(ship)-1 binds to the multifunctional docking site of c-met and

The SH2-containing inositol 5´phosphatase (SHIP)-1 binds to the multifunctional

docking site of c-Met and potentiates hepatocyte-growth factor (HGF) induced

branching tubulogenesis

Monica Stefan, Alexandra Koch, Annalisa Mancini, Andrea Mohr, K. Michael Weidner1, Heiner

Niemann, and Teruko Tamura*

Institut für Biochemie, OE 4310, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-

30623 Hannover,1 Roche Pharma Research Werk Penzberg, Nonnenwald 2, D-82372, Penzberg,

FRG.

Running title: Interaction of c-Met tyrosine kinase with SHIP-1.

*Corresponding author Tel.: 0049-511-(532)-2857, Fax: 0049-

511-(532)-2827

E-mail: [email protected]

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 7, 2000 as Manuscript M009333200 by guest on February 11, 2018

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Key words: branching tubulogenesis/cell scattering /c-Met tyrosine kinase/ SH2 domain

containing inositol 5´phosphatase/SHIP/ signal transduction

Summary.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that

induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells.

The receptor for HGF/SF was identified as c-Met tyrosine kinase and activation of the receptor

induces multiple signaling cascades. To gain further insight into c-Met mediated multiple events

at a molecular level, we isolated several signaling molecules including a novel binding partner of

c-Met, the SH2-domain-containing inositol 5´phosphatase (SHIP)-1. Western blot analysis

revealed that SHIP-1 is expressed in the epithelial cell line, MDCK cells. SHIP-1 binds at

phosphotyrosine-1356 at the multifunctional docking site. Since a number of signaling

molecules such as the Grb2, phosphatidylinositol 3´-kinase (PI-3´ kinase) and Gab1 bind to the

multifunctional docking site, we further performed an in vitro competition study using

glutathione S-transferase (GST)- or His-tagged signaling molecules with c-Met tyrosine

kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over PI-

3´ kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching

tubulogenesis within 2 days after HGF treatment, while wild-type MDCK cells showed

tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth

potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired the

HGF mediated branching tubulogenesis.

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Introduction

Met tyrosine kinase is the receptor for hepatocyte growth factor/scatter factor (HGF/SF)

(1,2), and signaling via the binding of this receptor to ligand has been shown to affect a wide

range of biological activities, including angiogenesis (3,4), cellular motility (5), growth (6,7),

invasion (8-10) and morphological differentiation including neuronal branching (11-14). c-Met

also plays a role in normal embryological development (15-17), tissue regeneration (18), and

wound healing (19). Furthermore, HGF also cooperates with nerve growth factor (NGF) to

enhance axonal outgrowth from cultured dorsal root ganglion (DRG) neurons (14).

Binding of the growth factors causes activation of their inherent receptor tyrosine kinases

leading to autophosphorylation of the cytoplasmic domains at multiple tyrosine residues. The

newly formed phosphotyrosines constitute binding sites for the Src homology 2 (SH2) domain-

and/or phosphotyrosine binding (PTB) domain-containing cytoplasmic proteins. Met tyrosine

kinase was reported to interact with several substrates including the growth factor receptor bound

protein 2 (Grb2) (20), STAT3 (21), the p85 subunit of phosphatidyl-inositol-3’(PI 3’) kinase

(22), Shc (23), phospholipase C-γ (PLCγ) (20), c-Src (20) and Gab1 (24). Interestingly, all these

proteins associate with c-Met via a multiple substrate binding site. The c-Met mediated

signaling was well studied in the epithelial cell system, MDCK cells. On planar culture surfaces,

sub-confluent MDCK cells, normally form coherent islands of relatively flattened cells. HGF

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has been shown to alter this morphology by promoting the initial expansion of colonies followed

by dispersion of the cells that make up these colonies, and an increase in their motility (cell

scattering) in liquid cell culture medium (25). The activation of a number of tyrosine kinase

receptors including Neu, Ros epidermal growth factor (EGF) receptor and keratinocyte growth

factor (KGF) receptor, induced the cell scattering in MDCK cells (26). Royal and Park (27)

reported that PI-3´ kinase and Ras are required for cell scattering. In addition, stimulation with

HGF induces the formation of tubular structures from MDCK cells grown in a three-dimensional

collagen matrix (11). For this, the epithelial cells are grown for several days in collagen, in which

they form cysts. When SF/HGF is added, individual cells dissociate and form continuous tubules

(28). It has been reported that a number of signaling molecules including Gab1(24), Grb2 (29),

phospholipase Cγ (30) , and STAT3 (21) are required for this process.

The underlying mechanisms of these multiple molecular events, however, are still poorly

understood. In the present study we identified the SH2-containing inositol 5´phosphatase

(SHIP)-1 as a novel binding partner of c-Met by yeast two-hybrid screening using a rat brain

library. SHIP-1 binds to one of the tyrosine residues at the multiple-substrate binding site,

pY1356VNV, which is also the binding site for Grb2. Interestingly, the YVNV motif is identical to

the common binding site of Shc to SHIP and Grb2. Furthermore, we show that PI-3´ kinase was

not able to bind c-Met in the presence of Grb2 , Gab1 or SHIP-1. Overexpression of SHIP-1 in

MDCK cells drastically enhanced the branching potency of c-Met without affecting of the

mitogenic and scattering potency. Furthermore, overexpression of a mutant SHIP-1 lacking

catalytic activity impaired HGF mediated branching tubulogenesis.

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Experimental Procedures.

Plasmid constructions, transfection, and yeast two-hybrid screening

The construction of LexA fusion genes encoding the cytoplasmic domains of c-Met, c-

Fms, TrkA, insulin receptor and c-Kit downstream of LexA, and the expression in S. cerevisiae

strain L40 were described previously (24,31,32). Met-mutants containing tyrosine/phenylalanine

replacements have been described (24). A single colony, selected for expression of the LexA-

fusion protein, was tested for autophosphorylation and used for transformation with the VP16

cDNA library derived from a 8 day rat brain (33). GST-Grb2, GST-SHIP, GST-Gab1, GST-

PI-3´ kinase fusion proteins were generated in the pGex system (Pharmacia, Freiburg,

Germany). His-tagged SHIP, Grb2 and PI-3´ kinase were generated in pQE30 (Qiagen, Hilden,

Germany). Myc-tagged wild-type and mutant SHIP-1 were generated using pcDNA3.1Myc-

His (Invitrogen, Carlsbad, CA).

Binding assay using the two-hybrid system

The qualitative and quantitative evaluations of various two-hybrid protein/protein

interaction were described previously (31).

Cells and antibodies.

MDCK cells were grown in Dulbecco´s modified Eagle’s medium supplemented with

10% FCS. HGF was from Sigma (Munich, Germany). Scattering and branching tubulogenesis

assays were performed after Gual et al., (30). Monoclonal antibodies against phosphotyrosine

(4G10) and c-Met were from Upstate Biotechnology Incorporated (Lake Placid, NY, USA).

Monoclonal antibody against Myc and rabbit IgG against c-Met and SHIP-1 were from Santa

Cruz Biotechnology (Santa Cruz, USA).

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Tyrosine kinase assays, immunoblotting.

These assays were performed as published (31,34).

Co-immunoprecipitation study.

After incubation for 8 h with medium containing 0.02 % FCS, MDCK cells were

stimulated with HGF (60 ng/ml) for 5, 10 or 30 min. Cells were extracted with lysis buffer

containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 % Triton X-100, 1 % Trasylol, 10 %

Glycerol, 1 mM phenylmethylsulfonyl fluoride and 400 µM sodium orthovanadate. One aliquot

of each clarified lysate (5 x 106 cells) was incubated for 16 h at 4oC with anti-SHIP or anti-Met

antibody preabsorbed on Staphylococcus aureus Cowan I strain. After washing, materials were

analyzed by SDS-PAGE, and by immunoblot analysis using the appropriate antibodies.

Binding of cellular proteins to GST-fusion proteins and His-tagged proteins.

GST-fusion proteins and His-tagged proteins were produced as recommended by the

manufacturer. Purified GST-fusion proteins were bound for 1h at 4°C to glutathione (GT)

agarose beads (40 µl slurry; Pharmacia) suspended in the binding buffer (50 mM HEPES, pH

7.5, 150 mM NaCl, 1 % TritonX-100, 1 % Trasylol, 10 % Glycerol, 1 mM

phenylmethylsulfonyl fluoride, 20 mg/ml BSA and 200 µM sodium orthovanadate). After

incubation with [32P]-labeled autophosphorylated c-Met overnight, beads were washed five

times with binding buffer and pellets were analyzed by SDS-PAGE.

Results.

SHIP-1 associates with c-Met tyrosine kinase.

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To identify neuronal proteins which specifically interact with the cytoplasmic domain of

c-Met, we employed a yeast two-hybrid screening using an 8 days-old rat brain library

(24,33,35). A total of 800 cDNA clones of various length were obtained together encoding seven

different proteins. Five of these proteins were previously identified as c-Met binding proteins

including Gab1, PI-3’ kinase, Grb2, PLC-γ, and c-Src. Furthermore, we detected the interaction

with a novel c-Met-interactive zinc-finger-containing protein, however, the biological

significance of this association remains to be studied. In addition to these molecules, we isolated

the SHIP-1 as a novel c-Met binding partner.

SHIP-1 was originally identified as a signaling molecule in cytokine-stimulated

hematopoietic cells such as macrophage colony stimulating factor (M-CSF) stimulated cells (36)

and has been clearly demonstrated to participate in the signaling pathways in hematopoietic cell

systems (37). To determine whether SHIP-1 is a relevant signaling molecule in other cell

systems, we next analyzed SHIP-1 expression in various cell lines such as colon carcinoma

CACO-2, MDCK cells, two human breast carcinoma cell lines EFM-19 and EFM-192A, and

Raji cells. As control, we applied a cell lysate derived from FDC-P1Mac11 cells (38). In

agreement with a previous report (36), SHIP-1 is expressed at high levels in FDC-P1Mac11

cells. In addition, SHIP-1 was also detected in the epithelial cell lines, MDCK, both human

breast carcinoma cell lines and Raji cells (Fig.1).

To demonstrate the protein/protein interaction between SHIP and c-Met in vivo, MDCK

cells were incubated for 8 h with medium containing 0.02 % FCS, then stimulated with HGF

(60ng/ml) for 5, 10 and 30 min and studied by immunoprecipitation. Aliquots of 5 x 106 cells

from each preparation were lysed for immunoprecipitations using the SHIP-specific or c-Met

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specific antibodies. Precipitated material was analyzed for the presence of c-Met or SHIP by

SDS-PAGE and Western-blotting using SHIP-, c-Met-, or phosphotyrosine (4G10)-specific

antibodies (Fig. 2). Following HGF stimulation, tyrosine phosphorylation of c-Met was

observed throughout the experiment. Prior to HGF stimulation (0 min), no c-Met was detectable

in the SHIP-specific immune complexes. In contrast, between 5 and 30 min after HGF

stimulation, c-Met was co-precipitated with SHIP (Fig.2).

The phosphorylated tyrosine 1356 provides the binding site for SHIP-1.

To determine whether the SHIP-1/c-Met interaction relied on the presence of particular

phosphotyrosine residues of c-Met, we employed a set of c-Met mutants in which established

binding sites for defined binding partners were destroyed (24). These mutants included Y1349F

(Y14F), Y1356F (Y15F), the double mutant Y14/15F, and the kinase negative mutant K1110A.

We examined the binding of these mutants with the SH2 domain of SHIP-1, PI-3’ kinase, Grb2,

Grb10, PLCγ, and c-Src, Met binding domain (MBD) of Gab1, and the PTB domain of Shc. As

shown in Fig. 3, Grb2, Grb10 and SHIP-1 did not associate with the Y15F mutant, suggesting

that phosphotyrosine-1356 at the multifunctional docking site is the only binding site for these

proteins. On the other hand, PI-3´ kinase, Gab1, PLCγ, c-Src and Shc bind to both Y14(1349)

and Y15(1356) (Fig. 3).

SHIP binds to c-Met and c-Fms, but not to c-Kit, TrkA or the insulin receptor.

Signaling molecules that bind to the multifunctional docking site such as PI-3´ kinase,

Shc, and PLCγ associate with most receptor tyrosine kinases. Since SHIP-1 binds at the same

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site in c-Met with these molecules, we performed similar binding analyses with four additional

activated receptor tyrosine kinases including c-Fms, c-Kit, TrkA, and the insulin receptor.

Western blotting with anti-phosphotyrosine antibody clearly demonstrated that these receptor

moieties were phosphorylated on tyrosine in the yeast two-hybrid assay (data not shown). As

expected, c-Fms binds to SHIP-1, however, SHIP-1 did not bind other tyrosine kinase receptors

such as c-Kit, TrkA, or the insulin receptor (Fig. 4). In agreement with previous data (39-41),

PI-3´ kinase binds to c-Fms, c-Kit and the insulin receptor, the sequences of which contain the

typical consensus motif, p-YXXM, for the PI-3´ kinase binding site (42). It is noteworthy that

both of the PI-3´ kinase binding sites of c-Met, tyrosine 1349 and 1356, and their following

amino acid sequences are different than this motif (Table 1).

Hierarchical binding of Grb2, Gab1, SHIP and PI-3´ kinase to the multifunctional docking site

of c-Met.

The multifunctional docking site of c-Met provides binding sites for many substrates

including PI-3´ kinase, Grb2, Gab1 and SHIP. As shown in Fig. 3, PI-3´ kinase and Gab1 bind

at both pY1349 and pY1356, while SHIP and Grb2 bind only at pY1356. This observation raises

the question of whether two of these molecules bind to c-Met simultaneously or sequentially. To

answer this question, we have generated the GST-SH2 domain of Grb2 (Grb2[SH2]), p85 of PI-

3´ kinase (PI-3´ K[SH2]) and SHIP (SHIP[SH2]) fusion proteins and GST-Met binding domain

(MBD) of Gab1 (Gab1[MBD]) which were incubated with tyrosine-autophosphorylated c-Met

in the presence and absence of His-tagged PI-3´ K[SH2], Grb2[SH2] or SHIP[SH2]. Firstly, we

compared the binding of c-Met to PI-3´ kinase and SHIP. When equal amounts of both

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molecules were incubated with c-Met, SHIP bound predominantly to c-Met. Secondly, Grb2

bound to c-Met preferentially over PI-3´ kinase and Gab1 (Fig. 5). Thirdly, SHIP did not

compete with the c-Met/Gab1 interaction. Taken together, these results indicate that when

phosphotyrosine 1356 binds to either Grb2 or SHIP, phosphotyrosine 1349 does not provide a

binding site for PI-3’ kinase, and when Grb2 binds to phosphotyrosine 1356, even Gab1 does

not bind to c-Met, suggesting that in vivo these molecules may bind to a single molecule of c-

Met sequentially.

Overexpression of SHIP-1 in MDCK cells drastically potentiated branching tubulogenesis

induced by c-Met without altering HGF stimulated cell proliferation and scattering.

To investigate the biological role of SHIP-1 in c-Met mediated signaling, we

overexpressed the myc-tagged SHIP-1 gene in MDCK cells and isolated 19 different clones,

each constitutively expressing distinct levels of a myc-tagged SHIP-1. The two clones (clones

15 and 21) employed in these studies expressed equally high levels of SHIP-1 (Fig. 6A). Using

these mutant cell lines together with the wild-type MDCK cells as control, we examined cell

dissociation (scattering), cell proliferation and branching tubulogenesis induced by HGF

stimulation. Firstly, the scattering effect of HGF to both transfectants was indistinguishable from

that of the wild-type (Fig. 6B). Secondly, in agreement with data obtained from the fibroblast

system (36), the over-expression of SHIP-1 showed no influence on [3H]-thymidine

incorporation, suggesting that overexpression of SHIP-1 did not affect cell growth of MDCK

cells (Fig. 6C). Finally, we tested the ability of these cells to form tubules in semi-solid collagen.

To our surprise, both transfectants which over-expressed Myc-tagged SHIP started to form

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branching tubules within 24 hours after HGF treatment. Two days after HGF-stimulation,

transfectants formed long branching tubules (Fig. 7), while wild-type MDCK cells formed

branching tubules only 6-7 days after HGF-treatment (Fig. 7). Furthermore, in agreement with

previous data, (43) even 7 days after HGF-treatment, about 70 % of wild-type cells formed

tubules, however, almost all transfectants formed tubules. In the absence of HGF, none of the

transfectants formed any branching tubules throughout a time period of 2 weeks (data not

shown). Taken together, these observations suggest that overexpression of SHIP clearly enhances

HGF-mediated tubulogenesis.

Overexpression of a mutant SHIP-1 lacking catalytic activity in MDCK cells impaired HGF-

mediated branching tubulogenesis.

As shown above, SHIP-1 overexpression accelerated tubulogenesis. This fact raised the

question of whether this phenotype results from enhanced phosphoinositol phosphatase activity

or from displacement of protein binding to the receptor at the SHIP-1 binding site. To answer

this question, we next generated a mutant SHIP-1 lacking catalytic activity by deleting amino

acid residues 666 to 680 (44) using pcDNA3.1Myc-His and overexpressed in MDCK cells.

Twelve different clones were isolated and the two clones (clones 1 and 4) employed in this study

expressed equally high levels of mutant SHIP-1 (Fig. 8A). Using these mutant cell lines together

with the pcDNA3.1Myc-His transfected MDCK cells as control, we examined branching

tubulogenesis induced by the HGF stimulation. In agreement with previous experiments using

wild-type MDCK cells, the control transfectant formed branching tubules within 7 days after the

HGF treatment. Two clones which overexpressed a mutant SHIP-1, however, failed to form

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branching tubules over 9 days (Fig. 8B), suggesting that the effects of SHIP-1 overexpression to

accelerate tubulogenesis are due to the enhanced phosphoinositide phosphatase activity.

Discussion

The results presented above can be summarized as follows: Firstly, we have identified

SHIP as a novel binding partner of c-Met. Secondly, SHIP binds to Y1356 at the multifunctional

docking site of c-Met. Thirdly, we showed here that in vitro this site which contains two

phosphotyrosine residues, binds to only one signaling molecule at a time. Fourthly,

overexpression of SHIP drastically enhanced the tubulogenesis potency of c-Met without

altering the c-Met mediated cell scattering and cell proliferation. Cells that overexpressed a

mutant SHIP-1 lacking catalytic activity, however, failed to form branching tubules in the

presence of HGF, suggesting that the effects of SHIP-1 overexpression to accelerate

tubulogenesis are due to the enhanced phosphoinositide phosphatase activity.

c-Met signaling is mediated by a multifunctional docking site comprising two

phosphotyrosines arranged in tandem (20). Most tyrosine kinase receptors including c-Fms, c-

Kit or epidermal growth factor receptor, autophosphorylate at multiple sites which bind several

signaling molecules, suggesting that one receptor molecule is able to simultaneously associate

with multiple signaling molecules. In addition, we showed here that the multifunctional docking

site of c-Met binds to only one signaling molecule and when several effector molecules are

present simultaneously, one can observe in vitro the hierarchical binding of proteins, such as

Grb2, Gab1, SHIP and PI-3´ kinase. Our competition assay reveals that PI-3´ kinase did not

compete with c-Met/Grb2 and c-Met/SHIP interactions, however, Grb2 competed with all of

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these interactions, suggesting that the binding order of these effector molecules is crucial for the

c-Met mediated multiple signal transduction. In vivo binding of full-length molecules, however,

may be dependent upon their allosteric interactions. It is therefore also possible that multiple

complexes of signaling molecules are assembled to the activated receptor. Furthermore, we have

to take into account that the number and the subcellular distribution of each of the signaling

molecules expressed in a given cell are different. For instance, when HGF treated cells are grown

in a three-dimensional collagen matrix, cells form tubules which have a lumen surrounded by

well-polarized epithelial cells with a smooth basal surface in contact with the collagen matrix

and an apical surface rich in microvilli that faces the lumen (11). The subcellular localization of

c-Met and substrates differs in these cells from non-polarized cells which are tested for cell

scattering. For the cell-scattering assay, cells were incubated in liquid culture medium without

collagen.

We show here that the SHIP-1 binding site, tyrosine 1356 is also a binding site for Grb2

with YXNX motif. Does SHIP-1 regularly share the binding site with Grb2? In the case of c-

Fms, Y696 and Y921 provide the binding sites for Grb2 with YXNX motif, Y696KNI and

Y921TNL, respectively (31). Mutation of both sites, however, did not exert an influence on the SHIP-

1/c-Fms association, indicating that none of these sites acts as a binding site for SHIP-1 (data

not shown). These data suggest that the second or fourth flanking position is also important for

binding. Interestingly, the SH2 domains of SHIP and Grb2 also share the binding site,

pY317VNV of Shc (45), the sequence of which is identical to the pY1356VNV of c-Met, indicating

YVNV is a consensus sequence for the SHIP binding site (Table 1). Interestingly, the point

mutation of N1358 into H abolished the branching potential of c-Met (29).

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A most striking observation of our studies regards the fact that overexpression of SHIP-1

drastically enhanced the HGF mediated branching tubulogenesis without altering cell growth and

cell scattering in response to HGF (Fig. 6, 7). Here, both transfectants which overexpressed

Myc-tagged SHIP started to form branching tubules within 24 hours after the HGF treatment.

SHIP plays a role in inositol phosphate and phosphatidylinositol phosphate metabolism (46).

SHIP-1 displays 5´-phosphatase activity specifically with both PtdIns-(3,4,5)P3 and Ins-

(1,3,4,5)P4 as substrate. Phee et al., (47) reported that enzymatic activity of SHIP is regulated by

a plasma membrane localization and as a result, significant reduction in the cellular PtdIns-

(3,4,5)P3 level was observed. Furthermore, a striking correlation was observed between PtdIns-

(3,4) P2 production and tyrosine phosphorylation of SHIP-1, as well as its relocation to the

cytoskeleton upon thrombin stimulation in human blood platelets (48). Here, PtdIns-(3,4)P2 may

bind to pleckstrin homolog (PH) domains of still undefined enzymes which may promote re-

localization to the membrane and provide enzyme access to new lipid substrates or regulatory

kinases. Indeed, the serine/ threonine kinase B, also known as Akt has been shown to bind

PtdIns-(3,4) P2. In addition, the members of protein kinase C family, PKCε, and PKCζ are

activated by PtdIns-(3,4) P2 (49). Interestingly, in the platelet system, Hartwig et al., (50)

reported that PtdIns-(3,4,)P2 inhibits actin filament severing and capping by human gelsolin in

vitro, suggesting the product of SHIP-1 plays a role in cytoskeleton rearrangement which is

important for morphogenesis. On the other hand, Maroun et al., (51) reported that the ability of

Gab1 to bind PtdIns-(3,4,5)P3 is crucial for subcellular localization of Gab1 and for efficient

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morphogenesis mediated by c-Met. However, the same authors (43) reported that Gab1

phosphorylation per se is not sufficient to induce branching tubulogenesis and suggested that a

Met-specific substrate, in addition to Gab1, is required for branching tubulogenesis. Recently, it

has been reported that SHIP interacted with the protein inhibitor of activated STAT1, PIAS1

(52). Another member of STAT family, STAT3 was reported to be an important molecule for c-

Met mediated branching tubulogenesis (21). Here, SHIP-1 may also participate in the STAT

pathway. Recently, both amplification of PI-3´ kinase and loss of function of a 3´lipid

phosphatase PTEN have been shown in multiple human tumors (53-55), demonstrating that the

regulation of PtdIns-(3,4,5)P3 at the cellular membrane may be critical for the control of

multiple biological processes including tumorigenesis. At the moment, the molecular mechanism

of potentiation of HGF mediated branching by SHIP-1 overexpression remains unclear,

however, inositol phosphate and/or phosphatidylinositol phosphate metabolisms play a key role

in epithelial cell-dissociation, reassociation and formation of continuous tubules.

ACKNOWLEDGEMENTS

We thank Karsten Heidrich and Regina Wilms (MHH) for technical assistance, Walter

Birchmeier (MDC, Berlin) for generously providing LexA-Met and its mutants, LexA-insulin

receptor, LexA-TrkA, and LexA-Kit cDNAs, Thomas Südhof (Univ. Texas) for rat brain cDNA

library and Larry Rohrschneider (Fred Hutchinson Cancer center, Seattle) for providing the SHIP

cDNA. This research was supported by the Deutsche Forschungsgemeinschaft (Ta-111/7/-1, 2),

IHFSP, and Fonds der Chemischen Industrie to HN. This work is a part of PhD thesis for MS.

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Figure Legends.

Fig. 1. SHIP is expressed in MDCK, hematopoietic cells, and human breast carcinoma cell lines.

Cell lysates from FDC-P1Mac11, the epithelial cells MDCK, the colon carcinoma cells CACO-2

(CaCo), the human Burkitt lymphoma Raji, and two human breast carcinoma cells EFM-19 and

EFM-192A (5 x 105 cells for each sample) were analyzed by SDS-PAGE and immunoblotting

using the anti SHIP-1 rabbit IgG.

Fig. 2. SHIP binds to c-Met tyrosine kinase.

MDCK cells were stimulated with HGF for 5, 10 and 30 min. Cell lysates obtained from 5 x 106

cells were subjected to immunoprecipitation using SHIP-1 or anti c-Met antibodies.

Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting using the anti-Met anti-

SHIP and anti-phosphotyrosine (pY) antibodies. As control, an aliquot of each lysate (5 x 104

cells) was analyzed by SDS-PAGE.

Fig. 3. Phosphotyrosine 1356 of c-Met is a binding site of SHIP.

Interaction of SHIP, PI-3 kinase, Grb2 and Gab1 with c-Met mutants carrying

tyrosine/phenylalanine or lysine/alanine replacements as Y1349F(Y14F), Y1356F (Y15F),

double mutant Y14/15F, and kinase negative mutant K1110A. These mutants were co-expressed

with the SH2 domain of SHIP, PI-3´ kinase, Grb2, Grb10, c-Src, or PLCγ, MBD domain of

Gab1, or PTB domain of Shc, as VP16 fusion proteins in YRN974 (31). Aliquots of 10,000 cells

of four independent transformants were analyzed for fluorescence intensity using a Becton

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Dickinson FACScan flow cytometer. Values represent the means obtained in four independent

experiments.

Fig. 4. SHIP binds to activated c-Met and M-CSF receptor but not to c-Kit, TrkA, or the insulin

receptor. pBTM116 carrying cloned cDNAs encoding the cytoplasmic domains of either c-Met,

c-Fms, c-Kit, TrkA, or the insulin receptor (IR) were co-transformed with pVP16 carrying the

cDNA of the SH2 domains of SHIP, Grb2, PLCγ, or PI-3´ kinase or the MBD domain of Gab1.

Ten-thousand cells of four independent transformants were analyzed for fluorescence intensity

using a Becton Dickinson FACScan flow cytometer.

Fig. 5. Hierarchical binding of Grb2, Gab1, SHIP and PI-3´ kinase to the multiple substrate-

binding site of c-Met.

GST-fusion proteins containing the SH2 domain of SHIP, Grb2 and p85 of PI-3´ kinase and the

MBD domain of Gab1 were purified from E. coli strain DH5α using GT-sepharose. The same

proteins, but His tagged, were purified from E. coli strain M15. GST-fusion protein or GST (2

µg each) were bound to GT-sepharose beads and incubated with [32P]-labeled c-Met, as

obtained by an in vitro kinase reaction with or without corresponding amounts of His-tagged

proteins. Following washing, bound protein was analyzed by SDS-PAGE (B). As a control,

aliquots were analyzed by SDS-PAGE and staining with Coomassie brilliant blue (A).

Fig. 6. Overexpression of SHIP did not alter cell growth and scattering in response to HGF.

A: Myc-tagged SHIP was transfected into MDCK cells. The established cell lines, clones 15

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and 21, were analyzed by immunoblot using anti Myc antibody.

B: Cells were stimulated with HGF (20 ng/ml) and chemically fixed 24 h after HGF treatment.

Cells were visualized by Giemsa staining.

C: The 3H-thymidine incorporation in wt and SHIP overexpressing MDCK (clones 15 and 21)

cells in the presence of FCS or HGF. The 3H-thymidine incorporation study was performed as

previously described (21). Sister cell cultures (8 x 103 cells per well) were starved for 24 h in

medium containing 0.1 % FCS before stimulation with HGF. [3H]thymidine (100µCi/ml) was

added for 4 h. Trichloroacetic acid-precipitable radioactivity was determined and expressed as

the mean value of four independent experiments.

Fig. 7. Overexpression of SHIP in MDCK cells potentiates HGF-mediated branching

tubulogenesis. Wild-type and SHIP overexpressing MDCK cell lines, clones 15 and 21 were

grown in collagen for 5 days, then medium containing HGF (40 ng/ml) was added and changed

every two days. After 2, 4 and 7 days, the branching tubulogenesis response was evaluated.

Photomicrographs by Hoffman modulation contrast microphotometry show a representative

experiment that had been performed in triplicate.

Fig. 8. Overexpression of a mutant SHIP-1 lacking catalytic activity in MDCK cells impaired

HGF-mediated branching tubulogenesis. (A) MDCK cells transfected with pcDNA3.1MycHis

(Control) and a mutant SHIP-1 lacking catalytic activity (SHIP∆666-680) (44), overexpressing

MDCK cell lines, clones 1 and 4 , were analyzed by immunoblot using anti Myc antibody. (B)

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Cells were grown in collagen for 5 days and then medium containing HGF (40 ng/ml) was added

and changed every two days. After 5, 7 and 9 days, the branching tubulogenesis response was

evaluated. Photomicrographs by Hoffman modulation contrast microphotometry show a

representative experiment that had been performed in triplicate.

Table 1: p-YVNV in c-Met and Shc binds to both SHIP and Grb2.

Tyrosine kinase Substrate

SHIP binding site PI-3´ kinase binding site Grb2 binding site

c-Met p-YVNV p-YVHV (20)*p-YVNV (20)

p-YVNV (20,56)

Shc p-YVNV (44) p-YVNV (57)c-Fms ? p-YVEM (39) p-YKNI (58)

p-YTNL (31)c-Kit - p-YMDM (40) p-YKNL (59)

p-YSNL (59)IR - p-YHTM (41)

(*): References

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Heiner Niemann and Teruko TamuraMonica Stefan, Alexandra Koch, Annalisa Mancini, Andrea Mohr, K. Michael Weidner,

branching tubulogenesisdocking site of c-Met and potentiates hepatocyte-growth factor (HGF) induced

The SH2-containing inositol 5´phosphatase (SHIP)-1 binds to the multifunc-tional

published online November 7, 2000J. Biol. Chem.

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