and 4D5. The supematants were split again andsubjected to anti-SHP-2 immunoprecipitation. The coimmunoprecipitation of Grb2 was detected by probing the Western blots with specific antibodies (A).Immunoprecipitations of Grb2 were analyzed by Western blotting with antibodies against SHP-2 (B). The blot with anti-SHP-2 immunoprecipitants (A) wasstripped and reprobed with a mixture of anti-SHP-2 and anti-HER2-specific antibodies (C).

minal tail). One isoform of SHP-2, thus far observed only inthe mouse, exhibited a truncated tail, probably generated by

alternative splicing sharply after tyrosine 546 (7). This iso-

form, which lacks phosphotyrosine 584, may contribute to

surplus modulation of the catalytic activity and Grb2-binding

capacity of SHP-2.

For the 5H2 domain of Sem5 (the Caenorhabditis elegans

homologue of Grb2) and for Drosophila and mouse Grb2, a

recognition sequence flanking the phosphotyrosine C-termi-nal with an asparagine at position +2 as major determinant

was predicted (12, 1 3). The positions + 1 and +3 were ap-

proximately three times less selective, allowing the amino

acids L, V, I, or M at position +1 and V or P at position +3

to be present (12). The phosphotyrosines 546 and 584 are, in

fact, both followed by XNX motives, namely V546 by TNI and

V584 by ENV. The valine within the phosphotyrosine 584

motive fits the predicted sequence precisely. This makes it

increasingly likely that this site is preferably recognized by

the SH2 domain of Grb2 under physiological conditions. It is

interesting that Lorenz et a!. (21) have reported the phospho-

rylation of the closely related SHP-1 by the src-Iike tyrosinekinase Ick at striking analogous positions. Whereas the over-

all C-terminal sequences of both 5H2-PTPases are highly

divergent, the position and recognition tails of the phospho-

rylation sites V538GN1 and V564ENL in SHP-1 are nearly iden-tical to the corresponding sites in SHP-2 (Y546TNI and

V5�ENV). Current observations in 293 cells have shown that

SHP-1 becomes phosphorylated at a single site (i.e., tyrosine

538; Ref. 22).

Another critical regulatory function may be the tyrosine

phosphorylation of the SH2 domains of SHP-2 at positions

1 00 and 1 97, achieved by activation of EGF receptor. Using

nuclear magnetic resonance studies of the N-terminal SH2

domain and the crystal structure of both SH2 domains of

SHP-2, it is now possible to locate these sites in the tertiary

structure of the protein (23, 24). Phosphotyrosine 1 00 is


4 w. Vogel and A. UlInch, unpublished data.

located in the last a-sheet (fiG) of the SH2 domain structure

at the opposite site of the phosphotyrosine binding pocket.

This allows no close contact between tyrosine 100 and the

SH2 domain ligand. Nevertheless, one can propose a long-range influence of the phosphorylation event by the forma-

tion of hydrogen bonds and by a change in the polarity of the

whole domain.

Furthermore, phosphotyrosine 197 of the C-terminal SH2domain is located in the middle of the aB-helix, again at adistance to the phosphotyrosine binding pocket (24). How-ever, cocrystallization of the SH2 domain with a PDGF re-

ceptor-derived peptide revealed that the aB-helix is able tomake contacts at positions 3, 4, and 5 of the peptide, allow-ing phosphorylation of tyrosine 197 to regulate the ligandspecificity of the SH2 domain. Because our data estimate

that this site is phosphorylated only in 5-1 0% of all mole-cules, fine-tuning of these interactions can be achieved, and

cell-specific responses can be defined. Phosphorylation ofSH2 domains is likely to be an important signal modulator,although only previously reported once for the SH2 domainof Ick, where one tyrosine and one senne was affected (25).

Recently, we were able to show that SHP-2 binds with thehighest affinity to HER2 compared with EGF receptor, PDGF

receptor, or c-kit (4). Now, we applied lysates of the human

breast carcinoma cell line SK-BR-3 in coprecipitation exper-iments, revealing that besides SHP-2, Grb2 is associatedwith the HER2 receptor.4 Despite the high overexpressionand strong autophosphorylation of HER2 in SK-BR-3 cells,the interaction of Grb2 and SHP-2 takes place distant to the

HER2 receptor, and no trimeric complex can be detected.This observation is in contrast to the PDGF receptor and c-kitsignaling pathways, where tyrosine-phosphorylated SHP-2acts as an adaptor between the receptor and Grb2 (26, 27).The PDGF receptor signaling complex seems to include

mSosl , and thereby enables Grb2, which is anchored by

PDGF receptor/SHP-2 linkage close to the plasma mem-brane, to stimulate the Ras pathway. Until now, a directinvolvement of SHP-2 in Ras activation had not been proven.Indeed, the affinity of Grb2 to various non-RTK proteins (e.g.,IRS-i , BCR-abl, Shc, and Large T-antigen) suggests that theactivation of the Ras pathway might be only one part of abroad diversity of Grb2 functions (3). This is supported by theobservation that Grb2 associates with the transmembrane

PTPase-a without detecting mSosi in the anti-PTPase im-munoprecipitation complex (28). Although only a fraction of

total SHP-2 and Grb2 are associated with each other, themultiple phosphorylation of SHP-2 may culminate in certain

other cellularfunctions than activation ofthe Ras pathway. Inthis context, it is of interest that the association of the p85

subunit of P13-kinase with SHP-2 after stimulation of a my-

eloid cell line with IL-3 or GM-CSF and that the association

of SHP-2 to the SH2 domain of c-src were found (29, 30). It

seems likely that another series of SH2 domain-containingproteins is able to interact with the phosphotyrosines of

SHP-2.

Furthermore, intramolecular interactions of SHP-2 can beproposed, in which the two N-terminal SH2 domains becomeoccupied by the C-terminal phosphotyrosines 546 and 584.Consequently, the conformational change in the overall pro-tein structure may alter the catalytic activity and disfavorintermolecular association of the SH2 domains to activatedRTK. A similar intramolecular-folding mechanism has beenproposed previously for c-src, Ick, and SHP-i (31-33). In thecase of c-src, the enzymatic activity is decreased after phos-phorylation of the COOH terminus (31); in the case of SHP-i,the intramolecular association resulted in autoinhibition (33).

Dunng Drosophila eye development, the formation of R7photoreceptors is initiated by stimulation of the RTK seven-

less (34). An essential component downstream of sevenlessis Csw, a SH2-containing phosphatase with striking homol-ogy to SHP-2 (35). A genetic screen for additional membersinvolved in this pathway recently resulted in the identificationof Dos, a tyrosine phosphorylated M� 1 15,000 protein (36,37). The data presented here allow us to hypothesize that thebroad band around Mr 1 1 5,000 coprecipitating with SHP-2

may contain the mammalian homologue of Dos. A catalyticinactive mutant of Csw binds hyperphosphorylated Dos,

suggesting that Dos serves as substrate for Csw (36). In ourexperiments, vanadate treatment led to inactivation ofSHP-2, thereby allowing us to detect coprecipitation of theMr 1 15,000 species equally well (Fig. 5). Further geneticanalysis of Dos mutant flies showed that Csw and Dos areacting upstream of Rasi and independent of Sos (37). There-

fore, it is tempting to speculate that SHP-2 is able to regulateRas activity, although using mammalian Dos instead of theGrb2-mSosi complex.

In mammalian cells, SHP-2 signals either in a positive ornegative regulatory manner, depending on different external

stimuli and the response measured. In insulin, prolactin, or

EGF signaling, the overexpression of a dominant-negativeform of SHP-2 blocked insulin- and prolactin-specific re-sponses or microtubule-associated protein kinase activa-tion, respectively (38-47). However, overexpression of dom-

inant-negative SHP-2 resulted in sustained formation ofmembrane ruffles after PDGF stimulation, claiming a nega-tive role for SHP-2 in the rearrangement of the cytoskeleton,before cell proliferation (48). Microinjection of the catalytic-impaired construct into Xenopus oocytes induced severeposterior truncation of the embryo (49). It is surprising thatthe microinjection of a SHP-2 construct with Y546F andV584F mutations resulted in normal development of the Xe-nopus oocyte, leaving the question of the functional signifi-cance of these tyrosine phosphorylation sites unanswered.

Materials and MethodsPlasmld Constructs, AntibodIes, and Cell Lines. The construction ofthe cytomegalovirus promotor-based expression plasmids for SHP-2,

PDGF receptor, EGF receptor, and EP receptor has been described

previously (4, 50). The full-length coding sequence of Grb2 was clonedinto a similar expression vector. The c-src expression construct was

kindly provided by S. Courtneidge (Sugen, Inc., Redwood City, CA). All

mutants of the SHP-2 cDNA were obtained using the site-directed mu-tagenesis protocol of Kunkel (51). For tyrosine to phenylalanine mutation,primers were 1 9-mers that generate single A-to-T transversions. For the

N21 7* mutant, an AAC-to-TAG exchange was introduced.


Chinchilla rabbits were used to raise polyclonal antibodies against

synthetic peptides corresponding to the 15 most C-terminal amino acids

of HER2, Grb2, and SHP-2 (2, 4, 52). These antibodies were used forimmunoprecipitation and blotting, except for anti-SHP-2 Western blots

and anti-HER2 immunoprecipitation. Here, an affinity-purified antibodyagainst part of the N-terminal sequence of SHP-2 (amino acids 29-217)

and the anti-HER2 monoclonal antibodies 4D5 and 2C4 were used, re-spectively (17). Tyrosine phosphorylated proteins were detected with 5E2

mouse monoclonal antibody (53).The cell lines SK-BR-3, A293, 6590, 6599, 6363, 6602, 6378, 6380, and

6374 were obtained from the American Tissue Culture Collection and

cultivated under the recommended conditions.

Transient Expression. Human embryonic kidney fibroblast 293 cells

(American Tissue Cufture Collection CAL 1573) were grown and trans-

fected as described (50). In brief, 2 x 1O� cells were seeded per cm2 dish

and transfected 24 h later with 200 ng ofCsCl gradient-purified DNA using

a calcium precipitation protocol (54). For cotransfection of plasmids con-taming the cDNA of different proteins, the amount of DNA was divided,

and empty expression vector was added if necessary. Sixteen h later, the

serum concentration in the medium was changed from 10 to 0.5%. After24 h, cells were stimulated with appropriate ligands for 5 mm or treated

with 1 mM orthovanadate (pH 10.0) for 90 mm before lysis.

Immunopreclpitatlon and Western Blothng. Transfected 293 cellswere lysed in Triton X-100 buffer containing 50 m� HEPES (pH 7.5), 150

mM NaCI, 1 .5 mM MgCI2, 5 m� EGTA, 5 mp,i EDTA, 10% glycerol, 1%

Triton X-100, 10 mM NaF, 1 m,�i phenylmethylsulfonyl fluoride, 1 m�

orthovanadate, and 1 0 �g/ml aprotinin. After 5 mm incubation on ice, the

cells were harvested and centrifuged for 10 mm at 4#{176}Cand 17,000 rpm.Aliquots of the supematant were subjected directly to SDS-PAGE or

further analyzed by immunoprecipitation with specific antibodies for 4 h at4#{176}Con a rotating wheel. The protein A-Sepharose complex was washed

twice with each of the following buffers: (a) 500 m�i NaCI, 0.1 % SDS, 0.2

Triton X-100, 50 mM HEPES (pH 7.5), 5 mu EGTA, 5 m� EDTA, 20 mp�i NaF,and 2 m� orthovanadate; (b) 150 m�a NaCI, 0.1 % SDS, 0.2% Triton X-100,

50 m�.i HEPES (pH 7.5), 5 mM EGTA, 5 mi.i EDTA, 20 m� NaF, and 2 muorthovanadate; and (c) 10 mu Tns-HCI (pH 7.8), 0.1 % Triton X-100, 20 mui

NaF, and 2 mM orthovanadate. In the case of SK-BR-3 cells, proteinassociation experiments were performed using NP4O buffer [20 mu Tns-HCI (pH 8.0), 150 mu NaCI, 2 mu EDTA, 1 % NP4O, and proteinase

inhibitors as described above] to lyse cells and to wash the immunocom-plex. After SDS-PAGE, proteins were transferred to a nitrocellulose mem-brane (Schleicher & Schuell, Dassel, Germany) and immunoblotted with

antibodies diluted 1 :1000 in 50 mu Tns-HCI (pH 7.5), 150 mu NaCI, 5 mu

EDTA, 0.05% Triton X-100, and 0.25% gelatin ovemight. After incubationwith a horseradish peroxidase-coupled secondary goat anti-rabbit anti-

body or goat anti-mouse antibody (Bio-Rad) for 1 h, the Western blot wasdeveloped using enhanced chemiluminescence (Amersham). For reprob-ing, the membrane was stripped in 70 mui Tns-HCI (pH 6.8), 2% SDS, and0.1 % �3-mercaptoethanol at 50#{176}Cfor 30 mm.

Phosphoamino Acid Analysis and Tryptic Peptide Mapping. For in

vivo labeling of phosphorylated proteins, cells were grown in phosphate-free DMEM-medium supplemented with 0.5 mCVml (�P]P� (Amersham)for 4 h. After growth factor stimulation and lysis in Triton X-100 buffer,SHP-2 was immunoprecipitated and analyzed by SDS-PAGE. The gel was

fixed in 30% methanol, 10% acidic acid, and exposed to an X-ray film(Kodak) for 2 h. The labeled SHP-2 protein was excised from the gel and

eluted into 50 mM ammonium hydrogencarbonate, 0.1 % SDS, and 0.5%

j3-mercaptoethanol. After trichloracidic acid precipitation and oxidation

with performic acid, one-tenth of the protein was hydrolyzed in 5.7 u HCIat 1 10#{176}Cfor 1 h and resolved with an HTLE 7000-apparatus (C.B.S. Inc.,Del Mar, CA) by cellulose thin-layer chromatography (1OO-�un plates,

Merck, Darmstadt, Germany) in two dimensions with pH 1 .9 buffer (2.5%formic acid and 7.8% acidic acid) and pH 3.5 buffer (5% acidic acid and0.5% pyridine), respectively. The remaining protein was exhaustively di-gested with N-p-tosyl-L-lysine chloromethyl ketone-treated trypsin (Sig-

ma) for 16 h, stepwise-desalted, and concentrated by lyophilization. Pep-

tides were analyzed in the first dimension by electrophoresis (pH 1.9-buffer) and in the second dimension by ascending chromatography(37.5% n-butyl alcohol, 25% pyndine, 7.5% acetic acid, and 30% H20).Thin-layer chromatography plates were dried and exposed to X-ray film at

-70#{176}Cusing an intensifying screen.

- -- �--a� � �

We especially thank Irmi Sures for continuous experimental advice. We

thank Frauke Alves, Ritu Dhand, and Vanita Jassal for critically comment-ing on this manuscript.

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