Báo cáo khoa học: Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling docx

Results Isolation of MVP as Src–SH2 interacting protein by GS–SH2 fusion pull-down assay To isolate proteins that regulate cancer-specific cell sig-nalling, we incubated GST fusion–SH2 do

epidermal growth factor-dependent cell signalling

Euikyung Kim1*, Seunghwan Lee1, Md Firoz Mian2, Sang Uk Yun2, Minseok Song2, Kye-Sook Yi2, Sung Ho Ryu2and Pann-Ghill Suh2*

1 Institue of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea

2 Department of Life Science, Pohang University of Science and Technology, Pohang, Korea

The major vault protein (MVP) is the predominant

component of a large cytoplasmic ribonucleoprotein

particle, the vault complex [1,2] The vault particle was

originally identified as a barrel shaped body in

prepa-rations of clathrin-coated vesicles and named after its

morphology reminiscent of the vaulted ceilings of

cathedrals [3] Vaults exist in thousands of copies per

cell and are widely expressed in all eukaryotic

organ-isms [4–8] In both structure and composition vaults

are highly conserved throughout evolution in diverse phylogenetic lineages including mammals, avians, amphibians and slime moulds [9] They represent multimeric protein complexes with one predominant member, the MVP which constitutes more than 70%

of the total complex The remaining mass comprises vault RNA and two high molecular weight proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1) [10,11] The

Keywords

ERK signaling pathway; MVP; Src; Src

activity; tyrosine phophorylation

Correspondence

E Kim, Institue of Animal Medicine, College

of Veterinary Medicine, Gyeongsang

National University, Jinju, 660-701, Korea

Fax: +82 55 751 5803

Tel: +82 55 751 5812

E-mail: ekim@nongae.gsnu.ac.kr

P.-G Suh, Department of Life Science,

Pohang University of Science and

Technology, 790-784, Korea

Fax: +82 54 283 4613

Tel: +82 54 279 2293

E-mail: pgs@postech.ac.kr

*Note

E Kim and P.-G Suh contributed equally to

this work.

(Received 14 November 2005, revised

13 December 2005, accepted 19 December

2005)

doi:10.1111/j.1742-4658.2006.05112.x

Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST–Src–SH2 fusion proteins

We found MVP as a Src–SH2 binding protein in human stomach tissue Interaction of Src and MVP was also observed in 253J stomach cancer cells A subcellular localization study using immunofluorescence micros-copy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where

it colocalizes with Src We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner MVP overexpression downregulates EGF-dependent ERK activation in Src over-expressing cells To our knowledge, this is the first report of MVP interact-ing with a protein tyrosine kinase involved in a distinct cell signallinteract-ing pathway It appears that MVP is a novel regulator of Src-mediated signal-ling cascades

Abbreviations

EGF, epidermal growth factor; GST, glutathione S-transferase; MVP, major vault protein; PAP, potato acid phosphatase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SH2, Src homology 2; TCL, total cell lysate; TEP1, telomerase-associated protein 1; VPARP, vault poly(ADP-ribose) polymerase.

expression of the unique 100 kDa MVP is sufficient

to form the basic vault structure Although many

molecular features of vault particles have been

charac-terized, the function of this large ribonucleoprotein

particle remains enigmatic The identification of lung

resistance-related protein (LRP) as the human MVP

shed new light on putative cellular functions of vaults

[7] Numerous multidrug resistance cancer cells

fre-quently overexpress MVP and increased MVP mRNA

expression was found to correlate strongly with a

pre-dictive value of a multidrug resistance phenotype

[12,13] An early postulate of vault function was

nucle-ocytoplasmic transport [1,14] A recent study using

MVP knockout mice has shown that MVP⁄ vaults are

not directly involved in the resistance to cytostatic

agents [15] Vaults have been proposed to constitute

the transporter or central plug of the nuclear pore

complex, controlling bi-directional exchange between

nucleus and cytoplasm [16] Major vault protein has

been coimmunoprecipitated with human oestrogen

receptor in oestradiol dependent interaction and might

be involved in nucleocytoplasmic shuttle for

modula-tion of signal transducmodula-tion of steroid hormone [17]

Another recent study showed that MVP physically

interacts with phosphatase and tensin homologue

deleted on chromosome 10 (PTEN) and the interaction

is Ca2+dependent [18] However the physiological role

of MVP hitherto remains elusive

The Src tyrosine kinase participates in multiple

sig-nalling pathways that regulate diverse cellular

func-tions, including proliferation, differentiation, motility,

adhesion and architecture [19,20] The subcellular

localization of Src in part determines its substrate

specificity and function One example of a Src

sub-strate, Sam68, an RNA binding protein [21], whose

phosphorylation by Src appears to determine specific

functions of Src Src phosphorylates Sam68 during

mitosis, presumably after breakdown of the nuclear

envelope Src appears to be important for cell cycle

progression via Sam68, particularly during the late

mitosis and possibly during G1⁄ S transition

Identifi-cation of Src binding proteins has led to a better

understanding of Src regulation and has provided

clues about the function of Src in normal and

trans-formed cells [22] Compelling evidence indicates that

Src-binding proteins can regulate Src activity [23]

While a number of interacting proteins that

upregu-late Src activity have been identified; however, only

a few that downregulate Src activity have been

known It is important to elucidate the molecular

mechanisms that inactivate c-Src Recently Caveolin,

a 22 kDa integral membrane protein [24–26] and a

receptor for activated C kinase (RACK1) [27] were

shown to bind Src and suppress its tyrosine kinase activity Domains within Src kinases target the enzyme to specific subcellular locations where they bind to regulatory and⁄ or substrate proteins and are integrated into cell signalling pathways and cell cycle events [23] The UD, Src homology 3 and Src homology 2 (SH2) domains in Src are key binding sites for proteins that regulate Src activity and integ-rate Src into important signalling pathways and cell cycle events The aim of the present study was to identify and characterize Src interacting proteins that potentially regulate Src activity We focused on pro-tein interactions that involve the SH2 domain of Src using a glutathione S-transferase (GST)–SH2 fusion pull-down assay and identified MVP as a Src–SH2 binding protein We observed that MVP interacts with Src in mammalian cells and inhibits the activity

of Src tyrosine kinase

Results Isolation of MVP as Src–SH2 interacting protein

by GS–SH2 fusion pull-down assay

To isolate proteins that regulate cancer-specific cell sig-nalling, we incubated GST fusion–SH2 domains of various Src SH2 domain-containing proteins with cell lysates from human stomach cancer tissues or normal stomach tissues The protein complexes were collected

on glutathione-agarose beads and resolved in SDS⁄ PAGE followed by silver staining (Fig 1A) The targeted protein bands were then analysed by MALDI-TOF MS A  100 kDa protein that bound strongly with the SH2 domain was identified as MVP (Fig 1B, Table 1) It was also verified by immunoblot-ting with polyclonal anti-MVP IgG (Fig 1C) Both the

MS analysis and immunoblotting with polyclonal anti-MVP IgG showed that anti-MVP strongly bound to the SH2 domain of Src, but not to the SH2 domains of other proteins tested (Fig 1C) Thus MVP interacted specifically with the SH2 domain of Src, but not with the SH2 domains of PLCc1, Grb2, STAT3 or Crk

MVP associates with Src endogenously in 253J cells and exogenously in cotransfected 293T cells

MVP constitutes about 70% of the total molecular mass of vault particles and is capable of assembling into the characteristic vault structure in the absence of other vault components (TEP1, VPARP or vRNA)

To examine whether the MVP can interact with full-length Src in vivo, we prepared MVP containing lysates

from 253J cells and immunoprecipitated c-Src using

a polyclonal antibody Western blot analysis of the

Src-immunoprecipitates with MVP antibody showed

that MVP⁄ vault interacted with Src in vivo in 253J

cells (Fig 2A) If MVP interacts with Src in other

established mammalian cell lines was examined by

cotransfecting flag-tagged MVP and c-Src into 293T

cells Coimmunoprecipitation and western blot analyses

of the immunoprecipitates were performed using anti-FLAG IgG or Src antibody Figure 2B shows that Flag–MVP immune complex contains c-Src (lane 2) The reciprocal experiment confirmed the interaction as shown in Fig 2B, lane-3 that MVP was coimmunopre-cipitated with Src

EGF enhances the MVP–Src interaction, which can be blocked by src kinase inhibitor, PP2

To determine whether epidermal growth factor (EGF) can activate Src and influences the association between MVP and Src, we treated serum starved fibroblasts

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Fig 1 Major vault protein interacts with c-Src through the Src SH2 domain (A) Stomach cancer tissue (C) and normal stomach tissue (N) were obtained from cancer patients in a local hospital (Dongguk University Pohang Hospital) and stored at )70
C until use The tis-sue lysates were prepared and incubated with GST fusion proteins

of various SH2 domains Formed protein complexes were isolated

by glutathione beads and washed three times with fresh TBS, and analysed by SDS ⁄ PAGE and subsequent silver staining as des-cribed in Experimental procedures (B) p100 isolated from proteins that markedly coprecipitated with the GST–Src-SH2 fusion protein was in-gel digested with trypsin, and the resulting peptide mixture was analysed by MALDI-TOF MS The arrows indicate matched peaks among the measured tryptic peaks of p100 with calculated molecular masses of MVP within 50 p.p.m The detailed descrip-tions of each peptide analysed and used for protein identification are shown in Table 1 (C) Binding proteins in stomach cancer tissue

to the GST–SH2 of various signaling proteins (Src, PLCc1, STAT3, Grb2, Crk) which were tested were immunoblotted with polyclonal anti-MVP IgG (from Dr Rome, UCLA, CA), confirming that MVP specifically interacts with the SH2 domain of Src, and not with SH2 domains of other proteins The input shows approximately 10% of the tissue lysate that was applied for GST-fusion pulldown.

Table 1 Peptide sequences and masses from p100 by MALDI-TOF MS.

M + H + (Da) Observed Calculated

P9 KEVEVVEIIQATIIR (155–169) 1738.998 1739.018 P10 AQDPFPLYPGEVLEK (92–107) 1814.926 1814.944 P11 VAGDEWLFEGPGTYIPR (137–154) 2004.986 2004.994 P12 QLQLAYNWHFEVNDR (537–552) 2044.936 2045.011 P13 VIGSTYMLTQDEVLWEK (400–417) 2081.970 2082.033 P14 PPYHYIHVLDQNSNVSR (10–27) 2151.017 2151.085

that overexpress FLAG–MVP and Src with EGF

(100 ngÆmL)1) for various time periods Then the cell

lysates were immunoprecipitated with anti-FLAG IgG

and the immune complexes were resolved by

SDS⁄ PAGE followed by immunoblotting with anti-Src

IgG (Fig 3A) We observed that EGF enhanced the

interaction between MVP and Src in time-dependent

manner with a peak after 3 min followed by gradual

decline and return to the basal level after 15 min

(Fig 3A) The effect of EGF on MVP–Src interaction

was concentration dependent, with a maximal effect

achieved at 100 ngÆmL)1 (data not shown) From the

current results, however, it is not clear whether only

the SH2 domain of Src is important for the Src–MVP

association in vivo This could be addressed by examin-ing whether an SH2 domain deletion mutant of Src can still associate with MVP in vivo from a further study We also examined the effect of Src specific tyro-sine kinase inhibitor (PP2) on EGF dependent Src– MVP interaction (Fig 3B) We treated serum starved 253J cells that express high levels of Src and MVP pro-teins endogenously, with EGF for various time periods and one group was pretreated with PP2 for 45 min before EGF stimulation We could observe that endo-genous interaction between Src and MVP after EGF stimulation was almost completely inhibited by PP2 These results clearly show that EGF potentiates the interactions between Src and MVP in a time-dependent manner, which is abrogated by specific Src kinase inhibitor

Epidermal growth factor-dependent coimmunopre-cipitation of Src and MVP prompted us to test if they colocalize in any subcellular compartment on EGF sti-mulation As we expected, immunofluorescence micros-copy showed EGF-dependent transient colocalization

of MVP and Src in the cytoplasmic region of 253J cells that express high levels of Src and MVP proteins endogenously (Fig 3C) Interestingly, MVP predomin-antly localized in the nucleus of quiescent cell seems to translocate onto perinuclear and cytoskeletal compart-ment where it overlaps with Src upon EGF treatcompart-ment The kinetics of Src–MVP colocalization correlated well with the biochemical data of protein complex forma-tion as shown earlier This result suggests that mole-cular interaction between Src and MVP may play an important role in EGF-dependent colocalization of the two proteins However, the detailed mechanism of MVP translocation from nucleus to cytoplasm should

be further elucidated

Tyrosine phosphorylation of MVP is important for the binding of MVP with Src

MVP is known as a phosphoprotein as it is tyrosine phosphorylated in vivo and phosphorylated by protein kinase C (PKC) and casein kinase II (CKII) in vitro [28,29] To investigate the significance of MVP tyrosine phosphorylation for the MVP–Src interaction, 293T cells transfected with Src and FLAG-MVP were serum starved, then treated with EGF for the indicated time periods (Fig 4A) Cell lysates were then immunopre-cipitated with anti-FLAG IgG followed by immuno-blotting with antiphosphotyrosine IgG (PY 20) We observed that MVP phosphorylation reached the peak level upon EGF stimulation for 3 min (Fig 4A) that was comparable to the time kinetics of Src–MVP inter-action upon EGF stimulation as shown in Fig 3 This

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Fig 2 MVP interacts with Src in vivo in 253J cells and in

cotrans-fected 293T cells (A) To confirm whether MVP interacts

endogen-ously with full-length Src, we prepared 253J (stomach cancer cell

line) cell lysates and immunoprecipitated with c-Src mAb or

non-immune serum, followed by immunoblotting with anti-MVP IgG or

anti-c-Src IgG Thw upper panel indicates MVP that had been

asso-ciated with Src and the lower panel indicates immunoprecipitated

c-Src protein The input shows approximately 10% of the tissue

lysate that was applied for immunoprecipitation (B) Flag-tagged

MVP was prepared by generating the rat MVP cDNA construct

encoding Flag sequence at the N terminus The flag-tagged MVP

cDNA and c-Src cDNA were cotransfected into 293T cells as

indica-ted (Tfx) in the result The total cell lysates (TCL) were prepared

and immunoprecipitated with anti-Flag IgG or anti-Src IgG The

immunoprecipitated complex and total cell lysates were run on

SDS ⁄ PAGE and transferred to nitrocellulose membrane, and

west-ern blotting was performed using anti-Flag IgG or anti-Src IgG The

TCL show the overexpression of Flag-MVP and Src in transfected

cells, respectively.

finding suggests that MVP tyrosine phosphorylation

might be required for MVP–Src interaction

Interest-ingly, in almost all cases, MVP seems to have some

basal level of tyrosine phosphorylation in our system and it should be clarified in a further study To further address this result, we examined whether MVP

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Fig 3 EGF substantially enhances Src–MVP

interaction that was blocked by Src tyrosine

kinase inhibitor (PP2) (A) To determine

whether Src–MVP interaction is EGF

signal-dependent, we starved 293T cells which

were transfected with c-Src and⁄ or

Flag-tagged MVP as indicated After 24 h, the

293T cells were treated with EGF

(100 ngÆmL)1) for the indicated times, and

the prepared cell lysates were then

immu-noprecipitated with anti-Flag mAb The

sam-ples were immunoblotted with anti-Flag IgG

or anti-c-Src IgG, showing that the

inter-action is EGF-signal dependently increased

then rapidly declined (B) To see the effect

of EGF on endogenous MVP–Src

inter-action, 253J cells were serum starved and

stimulated with EGF for the indicated time

periods One group after serum starvation

was pretreated with PP2 for 45 min

fol-lowed by EGF stimulation for 6 min The

results showed that in vivo Src–MVP

inter-action was also EGF signal dependent and

Src tyrosine kinase inhibitor (PP2) blocked

the EGF induced interaction (C) 253J cells

seeded onto coverslips in DMEM with 10%

heat-inactivated fetal bovine serum were

serum starved for 24 h in serum-free

DMEM media After serum starvation, the

cells were treated with EGF (100 ngÆmL)1

final concentration) at 37
C for the

indica-ted times, then fixed and permeabilized as

described in Experimental procedures

Non-specific bindings were blocked by incubating

the coverslips with 4% BSA in NaCl ⁄ Pi ,

then the coverslips were incubated with

mouse monoclonal anti-Src IgG and rabbit

polyclonal anti-MVP IgG After washing

three times with NaCl ⁄ Pi, the coverslips

were incubated with fluorescent

probe-con-jugated secondary antibodies (fluoresceine

isothiocyanate-conjugated goat anti-rabbit

IgG and rhodamine-conjugated goat

anti-mouse IgG) for another 1 h After washing

with NaCl ⁄ Pi, the coverslips were mounted

face down onto slides and examined under

confocal fluorescence microscopy.

phorylation is a prerequisite for MVP–Src association.

We overexpressed FLAG–MVP in 293T cells and the

cell lysates were incubated with potato acid

phospha-tase (PAP), a phosphotyrosyl-protein phosphaphospha-tase, for

the indicated time periods Then lysates were

immuno-precipitated with anti-Src IgG and immunoblotted

with anti-MVP IgG Potato acid phosphatase

treat-ment resulted in a marked decrease in MVP–Src

complex formation on 45-min pretreated lysates This result provides proof that the Src–MVP interaction is dependent on MVP tyrosine phosphorylation

MVP inhibits Src kinase activity

To assess the effect of MVP on Src protein kinase activity, we performed an in vitro Src kinase activity assay We overexpressed FLAG-tagged MVP in 293T cells, immunoprecipitated cell lysates with anti-FLAG monoclonal IgG (mAb) and MVP was eluted from FLAG-immunoprecipitates by the addition of excess FLAG peptides and used as purified MVP We incuba-ted rabbit muscle enolase, an exogenous Src substrate and purified Src kinase (Santa Cruz Biotechnologies Inc., Santa Cruz, CA) with [32P]ATP and MnCl2 in the presence or absence of purified MVP and measured phosphorylation from in vitro Src kinase assay (Fig 5A) We observed autophosphorylation of Src in the absence of MVP (Fig 5A, lane 2) Interestingly,

we found that Src autophosphorylation was dramatic-ally reduced by MVP in a dose-dependent manner (Fig 5A, lane 3 and 4) Enolase phosphorylation fol-lowed the same trend as Src and acted as an excellent control substrate for Src The addition of 0.5-lg MVP inhibited Src activity by  60% (measured from the autoradiogram), whereas the addition of 1.0 lg of MVP inhibited Src activity almost completely These results suggest that MVP has an intrinsic activity sup-pressing Src kinase enzymatic activity Next, we inves-tigated whether MVP can be a substrate of and phosphorylated by Src We incubated purified MVP and commercially obtained purified Src with [32P]ATP

in a kinase reaction mixture without enolase addition, then examined the phosphorylation status of MVP Autoradiogram results showed that MVP was highly phosphorylated by Src in vitro (Fig 5B, lane 2) The slight phosphorylation modification of MVP in the absence of exogenous Src kinase (Fig 5B, lane 3) seems to be by endogenous Src, which is basally inter-acting with and copurified with MVP in the immuno-precipitation procedure

MVP–Src interaction down regulates Src mediated ERK/MAPK pathway

Src mediates diverse signals to a number of down-stream effector molecules To explore the physiological significance of our finding that MVP inhibits Src kinase activity, we examined the EGF-dependent Src downstream signalling molecules 293T cells were tran-siently transfected with Src cDNA with or without Flag-tagged MVP and treated with EGF for the

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Fig 4 MVP–Src interaction is dependent on the tyrosine

phos-phorylation of MVP (A) 293T cells were transiently transfected with

c-Src cDNA, FLAG-MVP cDNA or c-Src and Flag-MVP cDNAs The

cells were then serum-starved and EGF stimulated as indicated.

Cell lysates were then immunoprecipitated with anti-FLAG IgG and

immunoblotted with an anti-phosphotyrosine IgG (PY20) The

results showed an EGF dependent MVP tyrosyl phosphorylation,

which consistently correlated with the EGF dependent interaction

between Src and MVP (B) We examined if the MVP–Src

interac-tion requires MVP tyrosine phosphorylainterac-tion For this, we performed

in vitro phosphatase treatment followed by coimmunoprecipitation

of the complex Briefly, 293T cells were transiently cotransfected

with Flag-tagged MVP cDNA and c-Src cDNA, then the cellular

phosphotyrosyl-proteins were dephosphorylated for the indicated

times by incubating with PAP, a phosphotyrosyl-protein

phospha-tase The dephosphorylated cell lysates were immunoprecipitated

with anti-Src monoclonal IgG The coimmunoprecipitated

com-plexes were run on SDS ⁄ PAGE, transferred to nitrocellulose, then

immunoblotted with anti-Flag mAb and anti-Src mAb The PAP

treatment markedly reduced the interaction between MVP and Src,

suggesting that the interaction is dependent on protein tyrosine

phosphorylation.

indicated time periods, cell lysates were immunoblotted

with phospho-ERK and phospho-Akt (S473)

antibod-ies Immunoblotting of the cell lysates with antic-Src

and ERK antibodies indicated loading control for

equal amounts of proteins in gels The results revealed

that MVP attenuated the EGF stimulated ERK

activa-tion which is probably mediated through inhibiting the

Src sinase activity (Fig 6) However the Src–MVP

complex apparently had no effect on Akt (Fig 6,

lower panel) Further studies will be required for the

detailed mechanism of MVP-mediated regulation of ERK signalling pathway in the near future

Discussion The present study shows that SH2 domain of Src but not the SH2 domains of STAT3, Grb2, Crk or PLCc1 interacts with MVP in tissue lysates from human stom-ach cancer and normal stomstom-ach (Fig 1) The Src– MVP interaction, which is mediated, at least in part,

by the SH2 domain of Src, is enhanced by EGF stimu-lation As shown in Figs 3 and 4, there is a correlation between tyrosine phosphorylation of MVP and its inter-action with Src: (a) MVP is tyrosine phosphorylated

by Src in an EGF-dependent manner; (b) the Src inhibitor, PP2 blocked the interaction between Src and MVP; (c) dephosphorylation of MVP reduced its affin-ity for Src These results prompted us to speculate that

a signal (like epidermal growth factor receptor activa-tion), which brings Src and MVP in close proximity to each other, results in phosphorylation of MVP by Src and, in turn, enhances binding of MVP to the SH2 Src domain We believe that tyrosine phosphorylation of MVP may be an important ‘switch’ that links this

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Fig 5 MVP inhibits Src kinase activity in a

concentration-depend-ent manner (A) The effect of MVP–Src interaction on Src kinase

activity was assessed by in vitro kinase assay Briefly, 293T cells

were transiently transfected with Flag-tagged MVP, and the cell

lysate was immunoprecipitated using anti-Flag mAb Then

Flag-tagged MVP proteins were eluted from FLAG-immunoprecipitates

by the addition of excess amounts of free Flag peptide to the

immunoprecipitation beads The eluted MVP was concentrated

using Centricon TM (cutoff molecular weight > 50 kDa) The src

tyro-sine kinase assay was performed by incubating enolase (substrate)

with [3H]ATP and purified Src proteins (Upstate Biotechnology Inc.)

in the presence or absence of MVP as indicated Src tyrosine

kin-ase activity was determined from the autoradiogram of the kinkin-ase

assay samples The result shows that MVP potently suppresses

the Src kinase activity in vitro (B) We examined if MVP is a

sub-strate of Src tyrosine kinase The experiment was performed using

the same Src kinase assay as in (A), without enolase The result

indicates that MVP is a substrate of Src tyrosine kinase in vitro as

well.

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Fig 6 MVP attenuates Src-mediated ERK signalling pathway To assess the functional significance of the MVP–Src interaction, we examined EGF-dependent Src downstream signalling pathways Briefly, 293T cells were transiently transfected by Src cDNA with

or without MVP cDNA Those cells were serum-starved for the next 24 h, then treated with EGF as indicated in the result The cell lysates were immunoblotted with anti-phospho ERK IgG, anti-ERK IgG, anti-pAkt or Src IgG as indicated The result suggests that MVP may downregulate EGF-dependent ERK activation by inhibit-ing Src activity via the EGF-dependent MVP–Src interaction.

molecule to other signalling molecules and relays

sig-nals across multiple pathways This is particularly

interesting in that both Src and MVP has been

inde-pendently reported to be overexpressed in various

kinds of cancer cells However, it is too premature to

speculate what is the clinical significance of the

interac-tion between Src and MVP in those cancer cells, which

are overexpressing these proteins Furthermore, Src

may not be the only tyrosine kinase that could

poten-tially phosphorylate MVP in cells There can be also

other factors, in addition to tyrosine phosphorylation

of MVP, which may regulate the interaction of Src

and MVP With all the uncertainty and lack of

infor-mation, we strongly believe that elucidation of the

interplays between Src and MVP can be very

import-ant for deciphering their pathophysiological roles in

normal cells as well as in anticancer drug resistance

and oncogenesis Two previous reports have indicated

that MVP is tyrosine phosphorylated in vivo in CHO

and PC12 cells and phosphorylated by PKC and casein

kinase II in vitro using specific kinase agonist and

inhibitors [31,32] Although MVP has been recently

reported to interact with oestrogen receptor [17] and

PTEN [18] and SHP-2 [33] and the La-autoantigen [34],

this is so far the first report of MVP interacting with a

tyrosine kinase (Src) and signal-dependently

modula-ting the function of its downstream effector molecule

In a variety of tumour types including those derived

from colon and breast, the Src nonreceptor tyrosine

kinase is either overexpressed or constitutively active

in a large percentage of the tumours The activity of

Src is strongly associated with malignant phenotype

changes [35–38], and increased expression or activity

of Src correlates with the stage and metastatic

poten-tial of some neoplasia [39] Although a number of

interacting proteins that upregulate Src activity have

been identified, only a few that downregulate Src

activ-ity have been known Here we report the identification

of a protein, MVP, which appears to be an inhibitor

of Src activity The likely explanation is that MVP

binds to the SH2 domain of Src and inhibits the

autophosphorylation at Tyr416 on Src, thereby

block-ing the enzymatic activity of Src How does MVP

inhi-bit Src activity? In the inactive state, Src folds up with

phosphorylated Tyr527 in the C-terminal tail binding

to the SH2 domain The ligand binding surfaces of the

SH2 and SH3 domains are tucked inside, thus

present-ing an inert surface to the outside environment [40–

42] Thus it is possible that MVP inhibits Src activity

by clamping down on Src and holding it in the closed,

inactive, conformational state Once MVP is tyrosine

phosphorylated, it binds to the SH2 domain of Src

and in turn, regulates its activity Once the precise

binding sites on Src and MVP have been identified, we may better understand the mechanism by which MVP inhibits Src activity

The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) for cell growth or proliferation To examine the downstream consequences of Src-dependent signalling in 293T cells,

we measured ERK activation using pERK antibody The finding that MVP inhibits Src kinase (Fig 5) and downregulates the EGF stimulated ERK pathway (Fig 6) suggests a role for MVP in Src-mediated mito-genic signalling A clear correlation exists between the suppression of Src activities by MVP and suppression

of Src-mediated ERK activation upon EGF stimula-tion Thus it is tempting to suggest that the two are linked, and that it is in part through the repression of Src kinases that MVP inhibits Erk phosphorylation It

is also likely to suggest that MVP exerts its influence

on Src activity at the G1⁄ S boundary, where the acti-vation of Src is required for EGF-induced G1⁄ S trans-ition and DNA synthesis [43,44] One recent study has shown that PTEN, a tumour suppressor gene, associ-ates with MVP [18] But the physiological function

of the association between PTEN and vault is not explored PTEN has been implicated in regulating many cellular events including growth, adhesion, migration, invasion and apoptosis [45] Therefore, elu-cidation of the physiological function of the PTEN– MVP interaction and effects on PTEN activity may shed new light on the role of MVP in cells In a more recent study, SH2 domain-containing tyrosine phos-phatase, SHP-2, was shown to be associated via its SH2 domains with tyrosyl phosphorylated MVP [34] They showed that MVP can be a substrate of SHP-2

in vitro and form enzyme–substrate complex in vivo The study suggested the function of MVP as a scaffold protein for both SHP-2 and Erk for the cell survival signalling This previous report and our current finding strongly suggest that MVP may have important roles

in ERK-related signalling pathways

Accumulated evidence showed that MVP and vault particles are frequently upregulated in multidrug resist-ant cancer cells [46] Several other studies have impli-cated that the vaults are involved in nucleocytoplasmic transport [47] However, a recent study using MVP knockout mice have clearly shown that MVP⁄ vaults are not directly involved in the resistance of cytostatic agents, and the activities of the ABC transporters P-glycoprotein, multidrug resistance-associated protein and breast cancer resistance protein were unaltered on MVP deletion in these cells [15] Our present study reveals that MVP downregulates Src-mediated ERK signalling pathways, indicate the role of MVP⁄ vaults

not as multidrug-resistant inducers rather implicating

the importance of MVP in cell growth regulation We

also examined the expression of MVP in various

cancer cells and drug-resistant cancer cells (cisplatin,

vincristin and adriamycin resistant leukaemia

lympho-blast cells) by immunoblotting with MVP antibody

However we could not observe MVP overexpression in

any of the drug-resistant cancer cell lines (data not

shown) Therefore, our data consistently correlates the

findings of Mossink et al [15] that MVP is not directly

related to drug resistance in cancer cells

In summary, we have shown that MVP interacts

with the SH2 domain of Src, as well as with full-length

Src kinase in mammalian cells The binding of MVP

to Src is enhanced by EGF stimulation and tyrosine

phosphorylation of MVP We believe that tyrosine

phosphorylated MVP plays an important role in

pro-tein–protein interactions and signal transduction

path-ways Moreover, MVP inhibits the activities of Src

tyrosine kinases and attenuates the Src-mediated

activ-ity of ERK pathways Thus MVP is involved in the

regulation of Src function and cell growth

Experimental procedures

Cell culture

253J cells were cultured in Dulbecco’s modified Eagle’s

medium (DMEM) (Biowhittaker, Baltimore, MD)

supple-mented with 10% heat-inactivated fetal bovine serum in a

maintained in DMEM containing 10% fetal bovine serum

under the same atmosphere as 253J cells

Antibodies and materials

Affinity-purified polyclonal antibody against rat MVP and

rat MVP cDNA clone were the generous gifts from Dr

L H Rome (UCLA, CA) Flag-tagged MVP was prepared

by generating the rat MVP cDNA construct encoding Flag

sequence at the N terminus Monoclonal antibody against

MVP (LRP56) was the generous gift of Dr G L Scheffer

(Free University Medical Center, Amsterdam, the

Nether-lands) Chicken c-Src cDNA was the generous gift of Dr

G S Martin (UC Berkeley, CA) Anti-Src mAb used for

immunoprecipitation was from Oncogene Research

Prod-ucts Inc and c-Src polyclonal antibody used for

immuno-blotting was from Santa Cruz Biotechnology Inc (Santa

Cruz, CA) Rabbit muscle enolase for in vitro Src kinase

assay, anti-FLAG monoclonal antibody and anti-FLAG

M2 agarose were from Sigma (St Louis, MO)

Anti-phos-photyrosine mAb (clone 4G10) and purified Src enzyme for

(Lake Placid, NY) Anti-phospho ERK polyclonal antibody was from Santa Cruz Biotechnology Inc

GST-fusion pull down assay

Cultures of Escherichia coli DH5a containing pGEX-Src– SH2, Grb2-SH2, PLCc1-nSH2 and cSH2, STAT3-SH2, and Crk-SH2 plasmids were induced with 0.1 mm isopro-pyl-b-d-thiogalactopyranoside (United States Biochemical,

resuspended in Tris-buffered saline (TBS) containing 1% Triton X-100, 100 mm EDTA, sonicated then lysed by soni-cation and clarified by centrifugation at 15 000 g for

20 min The GST fusion proteins were purified by incuba-ting the bacterial supernatants with glutathione–agarose beads (Pharmacia Biotech Inc., Piscataway, NJ) for 3 h at

TBS Stomach cancer tissue and normal stomach tissue were obtained from cancer patients in a local hospital (Dongguk University Pohang Hospital) and frozen stored

20 mm NaF, 200 m sodium orthovanadate, 1 mm

inhibitor cocktail (Sigma)] The tissue homogenates were centrifuged (100 000 g, 1 h), and the supernatants were incubated with purified GST fusion proteins (1–5 lg) immobilized on glutathione–agarose beads in a final volume

Associated protein complexes were dissociated by heating

proteins were visualized by silver staining, and the protein bands were analysed by MALDI-TOF MS

Protein identification by peptide mass fingerprinting analysis

Silver stained candidate bands were excised from the gel and digested with trypsin as described [28] A 1-lL aliquot

of the total digest (total volume, 30 lL) was used for pep-tide mass fingerprinting [29,30] The masses of the tryptic peptides were measured with a Bruker REFLEX III time-of-flight mass spectrometer (Bruker Daltonics Inc.,

was performed with -cyano-4-hydroxycinnamic acid as the matrix Trypsin autolysis products were used for internal calibration Delayed ion extraction resulted in peptide mas-ses with better than 50 p.p.m mass accuracy on average Comparison of the mass values against the NCBInr data-base was performed using peptide search

Protein extractions, transfection and

immuno-precipitations

cDNA encoding full length rat MVP with a FLAG epitope

at the N terminus (FLAG-tagged-MVP) was cloned into

vec-tor The plasmid DNA was transiently transfected into

293T cells by the use of Lipofectamine (Gibco-BRL,

Gaithersburg, MD) according to the manufacturer’s

cells were cultured in 60-mm dishes 16–20 h before transfection to obtain 40–50% confluency at

the time of transfection Transfections were performed with

1.0 lg Src and 12 lL lipofectamine After 36 h, the medium

was replaced with fresh DMEM containing 10% fetal

bovine serum For EGF treatment, cells were serum starved

for 24 h and then treated with EGF Then cells were

lysis Buffer (1% Triton X-100, 150 mm NaCl, 20 mm

The samples were vigorously vortexed for 15 s, kept on ice

resulting supernatants were harvested, the protein

concen-tration assayed by the Bradford method and subjected to

immunoprecipitation 253J cells were washed once with

15% glycerol, 10% sucrose, 1% Nonidet P-40 and EDTA

free protease inhibitor (mix)] and centrifuged at 20 000 g

with 20 lL anti-FLAG M2 agarose (Sigma) or with 2 lg

Src mAb coupled to 20 lL protein A–agarose beads

(Phar-macia) The protein complexes were then washed four times

with lysis buffer, eluted with SDS sample buffer and

achieve maximum separation of the 60 kDa Src and

55 kDa IgG heavy chain

Immunoblot analysis

(acrylamide–bisa-crylamide ratio, 20 : 1) Proteins were transferred to

polyvi-nylidene fluoride membranes (Millipore, Billerica, MA) in

15% methanol) with a transblot apparatus (Bio-Rad,

Her-cules, CA) for 1.5 h at 60 V The membrane was blocked

for 2 h or overnight in blocking buffer (5% skimmed milk

in TBS containing 0.05% Tween-20) Membranes were

polyclonal Ab, antiphosphotyrosine monoclonal antibody

(PY20), Anti-ERK or antiphospho-ERK IgG for 2–3 h,

washed in Tween 20 containing Tris-buffered saline (TTBS,

50 mm Tris-HCl, pH 7.4, 0.05% Tween 20, 150 mm NaCl),

with changes every 10 min for 45 min, and incubated with horseradish peroxidase-conjugated goat antimouse IgG (Bio-Rad) or goat antirabbit IgG Proteins were detected

by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol

Immunocytochemistry

253J cells were seeded on glass coverslips and cultured overnight then serum starved for the next 24 h in

times From this, the cells were washed three times on ice

treat-ment, they were fixed with 3% paraformaldehyde in

at room temperature After blocking nonspecific binding with

with primary antibodies for 1 h, then washed three times

probe-conju-gated secondary antibodies for another 1 h After washing

slides and examined by fluorescence microscopy

In vitro Src kinase activity assay

Flag-CMV plasmid containing MVP overexpressed 293T cell lysates were immunoprecipitated with anti-FLAG IgG The immunoprecipitates were washed three times with cell lysis buffer and once with kinase buffer (20 mm Pipes

ATP) The MVP was eluted from the immunoprecipitates

by adding excess FLAG peptide, and then concentrated

purified MVP Rabbit muscle enolase (Sigma), used as an exogenous substrate of Src, was denatured with 50 mm

pH 7.0 The kinase reaction mixture containing kinase

enolase as a substrate, 5 U purified recombinant human c-Src (Upstate Biotechnology) and purified MVP (0.5 lg or

was stopped by the addition of electrophoresis sample

and visualized by autoradiography

Acknowledgements

We are grateful for the generous gifts of polyclonal anti-MVP IgG and rat anti-MVP cDNA from Dr Rome (UCLA, CA) We also appreciate the generous gifts of