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

Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling Euikyung Kim1*, Seunghwan Lee1, Md Firoz Mian2, Sang Uk Yun2, Minseok Song2, Kye-Sook Yi2, Sung Ho Ryu2 and Pann-Ghill Suh2* Institue of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea Department of Life Science, Pohang University of Science and Technology, Pohang, Korea 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) 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 (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 microscopy 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 overexpressing cells To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway It appears that MVP is a novel regulator of Src-mediated signalling cascades doi:10.1111/j.1742-4658.2006.05112.x 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 preparations 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 organisms [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 (TEP1) [10,11] The 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 FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS 793 MVP interacts with Src tyrosine kinase E Kim et al expression of the unique 100 kDa MVP is sufficient to form the basic vault structure Although many molecular features of vault particles have been characterized, 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 frequently overexpress MVP and increased MVP mRNA expression was found to correlate strongly with a predictive value of a multidrug resistance phenotype [12,13] An early postulate of vault function was nucleocytoplasmic 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 modulation of signal transduction 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 signalling pathways that regulate diverse cellular functions, 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 substrate, 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 Identification 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 transformed cells [22] Compelling evidence indicates that Src-binding proteins can regulate Src activity [23] While a number of interacting proteins that upregulate 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 794 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 and Src homology (SH2) domains in Src are key binding sites for proteins that regulate Src activity and integrate 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 protein 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 signalling, 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 immunoblotting with polyclonal anti-MVP IgG (Fig 1C) Both the MS analysis and immunoblotting with polyclonal antiMVP IgG showed that 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 fulllength Src in vivo, we prepared MVP containing lysates FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS E Kim et al MVP interacts with Src tyrosine kinase 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 A GST onl y GSTPLC- γ1 nSH2 GS TPLC- γ1 cSH2 GSTPLC- γ1 SH22 GSTGrb2 SH2 GSTS t at SH2 GSTCrk SH2 GSTSrc SH2 - N C - N C - N C - N C - N C - N C - N C - N C Fusion : P r o te i n Tissue: 200 p100 97 68 43 GSTSrc-SH2 GST alone B 50 %Intensity p100 P2 500 P1 P6 P9 P1 P7 P1 P 10 P4 P5 1000 2000 1500 2500 (kDa) 97 68 IB: αMVP PLC γ SH22 PLC γ cSH2 PLC γ nSH2 Src SH2 Grb2 SH2 Crk SH2 Stat3 SH2 GST only GST-fusioned Input P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 VLFAPMR (43–49) SLQPLAPR (445–452) ELELVYAR (767–774) VSHQAGDHWLIR (349–360) VPHNAAVQVYDYR (462–474) AQALAIETEAELQR (748–761) EVEVVEIIQATIIR (156–169) DAQGLVLFDVTGQVR (68–82) KEVEVVEIIQATIIR (155–169) AQDPFPLYPGEVLEK (92–107) VAGDEWLFEGPGTYIPR (137–154) QLQLAYNWHFEVNDR (537–552) VIGSTYMLTQDEVLWEK (400–417) PPYHYIHVLDQNSNVSR (10–27) Calculated 848.426 880.513 991.518 1417.666 1530.741 1541.774 1610.853 1616.809 1738.998 1814.926 2004.986 2044.936 2081.970 2151.017 848.457 880.513 991.533 1417.721 1530.757 1541.804 1610.923 1616.851 1739.018 1814.944 2004.994 2045.011 2082.033 2151.085 To determine whether epidermal growth factor (EGF) can activate Src and influences the association between MVP and Src, we treated serum starved fibroblasts Mass (m/z) C Observed EGF enhances the MVP–Src interaction, which can be blocked by src kinase inhibitor, PP2 P 13 P8 P3 M + H+ (Da) of the immunoprecipitates were performed using antiFLAG 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 coimmunoprecipitated with Src Silver Staining P1 Table Peptide sequences and masses from p100 by MALDI-TOF MS Fig 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 tissue 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 described 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 descriptions of each peptide analysed and used for protein identification are shown in Table (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 FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS 795 MVP interacts with Src tyrosine kinase A IP: E Kim et al No n immune α c-Src ser um input α MVP IB α c-Src B TCL IP α f MVP α Src Tfx: f M VP c-Src + - + + + + + + + - + + α f MVP IB α c -S r c Fig MVP interacts with Src in vivo in 253J cells and in cotransfected 293T cells (A) To confirm whether MVP interacts endogenously with full-length Src, we prepared 253J (stomach cancer cell line) cell lysates and immunoprecipitated with c-Src mAb or nonimmune serum, followed by immunoblotting with anti-MVP IgG or anti-c-Src IgG Thw upper panel indicates MVP that had been associated 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 indicated (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 western 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 that overexpress FLAG–MVP and Src with EGF (100 ngỈmL)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 followed by gradual decline and return to the basal level after 15 (Fig 3A) The effect of EGF on MVP–Src interaction was concentration dependent, with a maximal effect achieved at 100 ngỈmL)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 796 association in vivo This could be addressed by examining 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 tyrosine 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 proteins endogenously, with EGF for various time periods and one group was pretreated with PP2 for 45 before EGF stimulation We could observe that endogenous 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 coimmunoprecipitation of Src and MVP prompted us to test if they colocalize in any subcellular compartment on EGF stimulation As we expected, immunofluorescence microscopy 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 predominantly localized in the nucleus of quiescent cell seems to translocate onto perinuclear and cytoskeletal compartment where it overlaps with Src upon EGF treatment The kinetics of Src–MVP colocalization correlated well with the biochemical data of protein complex formation as shown earlier This result suggests that molecular 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 immunoprecipitated with anti-FLAG IgG followed by immunoblotting with antiphosphotyrosine IgG (PY 20) We observed that MVP phosphorylation reached the peak level upon EGF stimulation for (Fig 4A) that was comparable to the time kinetics of Src–MVP interaction upon EGF stimulation as shown in Fig This FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS E Kim et al MVP interacts with Src tyrosine kinase A c-Src + - + + + + f MV P - + + + + + EGF (min): - - - 3’ 5’ 15’ Tfx α Flag MVP IB α c -S r c IP: α Flag MVP Fig EGF substantially enhances Src–MVP interaction that was blocked by Src tyrosine kinase inhibitor (PP2) (A) To determine whether Src–MVP interaction is EGF signaldependent, we starved 293T cells which were transfected with c-Src and ⁄ or Flagtagged MVP as indicated After 24 h, the 293T cells were treated with EGF (100 ngỈmL)1) for the indicated times, and the prepared cell lysates were then immunoprecipitated with anti-Flag mAb The samples were immunoblotted with anti-Flag IgG or anti-c-Src IgG, showing that the interaction is EGF-signal dependently increased then rapidly declined (B) To see the effect of EGF on endogenous MVP–Src interaction, 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 followed by EGF stimulation for The results showed that in vivo Src–MVP interaction 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 ngỈmL)1 final concentration) at 37 °C for the indicated times, then fixed and permeabilized as described in Experimental procedures Nonspecific 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-conjugated secondary antibodies (fluoresceine isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat antimouse IgG) for another h After washing with NaCl ⁄ Pi, the coverslips were mounted face down onto slides and examined under confocal fluorescence microscopy α Flag MVP α c - Sr c IB α G A PDH Tota l Ce ll Lysate B EGF (Min): PP2 (5 µM): - 1’ - 3’ - 6’ - 10’ 20’ 45’ - 6’ + MVP MVP Src Src IB IP: Src MVP IB Src T o tal C ell L ys ate C Src MVP Merged Starved EGF EGF 20 finding suggests that MVP tyrosine phosphorylation might be required for MVP–Src interaction Interestingly, 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 phos- FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS 797 MVP interacts with Src tyrosine kinase A E Kim et al c-Src + - + + f M VP - + + + + EGF (min): - - 1’ complex formation on 45-min pretreated lysates This result provides proof that the Src–MVP interaction is dependent on MVP tyrosine phosphorylation + 3’ 15’ Tfx MVP inhibits Src kinase activity α pTyr pMVP IB α Flag MVP IP: α F l a g M V P B PAP (Min): 15 ’ 45’ α pTyr IB pMVP α Flag MVP α c-Src IP: α c-Src Fig MVP–Src interaction is dependent on the tyrosine phosphorylation 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 interaction requires MVP tyrosine phosphorylation 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 phosphatase The dephosphorylated cell lysates were immunoprecipitated with anti-Src monoclonal IgG The coimmunoprecipitated complexes 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 phorylation is a prerequisite for MVP–Src association We overexpressed FLAG–MVP in 293T cells and the cell lysates were incubated with potato acid phosphatase (PAP), a phosphotyrosyl-protein phosphatase, for the indicated time periods Then lysates were immunoprecipitated with anti-Src IgG and immunoblotted with anti-MVP IgG Potato acid phosphatase treatment resulted in a marked decrease in MVP–Src 798 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 incubated 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 dramatically reduced by MVP in a dose-dependent manner (Fig 5A, lane and 4) Enolase phosphorylation followed 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 suppressing Src kinase enzymatic activity Next, we investigated 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 interacting with and copurified with MVP in the immunoprecipitation procedure MVP–Src interaction down regulates Src mediated ERK/MAPK pathway Src mediates diverse signals to a number of downstream 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 transiently transfected with Src cDNA with or without Flag-tagged MVP and treated with EGF for the FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS E Kim et al MVP interacts with Src tyrosine kinase A Enolase: S rc : MVP (µg): + - + + - + + c-Src + + + + - - + + + + EGF (min) : Src - 10’ - ’ ’ 20’ α p E RK / Enolase IB: α c-Src Ponceau S stain α ER K 1/ Src Enolase Src: MVP (0.5 µg): + Tfx Autoradiogram B + f MV P + + IB + - α pAkt α c -S r c + + α MVP + Total Cell Lysat e Autoradiogram MVP Ponceau S stain MVP Fig MVP inhibits Src kinase activity in a concentration-dependent 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 Flagtagged 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 CentriconTM (cutoff molecular weight > 50 kDa) The src tyrosine 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 kinase activity was determined from the autoradiogram of the kinase assay samples The result shows that MVP potently suppresses the Src kinase activity in vitro (B) We examined if MVP is a substrate 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 indicated time periods, cell lysates were immunoblotted with phospho-ERK and phospho-Akt (S473) antibodies 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 activation 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 Fig 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 inhibiting Src activity via the EGF-dependent MVP–Src interaction 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 stomach cancer and normal stomach (Fig 1) The Src– MVP interaction, which is mediated, at least in part, by the SH2 domain of Src, is enhanced by EGF stimulation As shown in Figs and 4, there is a correlation between tyrosine phosphorylation of MVP and its interaction 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 affinity for Src These results prompted us to speculate that a signal (like epidermal growth factor receptor activation), 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 FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS 799 MVP interacts with Src tyrosine kinase E Kim et al molecule to other signalling molecules and relays signals across multiple pathways This is particularly interesting in that both Src and MVP has been independently reported to be overexpressed in various kinds of cancer cells However, it is too premature to speculate what is the clinical significance of the interaction between Src and MVP in those cancer cells, which are overexpressing these proteins Furthermore, Src may not be the only tyrosine kinase that could potentially 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 information, we strongly believe that elucidation of the interplays between Src and MVP can be very important 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 modulating 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 potential of some neoplasia [39] Although a number of interacting proteins that upregulate Src activity have been identified, only a few that downregulate Src activity 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 blocking the enzymatic activity of Src How does MVP inhibit 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 presenting 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 800 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 mitogenic signalling A clear correlation exists between the suppression of Src activities by MVP and suppression of Src-mediated ERK activation upon EGF stimulation 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 activation of Src is required for EGF-induced G1 ⁄ S transition and DNA synthesis [43,44] One recent study has shown that PTEN, a tumour suppressor gene, associates 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, elucidation 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 phosphatase, 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 resistant cancer cells [46] Several other studies have implicated 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 FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS E Kim et al 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 lymphoblast 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 protein–protein interactions and signal transduction pathways Moreover, MVP inhibits the activities of Src tyrosine kinases and attenuates the Src-mediated activity 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) supplemented with 10% heat-inactivated fetal bovine serum in a humidified 5% CO2 atmosphere at 37 °C 293T cells were 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 Netherlands) 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 Products Inc and c-Src polyclonal antibody used for immunoblotting 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-phosphotyrosine mAb (clone 4G10) and purified Src enzyme for in vitro Src kinase assay were from Upstate Biotechnology MVP interacts with Src tyrosine kinase (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 isopropyl-b-d-thiogalactopyranoside (United States Biochemical, Cleveland, OH) for h at 30 °C Bacteria were harvested, resuspended in Tris-buffered saline (TBS) containing 1% Triton X-100, 100 mm EDTA, sonicated then lysed by sonication and clarified by centrifugation at 15 000 g for 20 The GST fusion proteins were purified by incubating the bacterial supernatants with glutathione–agarose beads (Pharmacia Biotech Inc., Piscataway, NJ) for h at °C, and the beads were washed three times with fresh TBS Stomach cancer tissue and normal stomach tissue were obtained from cancer patients in a local hospital (Dongguk University Pohang Hospital) and frozen stored at )70 °C until use The frozen tissues were thawed and chopped, then homogenized using Polytron (3 · 30 s with 1-min interval) in 10 · (v ⁄ w) ice cold Triton-X lysis buffer [1% Triton X-100, 150 mm NaCl, 20 mm Tris ⁄ HCl pH 7.4, 20 mm NaF, 200 m sodium orthovanadate, mm phenylmethylsulphonyl fluoride (PMSF), lgỈmL)1 protease inhibitor cocktail (Sigma)] The tissue homogenates were centrifuged (100 000 g, h), and the supernatants were incubated with purified GST fusion proteins (1–5 lg) immobilized on glutathione–agarose beads in a final volume of mL lysis buffer for h at °C The GST fusion protein ⁄ bead ⁄ cell lysate complexes were washed three times with fresh TBS prior to adding SDS ⁄ PAGE sample buffer Associated protein complexes were dissociated by heating in SDS sample buffer and resolved by SDS ⁄ PAGE The 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 peptide 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., Billerica, MA) Matrix-assisted laser desorption ⁄ ionization was performed with -cyano-4-hydroxycinnamic acid as the matrix Trypsin autolysis products were used for internal calibration Delayed ion extraction resulted in peptide masses with better than 50 p.p.m mass accuracy on average Comparison of the mass values against the NCBInr database was performed using peptide search FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS 801 MVP interacts with Src tyrosine kinase E Kim et al Protein extractions, transfection and immunoprecipitations cDNA encoding full length rat MVP with a FLAG epitope at the N terminus (FLAG-tagged-MVP) was cloned into the pFLAG CMVTM-2 (Sigma) mammalian expression vector The plasmid DNA was transiently transfected into 293T cells by the use of Lipofectamine (Gibco-BRL, Gaithersburg, MD) according to the manufacturer’s protocol Briefly, · 105 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 serum-free DMEM containing 1.0 lg FLAG-MVP and ⁄ or 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 washed twice with NaCl ⁄ Pi and lysed in ice cold Triton-X lysis Buffer (1% Triton X-100, 150 mm NaCl, 20 mm Tris ⁄ HCl pH 7.4, 20 mm NaF, 200 m sodium orthovanadate, mm PMSF, lgỈmL)1 protease inhibitor cocktail) The samples were vigorously vortexed for 15 s, kept on ice 20 and centrifuged at 20 000 g for 20 at °C The resulting supernatants were harvested, the protein concentration assayed by the Bradford method and subjected to immunoprecipitation 253J cells were washed once with icecold NaCl ⁄ Pi and lysed with Buffer B [20 mm Hepes pH 7.9, 100 mm KCl, mm MgCl2, mm dithiothreitol, 15% glycerol, 10% sucrose, 1% Nonidet P-40 and EDTA free protease inhibitor (mix)] and centrifuged at 20 000 g for 15 at °C The lysates were incubated for 2–3 h with 20 lL anti-FLAG M2 agarose (Sigma) or with lg Src mAb coupled to 20 lL protein A–agarose beads (Pharmacia) The protein complexes were then washed four times with lysis buffer, eluted with SDS sample buffer and resolved by SDS ⁄ PAGE on 7% polyacrylamide gels to achieve maximum separation of the 60 kDa Src and 55 kDa IgG heavy chain Immunoblot analysis FLAG or Src immunoprecipitates were resolved by SDS ⁄ PAGE on 8% polyacrylamide gels (acrylamide–bisacrylamide ratio, 20 : 1) Proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA) in transfer buffer (25 mm Tris ⁄ HCl pH 7.4, 192 mm glycine, 15% methanol) with a transblot apparatus (Bio-Rad, Hercules, CA) for 1.5 h at 60 V The membrane was blocked for h or overnight in blocking buffer (5% skimmed milk in TBS containing 0.05% Tween-20) Membranes were incubated with anti-FLAG mAb (0.2 lgỈmL)1), antic-Src 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), 802 with changes every 10 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 serumfree DMEM The cells were treated with EGF (100 ngỈmL)1 final concentration) at 37 °C for the indicated times From this, the cells were washed three times on ice with ice-cold NaCl ⁄ Pi between each step After EGF treatment, they were fixed with 3% paraformaldehyde in NaCl ⁄ Pi for 20 min, and then permeabilized in 0.1% Triton X-100 in NaCl ⁄ Pi for 20 The cells were then prepared at room temperature After blocking nonspecific binding with 4% BSA in NaCl ⁄ Pi for h, the cells were incubated with primary antibodies for h, then washed three times with NaCl ⁄ Pi and incubated with fluorescent probe-conjugated secondary antibodies for another h After washing with NaCl ⁄ Pi, the coverslips were mounted face down onto 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 pH 7.0, 10 mm MnCl2, 20 lgỈmL)1 aprotinin, 100 lm ATP) The MVP was eluted from the immunoprecipitates by adding excess FLAG peptide, and then concentrated using CentriconTM (Millipore, MA) This MVP was used as purified MVP Rabbit muscle enolase (Sigma), used as an exogenous substrate of Src, was denatured with 50 mm acetic acid for 10 at 30 °C and buffered with Pipes pH 7.0 The kinase reaction mixture containing kinase buffer (20 mm Pipes pH 7.0, 10 mm MnCl2, 20 lgỈmL)1 aprotinin, 100 lm ATP, lCi [c-32P]ATP, 2.0 lg acid denatured enolase as a substrate, U purified recombinant human c-Src (Upstate Biotechnology) and purified MVP (0.5 lg or 1.0 lg) were incubated at 30 °C for 20 The reaction was stopped by the addition of electrophoresis sample buffer Samples were then boiled, resolved by SDS ⁄ PAGE, and visualized by autoradiography Acknowledgements We are grateful for the generous gifts of polyclonal antiMVP IgG and rat MVP cDNA from Dr Rome (UCLA, CA) We also appreciate the generous gifts of FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS E Kim et al monoclonal anti-MVP IgG (LRP56) from Dr Scheffer (Free University Medical Center, Amsterdam, the Netherlands) and chicken c-Src cDNA from Dr G.S Martin (UC Berkeley, CA) We are also greatly indebted to Dr Wiemer (Erasmus Medical Center, the Netherlands) for his valuable comments and advice for this manuscript References Rome LH, Kedersh NL & Chugani D (1991) Unlocking vaults: organelles in search of a function Trends Cell Biol 1, 47–50 Kickhoefer VA, Vasu SK & Rome LR (1996) Vaults are the answer, what is the question? 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Oncogene 22, 7458–7467 47 Hamill DR & Supernant KA (1997) Characterization of the sea urchin major vault protein: a possible role of vault ribonucleoprotein particles in nucleocytoplasmic transport Dev Biol 190, 117–128 FEBS Journal 273 (2006) 793–804 ª 2006 The Authors Journal compilation ª 2006 FEBS ... [23] The UD, Src homology and Src homology (SH2) domains in Src are key binding sites for proteins that regulate Src activity and integrate Src into important signalling pathways and cell cycle... 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. .. understanding of Src regulation and has provided clues about the function of Src in normal and transformed cells [22] Compelling evidence indicates that Src- binding proteins can regulate Src activity