Purification and Molecular Cloning of SH2- and SH3 - Blood

Purification and Molecular Cloning of SH2- and SH3-Containing InositolPolyphosphate-5-Phosphatase, Which Is Involved in the Signaling Pathway

of Granulocyte-Macrophage Colony-Stimulating Factor, Erythropoietin,and Bcr-Abl

By Hideharu Odai, Ko Sasaki, Akihiro Iwamatsu, Tetsuya Nakamoto, Hiroo Ueno, Tetsuya Yamagata, Kinuko Mitani,Yoshio Yazaki, and Hisamaru Hirai

Grb2/Ash and Shc are the adapter proteins that link tyro- cDNA probe, which was obtained by the degenerate poly-merase chain reaction based on a peptide sequence of thesine-kinase receptors to Ras and make tyrosine-kinase func-purified pp135. The cloned cDNA has 3,958 nucleotides thattionally associated with receptors and Ras in fibroblasts andcontain a single long open reading frame of 3,567 nucleo-hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2,tides, encoding a 1,189 amino acid protein with a predictedor phosphotyrosine binding domains. These domains bindmolecular weight of approximately 133 kD. The deducedto proteins containing proline-rich regions or tyrosine-phos-amino acid sequence reveals that pp135 is a protein that hasphorylated proteins and contribute to the association ofone SH2, one SH3, and one proline-rich domain. The pp135,Grb2/Ash and Shc with other signaling molecules. However,which contains two motifs conserved among the inositolthere could remain unidentified signaling molecules thatpolyphosphate-5-phosphatase proteins, was shown to havephysically and functionally interact with these adapter pro-the inositol polyphosphate-5-phosphatase activity. Theteins and have biologically important roles in the signalingpp135 was revealed to associate constitutively with Grb2/pathways. By using the GST fusion protein including theAsh and inducibly with Shc using UT-7 cells stimulated withfull length of Grb2/Ash, we have found that c-Cbl and anGM-CSF. In the cell lines derived from human chronic my-

unidentified 135-kD protein (pp135) are associated with elogenous leukemia, pp135 was constitutively tyrosine-Grb2/Ash. We have also found that they become tyrosine- phosphorylated and associated with Shc and Bcr-Abl. Thesephosphorylated by treatment of a human leukemia cell line, facts suggest that pp135 is a signaling molecule that has aUT-7, with granulocyte-macrophage colony-stimulating fac- unique enzymatic activity and should play an important roletor (GM-CSF). We have purified the pp135 by using GST- in the signaling pathway triggered by GM-CSF and in theGrb2/Ash affinity column and have isolated the full-length transformation of hematopoietic cells caused by Bcr-Abl.complementary DNA (cDNA) encoding the pp135 using a q 1997 by The American Society of Hematology.

M C3G,32 which regulate Ras or Ras-related proteins. Recently,it was reported that the SH3 domain of Grb2/Ash is also

ANY GROWTH FACTORS, including hematopoieticgrowth factors, regulate the proliferation, differentia-

tion, and metabolic activities of their target cells by binding associated with c-Cbl, which becomes tyrosine-phosphory-lated by a variety of stimulations such as Epo, GM-CSF,to their receptors on the cell surface. These stimuli cause

tyrosine phosphorylation of the receptors including the com- TPO, T-cell receptor cross-linking, and EGF.17,33-36

However, there could remain unidentified signaling mole-mon b chain of granulocyte-macrophage colony-stimulatingfactor (GM-CSF)/interleukin-3 (IL-3)/IL-5 receptors and cules that physically and functionally interact with these

adapter proteins and have biologically important roles in theseveral cellular proteins such as Shc, Vav, Raf, and mitogen-activated protein (MAP) kinase by the activation of receptor signaling pathways. In this work, we have purified unidenti-

fied pp135, which is inducibly tyrosine-phosphorylated bytype and non–receptor type tyrosine kinases.1-3 It is consid-ered that the tyrosine phosphorylation of these proteins is the stimulation with GM-CSF and constitutively binds to

Grb2/Ash in a human leukemia cell line, UT-7. We haveneeded for the generation of the binding site for SH2 orphosphotyrosine binding (PTB) domains and to form signal cloned the complementary DNA (cDNA) for pp135 and de-

termined the complete nucleotide sequence, which hastransduction complexes including the adapter proteins.4,5

The adapter protein Shc is widely expressed in a variety shown that pp135 contains several known functional motifssuch as two motifs conserved among the inositol polyphos-of tissues and encodes three forms: p46, p52, and p66. The

Shc contains the SH2 and PTB domains and can bind tothe tyrosine-phosphorylated receptors such as receptors for

From the Third Department of Internal Medicine, Faculty of Med-epidermal growth factor (EGF), nerve growth factor, andicine, University of Tokyo, Tokyo, Japan; and Kirin Brewery Co

insulin.6-12 At the same time, Shc becomes tyrosine-phos- Ltd, Central Laboratory for Key Technology, Yokohama, Kanagawa,phorylated on Tyr-317 by stimulation with growth factors Japan.or cytokines (EGF, IL-2, erythropoietin [Epo], GM-CSF, Submitted August 22, 1996; accepted December 4, 1996.thrombopoietin [TPO], and others) and subsequently inter- Supported in part by Grants-in-Aids from the Ministry of Educa-

tion, Science and Culture of Japan, and from the Ministry of Healthacts with the SH2 domain of Grb2/Ash.13-19 This bindingand Welfare of Japan.leads to the activation of Ras pathway, via the guanine nucle-

Address reprint requests to Hisamaru Hirai, MD, Third Depart-otide exchange factor, Sos, which constitutively binds to thement of Internal Medicine, Faculty of Medicine, University of Tokyo,SH3 domains of Grb2/Ash.7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.Grb2/Ash is a 25-kD protein composed of one SH2 and

The publication costs of this article were defrayed in part by pagetwo SH3 domains in the following order: SH3-SH2-SH3.20,21

charge payment. This article must therefore be hereby markedThe SH2 domain of Grb2/Ash binds to tyrosine-phosphory- ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely tolated proteins such as EGF receptor, Shc, IRS-1, and indicate this fact.Syp.16,22-27 On the other hand, the SH3 domains of Grb2/Ash q 1997 by The American Society of Hematology.

0006-4971/97/8908-0021$3.00/0bind to the proline-rich domain of Sos,28,29 dynamin,30,31 and

2745Blood, Vol 89, No 8 (April 15), 1997: pp 2745-2756

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ODAI ET AL2746

with the lysis buffer before resuspension in Laemmli’s samplephate-5-phosphatase proteins as well as one SH2, one SH3,buffer.and one proline-rich domain. Moreover, pp135 became

Immunoblotting. Samples were subjected to sodium dodecyl sul-associated with Shc in response to GM-CSF stimulation inphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-UT-7 cells. In the cell lines derived from human chronictransferred onto polyvinylidene difluoride filters (Millipore, Bedford,

myelogenous leukemia (CML), pp135 was constitutively ty-MA). Filters were blocked with the buffer containing 10 mmol/L

rosine-phosphorylated and associated with Shc and Bcr-Abl. Tris/HCl (pH 7.4), 150 mmol/L NaCl, 5% BSA, and 0.05% TritonThese facts suggest that pp135 is a signaling molecule that X-100. Filters were sequentially incubated with the specific antibodyhas a unique enzymatic activity and may play an important and with horseradish peroxidase–coupled second antibodies (anti-role in the signaling pathway triggered by GM-CSF and in rabbit or antimouse Fc; Amersham, Buckinghamshire, UK). Devel-

opment by enhanced chemiluminescence was performed using thethe oncogenic transformation of hematopoietic cells causedstandard protocol.by Bcr-Abl.

Purification of pp135. After the lysates from 1 1 109 UT-7cells were pretreated with GST protein noncovalently coupled to

MATERIALS AND METHODS glutathione-agarose beads, the unbound fractions were mixed withthe GST-Grb2/Ash fusion protein noncovalently coupled to glutathi-Cell lines and growth factors. UT-7 cells were maintained inone-agarose beads. Successively, the eluent from the GST-Grb2/AshRPMI 1640 medium containing 8% bovine serum and 10 ng/mLcolumn containing pp135 was subjected to SDS-PAGE and blottedGM-CSF. Recombinant human GM-CSF and recombinant humanonto the polyvinylidene difluoride membrane. After being visualizedEpo were supplied by Kirin Brewery Co Ltd (Tokyo, Japan) andby ponceau-S, an isolated 135-kD band (approximately 20 pmol) wasChugai Pharmacy Co Ltd (Tokyo, Japan), respectively.cut out and in situ–digested with lysylendopeptidase AchromobactorAntibodies. Mouse monoclonal antibody to Grb2/Ash purchasedprotease I (Wako, Tokyo, Japan) or endproteinase Asp-N (Boeh-from MBL (Nagoya, Japan) was used for the immunoblotting ofringer Mannheim, Mannheim, Germany). Sequences of six digestedGrb2/Ash. Mouse monoclonal antiphosphotyrosine antibody (anti-fragments were determined by amino acid sequence analysis.Py) 4G10 and rabbit polyclonal antibody to Shc were purchased

Cloning of pp135. For preparing the screening probes, RNA-from UBI (Lake Placid, NY). The anti-pp135 antibodies (anti-P6)based PCR (reverse transcriptase PCR [RT-PCR]) was performedwere raised by immunizing rabbits with the GST-P6 fusion proteinby oligo(dT)-priming cDNA synthesis from the total RNA of UT-expressed in bacteria as described below and were used for immu-7 cells, followed by PCR with degenerate sense and antisense oligo-noblotting and immunoprecipitation.nucleotide primers synthesized based on the three-peptide sequence.GST fusion proteins. The bacterial expression plasmids codingOne pair of primers was DN-11 sense (5*-GAYCARYTIATHGAR-for GST fusion proteins containing the full-length, the N-terminalTTYTA-3*) and DN-9 antisense (5*-AAICCDATIACRTA-SH3 domain, or the SH2 domain of Grb2/Ash31 were generouslyDATRTC-3*), and the other was DN-11 sense and AP-6 antisense

provided by Dr T. Takenawa (University of Tokyo, Tokyo, Japan).(5*-TCIGCRAAIACRTAYTCYTT-3*). The PCR fragment 1 (1,117

The bacterial expression plasmids coding for GST fusion proteinsbp) and fragment 2 (898 bp) were subcloned into pCRTMII vector

containing the SH3 domains of Tec, Lyn, Fyn, or Abl were fromusing Original TA Cloning Kit (Invitrogen, San Diego, CA) and

Dr H. Mano, Dr T. Yamamoto, and Dr B. Mayer, respectively.sequenced, and the nucleotide sequences were confirmed to match

The bacterial expression plasmids coding for GST fusion proteinswith the corresponding original amino acid sequences. The EcoRI

containing the SH3 domains of Crk, Nck, Src, or Cas were also used fragment of pCRTMII vector containing PCR fragment 1 was labeledin this study. The cDNA sequence encoding a polypeptide of amino with Megaprime DNA labeling system (Amersham) and was usedacids 318 to 957 of pp135 was amplified by polymerase chain reac- as the probe to screen the cDNA library of SKH1, a human leukemiction (PCR) using the cDNA obtained as a template. The resulting cell line.37 Six overlapping clones were isolated, and nested deletionsPCR fragment was cloned into pGEX-2T vector (Pharmacia Inc, of two clones were generated using exonuclease III according to theUppsala, Sweden; GST-P6) and was expressed in XL-I Blue strain manufacturer’s instructions (Pharmacia) and sequenced by the cycleof Escherichia coli. Induction and purification of GST fusion pro- sequencing method using Thermo Sequenase Kit (Amersham), Cy5-teins were performed as described before.33

labeled M13-reversal and universal primers, and A.L.F. DNA Se-Preparation of cell lysates. UT-7 cells were incubated in RPMI quencer II (Pharmacia). The sequence in this report has been depos-

1640 medium containing 0.1% bovine serum albumin (BSA) without ited in the GenBank database (accession no. U53470).serum and growth factors for 8 to 15 hours before stimulation with Assay of inositol-5-phosphatase activity. Inositol-5-phosphatasegrowth factors and then were resuspended in RPMI 1640 medium activity was assayed at 377C for 15 minutes in 100 mL of 50 mmol/containing 100 mmol/L Na3VO4. The cells were treated with 10 ng/ L Tris/HCl buffer (pH 7.4)/3 mmol/L MgCl2/20 nCi [3H]-inositolmL GM-CSF or 20 U/mL Epo for 5 minutes at 377C and then lysed (1,3,4,5)-P4 (Dupont-New England Nuclear, Boston, MA)/10 mmol/at 47C for 20 minutes in the lysis buffer containing 20 mmol/L Tris/ L inositol (1,3,4,5)-P4 (Boehringer Mannheim) containing immuno-HCl (pH 8.0), 1% Nonidet P-40, 1 mmol/L phenylmethyl sulphonyl precipitates from UT-7 cells. The reaction was initiated by the addi-fluoride, 500 U/mL aprotinin, 2 mmol/L EDTA, 50 mmol/L NaF, tion of the substrate and was terminated by adding 1 mL of 0.1and 1 mmol/L Na3VO4. Unsolubilized materials were removed by mol/L ammonium formate/0.1 mol/L formic acid. The [3H]-inositolcentrifugation at 15,000g at 47C for 10 minutes. polyphosphates were then separated on Dowex 1 (BioRad Labora-

Affinity precipitation with GST fusion proteins and immunoprecip- tories, Richmond, CA) anion exchange chromatography columns38

itation. Lysates from 1 1 107 cells were mixed with 40 mg of the and quantified by liquid scintillation spectrometry.fusion protein noncovalently coupled to glutathione-agarose beads(Sigma Chemical Co, St Louis, MO) for 3 hours at 47C. Beads RESULTSwere washed with the lysis buffer before resuspension in Laemmli’s

As shown in Fig 1A, several tyrosine-phosphorylated pro-sample buffer. To immunoprecipitate the proteins, lysates from 1 1teins, including pp130 and pp135, in the lysates of GM-107 cells were incubated with the antibody for 3 hours at 47C. TheCSF–treated UT-7 cells were found to bind to GST-Grb2/immunoprecipitates were collected with protein A-sepharose (Sigma

Chemical Co). All the immunoprecipitates were washed intensively Ash in vitro, and their phosphorylation was dependent on





A NOVEL INOSITOL POLYPHOSPHATE-5-PHOSPHATASE 2747

GM-CSF stimulation. Because these tyrosine-phosphory- cytokines such as GM-CSF, Epo, IL-3, TPO, and EGF,17,33-

35 and binds to the many signaling molecules including EGF-lated bands were not observed when GST protein was usedinstead of GST-Grb2/Ash (data not shown), these tyrosine- receptor (EGFR), Grb2/Ash, Crk, Nck, p85 PI3 kinase, and

v-Src.17,33-36,39-44 Both c-Cbl and pp135 constitutively boundphosphorylated proteins were thought to bind specifically toGrb2/Ash protein in vitro. Using anti-Shc polyclonal anti- to the SH3 domain but not to the SH2 domain of Grb2/

Ash and became tyrosine-phosphorylated with stimulationbody, the 52-kD (pp52) and 66-kD proteins (pp66) wereidentified as Shc (data not shown), which is one of the well- of GM-CSF in UT-7 cells (Fig 1B). However, the nature of

pp135 was still unknown. To identify the Grb2/Ash-bindingknown molecules that binds to Grb2/Ash and has an im-portant role in the signal transduction of many cytokines protein, pp135, we purified this protein and cloned its cDNA.

The pp135 protein was purified from UT-7 cell lysatesincluding EGF, Epo, GM-CSF, TPO, and IL-3.13,16,17 As pre-viously reported, the 130-kD protein was identified as c-Cbl, using the GST-Grb2/Ash affinity column by the procedure

described in the Materials and Methods. The six peptidewhich becomes tyrosine-phosphorylated with stimulation ofsequences derived from the purified pp135 were determinedby the amino acid sequence analysis. None of the peptidesequences were identical to the proteins in the database of theNational Center of Biotechnology Information (N.C.B.I.).Using the DN-11, DN-9, and AP-6 oligonucleotide se-quences as PCR primers, we obtained two DNA fragmentsfrom the cDNA prepared from UT-7 cells. The amino acidsequences deduced from the nucleotide sequences of thetwo DNA fragments contained the sequences completelyidentical to the original peptide sequences. The longer DNAfragment was used as a probe to screen a cDNA library ofa human leukemic cell line, SKH1. Two cDNAs of the sixobtained clones were sequenced and revealed to contain3,958 nucleotides, 3,567 of which encoded an open readingframe of 1,189 amino acids with a predicted molecular sizeof approximately 133 kD (Fig 2). The deduced amino acidsequence contained the sequences of the six obtained peptidesequences. The cDNA sequence around the first methioninewas in good agreement with Kozak’s consensus sequence,and therefore, the first methionine is considered to be thetranslation initiation one.45,46 The homology search analysisbased on the deduced amino acid sequence revealed thatthere were several interesting motifs within pp135. Therewas a consensus sequence for the SH2 domain (amino acids5 to 102) that was most similar to that of Abl. The consensussequence for the SH3 domain was also identified withinpp135 (amino acids 861 to 909),4,47 but it is necessary toconfirm whether this region is, in fact, capable of binding

RFig 1. (A) Proteins associated with Grb2/Ash in vitro and their

tyrosine phosphorylation. The lysates from UT-7 cells unstimulated(lane 2) or stimulated with GM-CSF (lane 1) were mixed with theGST fusion protein including the full-length Grb2/Ash noncovalentlycoupled to glutathione-agarose beads. Beads were resuspended inLaemmli’s sample buffer, subjected to SDS-PAGE, and immunoblot-ted with anti-Py (4G10). Molecular weight markers, indicated at theleft, are given in kilodaltons. The arrows indicate the position of pp52,pp66, pp130, and pp135. (B) In vitro association of pp130 and pp135with the SH3 domain of Grb2/Ash and tyrosine phosphorylation ofpp130 and pp135. The lysates from UT-7 cells unstimulated (lanes 1and 3) or stimulated with GM-CSF (lanes 2 and 4) were mixed withGST fusion protein including the SH2 domain (lanes 1 and 2) or theN-terminal SH3 domain (lanes 3 and 4) of Grb2/Ash noncovalentlycoupled to glutathione-agarose beads. The resulting precipitateswere resuspended in Laemmli’s sample buffer, subjected to SDS-PAGE, and immunoblotted with anti-Py (4G10). Molecular weightmarkers, indicated at the left, are given in kilodaltons. The arrowsindicate the position of pp52, pp66, pp130, and pp135.





ODAI ET AL2748

Fig 2. (A) Amino acid sequence and known motifsof pp135. The underlined amino acids are the SH2domain, SH3 domain, 5-phosphatase conserved mo-tifs, and proline-rich domain. The hatched-under-lined amino acids are the two binding sites for PTBdomain. The hatched-overlined amino acids are thesix peptides obtained from the amino acid sequenceof purified pp135. (B) Diagrammatic representationof the various domains within pp135.

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to some proline-rich polypeptides. Two sequences, INPNY Ash antibody. Grb2/Ash was shown to be coimmunoprecipi-tated with pp135 from both lysates of GM-CSF–stimulatedand ENPLY, within pp135 matched the consensus NPXY

motif to which the PTB domain binds.6-8,48 In the proline- or –unstimulated cells (Fig 4C), suggesting the constitutiveassociation of pp135 and Grb2/Ash in vivo. From these find-rich domain near the C-terminus and at amino acids 123 to

128, there were several sequences that match the binding ings, we concluded that we had correctly cloned the cDNAfor the pp135 originally identified.sequences for the SH3 domain (eg, PRKEPPPCP for class

I and PELPPR, PISPKK, PKMPRK, and PRPPRR for class So far, it has been shown that the signaling pathwaysdownstream of GM-CSF receptor and Epo receptor areII)49,50 and were probably responsible for the purification

of pp135 with GST-Grb2/Ash affinity chromatography. The closely related to each other. These observations motivatedus to investigate the tyrosine-phosphorylation profile ofhomology search with the N.C.B.I. database revealed that

pp135 shares a similarity with human inositol polyphos- pp135 in the signaling pathway triggered by Epo. The tyro-sine phosphorylation of pp135 was also induced in UT-7phate-5-phosphatase (29.1% identity in 326 amino acid over-

lap)51 and OCRL (29.9% identity in 351 amino acid overlap), cells stimulated with Epo (data not shown), indicating thatpp135 has an important role in a variety of cytokine-trig-which is encoded by the causative gene for human Low’s

oculocerebrorenal syndrome and is a homologue of inositol gered signaling pathways.It is well known that Shc also associates with a highlypolyphosphate-5-phosphatase.52,53 The members of inositol

polyphosphate-5-phosphatase (5-phosphatase) proteins have tyrosine-phosphorylated protein of approximately 145-kD inseveral cell types activated by a variety of stimuli such astwo conserved motifs,54 and the amino acid sequences at 583

to 594 and 665 to 692 of pp135 were matched to these motifs. GM-CSF, Epo, IL-3, IL-2, Steel factor, EGF, and platelet-derived growth factor,9,55-61 or in cells transformed by acti-These results suggest that pp135 could have the inositol

polyphosphate-5-phosphatase activity. The various domains vated oncogenes.62 To assess whether pp135 also binds toShc, immunoprecipitates with anti-Shc from lysates of theof pp135 are shown in Fig 2B.

Northern blot analysis was performed with poly(A)/ UT-7 cells with or without the stimulation of GM-CSF weresubjected to SDS-PAGE and immunoblotted with anti-PyRNAs from various human tissues (human multiple tissue

Northern blot; Clontech, Palo Alto, CA). As shown in Fig or anti-P6 antibody (Fig 5). Anti-Shc immunoprecipitantscontained several tyrosine-phosphorylated proteins including3A, pp135 mRNA (5.4 kb) was obviously expressed in

spleen, thymus, and peripheral blood leukocytes, but in other pp135 in GM-CSF–treated cells, and pp135 in the anti-Shcimmunoprecipitants was also recognized with the anti-P6tissues, the expression of pp135 mRNA was very low or

undetectable. Next, we examined the expression of the antibody (Fig 5). These results have shown that the pp135associates with Shc, depending on GM-CSF stimulation inmRNA of pp135 in myelocytic/monocytic cell lines (HL60,

U937, KG1, KU812, and SKH1), erythroid cell lines (HEL vivo.As described above, pp135 has a proline-rich domain andand K562), and megakaryocytic cell lines (UT-7, CMK, and

MOLM1) as described previously.55 As shown in Fig 3B, associates with the SH3 domain of Grb2/Ash. Therefore, wenext examined whether pp135 binds to the SH3 domains ofpp135 mRNA was ubiquitously expressed in all these cell

lines. These results indicate that the pp135 might play an other proteins. The cell lysates from UT-7 cells unstimulatedor stimulated with GM-CSF were mixed with a variety ofimportant role in the signal transduction in hematopoietic

cells. GST-SH3 fusion proteins. The resulting complexes were re-trieved with glutathione-agarose beads, subjected to SDS-We then tried to confirm that we had indeed purified the

pp135 and cloned the cDNA for the pp135 originally identi- PAGE, and immunoblotted with anti-P6. As shown in Fig6, pp135 bound to the GST fusion proteins containing thefied. After the lysates from UT-7 cells stimulated with or

without GM-CSF were mixed with rabbit antiserum to pp135 SH3 domain of Abl, Lyn, and Src as well as Grb2/Ash. Theassociation with GST-SH3 fusion proteins was independent(anti-P6) generated against the GST-P6 fusion protein, the

resulting immunoprecipitates, in combination with the pre- of GM-CSF stimulation in UT-7 cells. These findings indi-cate that the proline-rich region of pp135 has some specific-cipitates bound to GST-SH3 domain of Grb2/Ash, were sub-

jected to SDS-PAGE and immunoblotted with anti-Py ity for the association with the SH3 domains.In some tumor cells, it is considered that the signal for(4G10) or anti-p6. The pp135 immunoprecipitated with anti-

P6 showed the same electrophoretic mobility as that of cell growth is constitutively transduced to the nucleus andthat many cellular proteins are constitutively tyrosine-phos-pp135 bound to GST-SH3 domain of Grb2/Ash, whether

probed with anti-Py or anti-P6 (Fig 4A and B). The pp135 phorylated. In the case of cell lines derived from CML, thesephenomena could be caused by the Bcr-Abl oncogene. Weimmunoprecipitated with anti-p6 and probed with anti-Py

was shown to become tyrosine-phosphorylated in vivo, de- then examined if pp135 was tyrosine-phosphorylated inCML cell lines. The cell lysates from K562, KCl22, SKH1pending on GM-CSF stimulation (lanes 3 and 4 of Fig 4A).

Reprobing with anti-P6 has shown that pp135 constitutively (CML), and HL-60 (acute myelogenous leukemia [AML])cell lines were immunoprecipitated with anti-P6 antibodyassociates with GST-Grb2/Ash-SH3 (lanes 1 and 2 in Fig

4B) and that the amount of pp135 was not altered with GM- and immunoblotted with anti-Py. As shown in Fig 7A andB, pp135 was constitutively tyrosine-phosphorylated in allCSF stimulation (lanes 3 and 4 in Fig 4B). To confirm the

association of pp135 and Grb2/Ash in vivo, immunoprecipi- three CML cell lines but not in HL-60. Moreover, pp135was coimmunoprecipitated with Shc and Bcr-Abl in Bcr-tates with anti-P6 from lysates of the UT-7 cells were sub-

jected to SDS-PAGE and immunoblotted with anti-Grb2/ Abl–expressing cells (Fig 7A and C). At the same time,





ODAI ET AL2750

Fig 3. The expression of the pp135 mRNA in hu-man tissues and leukemic cell lines. Poly (A)" RNAsfrom various human tissues (A) or leukemic cell lines(B) were blot-hybridized with the PCR fragmentprobe used for cloning cDNA. The migration positionof RNA size makers (in kilobases) is indicated on theleft.

tyrosine-phosphorylated 145-kD protein (pp145) was also inositol (1,3,4,5)-P4 as the substrate. The inositol-5-phos-phatase can remove the 5-phosphate and produce inositolrecognized by anti-P6 antibody and was found to be associ-

ated with Shc and Bcr-Abl. Although the nature of p145 is (1,3,4)-P3 from inositol (1,3,4,5)-P4. After the total lysates,anti-P6 immunoprecipitates, or anti-Shc immunoprecipitatesnot clear, pp145 may be an alternative–splicing form or a

posttranslationally modified form of pp135. These results were incubated with [3H]-inositol (1,3,4,5)-P4, the productsand substrates were separated with Dowex 1 ion-exchangesuggest that constitutive tyrosine phosphorylation of pp135

could be caused by Bcr-Abl in CML cells and might be resin and the radioactivity of the [3H]-inositol (1,3,4)-P3 wasmeasured. As shown in Fig 8, anti-P6 immunoprecipitatesinvolved in neoplastic transformation of hematopoietic cells.

As described above, pp135 has two motifs conserved hydrolyzed inositol (1,3,4,5)-P4 regardless of GM-CSF stim-ulation. Moreover, anti-Shc immunoprecipitates from GM-among the inositol polyphosphate-5-phosphatase proteins.

We next analyzed the phosphatase activity of pp135 using CSF–stimulated cell lysates but not from unstimulated cell






Fig 4. The pp135 constitutively binds to the SH3 domain of Grb2/Ash and inducibly becomes tyrosine-phosphorylated with the stimu-lation of GM-CSF. The lysates from UT-7 cells stimulated with GM-CSF (lanes 2 and 4) or unstimulated (lanes 1 and 3) were mixed withGST-Grb2/Ash SH3 (lanes 1 and 2) or with anti-P6 (lanes 3 and 4).The resulting precipitates were subjected to SDS-PAGE and immu-noblotted with anti-Py (A) and reprobed with anti-P6 (B). The arrowindicates the position of pp135. Molecular weight markers, indicatedat the left, are given in kilodaltons. (C) In vivo association of pp135with Grb2/Ash. The lysates from UT-7 cells stimulated with GM-CSF (lane 2) or unstimulated (lane 1) were mixed with anti-P6. Theresulting precipitates were subjected to SDS-PAGE and immunoblot-ted with anti-Grb2/Ash. The total UT-7 cell lysate (TCL, lane 3) is alsoapplied for reference. Molecular weight markers, indicated at the left,are given in kilodaltons. The arrow indicates the position of Grb2/Ash.

lysates have shown the inositol-5-phosphatase activity. 145- to 150-kD protein associated with Shc was cloned fromThese results suggest that pp135 has inositol-5-phosphatase the cDNA library of murine hematopoietic cell lines.63,64 Thisactivity and confirmed that pp135 associates with Shc, de- protein has an SH2 domain and the consensus motifs for thepending on the stimulation of GM-CSF. 5-phosphatase, as is the case with pp135, and was named

SHIP for SH2-containing inositol-5-phosphatase. We com-DISCUSSION pared the amino acid sequences of the two proteins and

found that overall 85% of the amino acids of pp135 wereIn this work, we have purified pp135, which bound GST-identical with those of SHIP. From this observation, ourGrb2/Ash and became tyrosine-phosphorylated with stimula-pp135 can be considered to be the human counterpart oftion of GM-CSF in UT-7 cells. Moreover, we cloned theSHIP. On the other hand, Kavanaugh et al65 have reportedfull-length cDNA for pp135, which has the SH2, SH3, and

inositol-5-phosphatase domains. Recently, cDNA for the that the partial amino acid sequence of human 130-kD pro-





ODAI ET AL2752

cipitates (Fig 4). On the other hand, anti-P6 antibody recog-nized both pp135 and pp145 in KCL22 and SKH1 cells, butmainly pp145 in K562 (Fig 8). The nature of pp145 is un-clear, but it was suggested that three alternative splicingforms of SIP (SIP-110, SIP-130, and SIP-140) were gener-ated.65 Therefore it may be reasonable to consider that pp145is an alternative splicing form of pp135 and that pp135 andpp145 are identical with SIP-130 and SIP-140, respectively.The differences in the expression patterns of pp135 andpp145 among the cell lines remain to be elucidated.

We have shown that pp135 associates with Shc, constitu-tively in CML cell lines and inducibly in UT-7 cells withGM-CSF stimulation. Although the mechanism for the asso-ciation between Shc and pp135 or SHIP has not been clari-fied, we could consider that tyrosine-phosphorylated proteinscan bind to the PTB domain of Shc,9,62 which may interactwith the tyrosine-phosphorylated NPXY motifs within SHIPor pp135. Our result that GST fusion protein with the SH2domain of Shc did not bind to pp135 (data not shown) andthe reported result that p150SHIP was cloned by the yeasttwo-hybrid screening using the PTB domain of Shc stronglysupport this mechanism.64

Although the SH3 domains of Grb2/Ash bind to the pro-line-rich domains of dynamin,30,31 C3G,32 and Sos,28,29 which

Fig 5. GM-CSF induces the association of pp135 with Shc. The regulate the small G-proteins, pp135 has no homologouslysates from UT-7 cells stimulated with GM-CSF (lanes 1 and 3) or regions with guanine nucleotide exchange factors orunstimulated (lanes 2 and 4) were mixed with anti-Shc. The resulting

GTPase-activating proteins. Therefore, pp135, which consti-precipitates were subjected to SDS-PAGE and immunoblotted withtutively associates with Grb2/Ash through its SH3 domain,anti-Py (lanes 1 and 2) and reprobed with anti-P6 (lanes 3 and 4).

Molecular weight markers, indicated at the left, are given in kilodal- could be involved in the signal transduction pathway differ-tons. The arrow indicates the position of pp135. ent from the Ras pathway. As shown in Fig 8, it has been

demonstrated that pp135 has the 5-phosphatase activity, aswas reported with SHIP/SIP.63-65 However, the 5-phospha-tase activity of pp135 was not altered by the GM-CSF stimu-tein (SIP-130)65 and this sequence was included within thatlation and the tyrosine phosphorylation of pp135. A numberof pp135. Moreover, SIP130 has the SH2 and inositol-5-of cellular proteins are tyrosine-phosphorylated, dependingphosphatase domains and shows inositol-5-phosphatase ac-on extracellular signals including growth factors, and begintivity as pp135 does. These results suggest that pp130 andto form complexes with other signaling molecules.4,5 In theSIP130 are probably identical.case of pp135, tyrosine-phosphorylated pp135 may form aIn UT-7 cells, anti-P6 antibody recognized mainly pp135

in GST-Grb2/Ash binding proteins and anti-Shc immunopre- complex with Grb2/Ash, Shc, and other molecules con-

Fig 6. pp135 associates with the SH3 domains of different proteins. The lysates from UT-7 cells stimulated with GM-CSF (lanes 2, 4, 6, 8,10, 12, 14, 16, and 18) or unstimulated (lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17) were mixed with each GST-SH3 fusion protein noncovalentlycoupled to glutathione-agarose beads. The resulting precipitates were subjected to SDS-PAGE and immunoblotted with anti-P6. The arrowindicates the position of pp135.






Fig 7. pp135 was constitutively tyrosine-phosphorylated and as-sociated with Shc and Bcr-Abl in CML cell lines. The lysates fromthree human CML (K562, Kcl22, and SKH1) and human AML (HL-60)cell lines were mixed with anti-Shc (A) or anti-P6 antibody (B). Theresulting precipitates were subjected to SDS-PAGE and immunoblot-ted with anti-Py (top panels) and reprobed with anti-P6 (bottom panelof A). The lysates from these CML cell lines were also mixed withanti-Abl antibody (C). The precipitates were subjected to SDS-PAGEand immunoblotted with anti-Py (top panel) and reprobed with anti-P6 (bottom panel). The arrow indicates the position of pp135 andpp145. Molecular weight markers, indicated at the left, are given inkilodaltons.

taining SH2, SH3, or PTB domain. Furthermore, pp135 asso- SHIP and SIP hydrolyzed only the polyphosphate-containingphosphate in the 3 position of the inositol ring.63-65 Theseciated with the SH3 domains of the different proteins in vitro

(Fig 6) and was coimmunoprecipitated with Bcr-Abl in CML substances are produced by activated PI3 kinase, dependingon the stimulation, such as growth factors, and the productioncell lines (Fig 7). The formation of such a complex is proba-

bly important for the regulation of the enzymatic activity of of these substances is correlated with Ca2/ mobilization, Ras,PKCl, or c-Akt regulation.67-71 These observations suggestpp135. Theoretically, the candidates for the substrates of 5-

phosphatases are inositol 1,4,5-trisphosphate, inositol that pp135 and SHIP could play an important role in thesignal transduction pathways triggered by hematopoietic1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphos-

phate, and phosphatidylinositol 3,4,5-trisphosphate,38,66 but growth factors, especially in the pathway in which PI3 kinase





ODAI ET AL2754

specific binding of the Shc phosphotyrosine-binding domain to phos-photyrosine sites. Proc Natl Acad Sci USA 93:963, 1996

7. Prigent SA, Pillay TS, Ravichandran KS, Gullick WJ: Bindingof Shc to the NPXY motif is mediated by its N-terminal domain. JBiol Chem 270:22097, 1995

8. Batzer AG, Blaikie P, Nelson K, Schlessinger J, Margolis B:The phosphotyrosine interaction domain of Shc binds an LXNPXYmotif on the epidermal growth factor receptor. Mol Cell Biol15:4403, 1995

9. Kavanaugh WM, Williams LT: An alternative to SH2 domainsfor binding tyrosine-phosphorylated proteins. Science 266:1862,1994

10. He W, O’Neill TJ, Gustafson TA: Distinct modes of interac-tion of SHC and insulin receptor substrate-1 with the insulin receptorNPEY region via non-SH2 domains. J Biol Chem 270:23258, 1995

11. Bork P, Margolis B: A phosphotyrosine interaction domain.Fig 8. Inositol-5-phosphatase activity of pp135. The lysates from Cell 80:693, 1995

UT-7 cells with (") or without (Ï) GM-CSF stimulation were mixed 12. Blaikie P, Immanuel D, Wu J, Li N, Yajnik V, Margolis B:with normal rabbit serum (NRS), anti-P6, or anti-Shc. The inositol-5- A region in Shc distinct from the SH2 domain can bind tyrosine-phosphatase activity of the resulting immunoprecipitates was mea-

phosphorylated growth factor receptors. J Biol Chem 269:32031,sured using [3H]inositol (1,3,4,5)-P4 as the substrate. Results are ex-1994pressed as picomoles of hydrolysis product per 15 minutes.

13. Pelicci G, Lanfrancone L, Grignani F, McGlade J, CavalloF, Forni G, Nicolett I, Grignani F, Pawson T, Pelicci PG: A noveltransforming protein (Shc) with an SH2 domain is implicated in

is involved and in hematopoietic cell transformation caused mitogenic signal transduction. Cell 70:93, 1992by Bcr-Abl. 14. Gotoh N, Tojo A, Muroya K, Hashimoto Y, Hattori S, Naka-

In summary, we have purified the 135-kD protein that mura S, Takenawa T, Yazaki Y, Shibuya M: Epidermal growthfactor-receptor mutant lacking the autophosphorylation sites inducesassociates constitutively with Grb2/Ash but inducibly withphosphorylation of Shc protein and Shc-Grb2/ASH association andShc. Moreover, pp135 becomes tyrosine-phosphorylatedretains mitogenic activity. Proc Natl Acad Sci USA 91:167, 1994with the stimulation of GM-CSF or Epo in hematopoietic

15. Pronk GJ, de Vries Smits AM, Buday L, Downward J, Maas-cells. In CML cell lines, pp135 was constitutively tyrosine-sen JA, Medema RH, Bos JL: Involvement of Shc in insulin- andphosphorylated and associated with Shc and Bcr-Abl. Theepidermal growth factor-induced activation of p21ras. Mol Cell Bioldeduced amino acid sequence has shown that pp135 has the14:1575, 1994SH2, SH3, and 5-phosphatase domains. Indeed, it has been

16. Cutler RL, Liu L, Damen JE, Krystal G: Multiple cytokinesshown that pp135 has the 5-phosphatase activity. We pro- induce the tyrosine phosphorylation of Shc and its association withpose that the cloned isoform pp135 should be designated as Grb2 in hemopoietic cells. J Biol Chem 268:21463, 1993SHIP-135 for Src homology containing inositol phosphatase, 17. Sasaki K, Odai H, Hanazono Y, Ueno H, Ogawa S, Langdon135-kD protein. WY, Tanaka T, Miyagawa K, Mitani K, Yazaki Y, Hirai H: TPO/

c-mpl ligand induces tyrosine phosphorylation of multiple cellularACKNOWLEDGMENT proteins including proto-oncogene products, Vav and c-Cbl, and Ras

signaling molecules. Biochem Biophys Res Commun 216:338, 1995UT-7 was a kind gift from Dr Norio Komatsu (Jichi Medical18. Ravichandran KS, Burakoff SJ: The adapter protein Shc inter-School, Tochigi, Japan). We thank Dr Tomoyuki Tanaka, Dr Seishi

acts with the interleukin-2 (IL-2) receptor upon IL-2 stimulation. JOgawa, and Dr Naoto Hirano for helpful discussions and NaokoBiol Chem 269:1599, 1994Nishimura and Rie Mitsumori for technical assistance.

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1997 89: 2745-2756

Mitani, Yoshio Yazaki and Hisamaru HiraiHideharu Odai, Ko Sasaki, Akihiro Iwamatsu, Tetsuya Nakamoto, Hiroo Ueno, Tetsuya Yamagata, Kinuko Bcr-Ablof Granulocyte-Macrophage Colony-Stimulating Factor, Erythropoietin, andPolyphosphate-5-Phosphatase, Which Is Involved in the Signaling Pathway Purification and Molecular Cloning of SH2- and SH3-Containing Inositol

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