Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M703222200 on May 11, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19321-19330, July 6, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/27/19321    most recent
M703222200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leng, Y.
Right arrow Articles by Kufe, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leng, Y.
Right arrow Articles by Kufe, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Nuclear Import of the MUC1-C Oncoprotein Is Mediated by Nucleoporin Nup62*

Yumei Leng, Cheng Cao1, Jian Ren, Lei Huang, Dongshu Chen, Masaki Ito, and Donald Kufe2

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, April 17, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The MUC1 heterodimeric transmembrane protein is aberrantly overexpressed by most human carcinomas. The MUC1 C-terminal subunit (MUC1-C) is devoid of a classical nuclear localization signal and is targeted to the nucleus by an unknown mechanism. The present results demonstrate that MUC1-C associates with importin β and not importin {alpha}. The results also show that, like importin β, MUC1-C binds to Nup62 (nucleoporin p62). MUC1-C binds directly to the Nup62 central domain and indirectly to the Nup62 C-terminal {alpha}-helical coiled-coil domain. We demonstrate that MUC1-C forms oligomers and that oligomerization is necessary for binding to Nup62. The MUC1-C cytoplasmic domain contains a CQC motif that when mutated to AQA abrogates oligomerization and binding to Nup62. Stable expression of MUC1 with the CQC -> AQA mutations was associated with targeting to the cell membrane and cytosol and attenuation of nuclear localization. The results further show that expression of MUC1(CQC-AQA) attenuates MUC1-induced (i) transcriptional coactivation, (ii) anchorage-independent growth, and (iii) tumorigenicity. These findings indicate that the MUC1-C oncoprotein is imported to the nucleus by a pathway involving Nup62.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human MUC1 heterodimeric glycoprotein is expressed on the apical borders of normal secretory epithelial cells (1). The MUC1 protein is translated as a single polypeptide and is cleaved into N- and C-terminal subunits in the endoplasmic reticulum (2–4). The MUC1 N-terminal subunit (MUC1-N)3 contains a signal sequence for cell membrane localization and includes variable numbers of conserved 20-amino acid tandem repeats (5, 6). The tandem repeats are extensively modified by O-linked glycans that contribute to a structure that extends beyond the glycocalyx of the cell. MUC1-N is tethered to the cell membrane as a heterodimer with the MUC1 C-terminal subunit (MUC1-C), which includes a 58-amino acid extracellular domain, a 28-amino acid transmembrane domain, and a 72-amino acid cytoplasmic tail (7). Aberrant overexpression of MUC1, as found in most human carcinomas (1), confers anchorage-independent growth and tumorigenicity (8–11). Other studies have demonstrated that overexpression of MUC1 confers resistance to apoptosis induced by oxidative stress and genotoxic anti-cancer agents (12–17).

The family of tethered and secreted mucins functions in providing a protective barrier of the epithelial cell surface. With damage to the epithelial layer, the tight junctions between neighboring cells are disrupted, and polarity is lost as the cells initiate a heregulin-induced repair program (18). MUC1-N is shed from the cell surface (19), leaving MUC1-C to function as a transducer of environmental stress signals to the interior of the cell. In this regard, MUC1-C forms cell surface complexes with members of the ErbB receptor family, and MUC1-C is targeted to the nucleus in the response to heregulin stimulation (20, 21). MUC1-C also functions in integrating the ErbB receptor and Wnt signaling pathways through direct interactions between the MUC1 cytoplasmic domain (CD) and members of the catenin family (11, 21–25). Other studies have demonstrated that MUC1-CD is phosphorylated by glycogen synthase kinase 3β, c-Src, protein kinase C{delta}, and c-Abl (16, 23, 24, 26).

The mechanisms responsible for nuclear targeting of MUC1-C are unclear. Proteins containing a classical nuclear localization signal (NLS) are imported into the nucleus by first binding to importin {alpha} and then, in turn, importin β (27, 28). The cargo-importin {alpha}/β complex docks to the nuclear pore by binding to nucleoporins and is transported through the pore by a mechanism dependent on the Ran GTPase. Classical NLSs are monopartite with a single cluster of 4–5 basic amino acids or bipartite with two clusters of basic amino acids separated by a linker of 10–12 amino acids. MUC1-CD contains a RRK motif that does not conform to a prototypical monopartite NLS (29). However, certain proteins containing nonclassical NLSs are transported through the nuclear pore by binding directly to importin β (30). Importin β associates with several nucleoporins (31), including Nup62, which is located on both the cytoplasmic and nucleoplasmic faces of nuclear pore complexes (32). Other studies have indicated that β-catenin is imported into the nucleus by an importin- and nucleoporin-independent mechanism (33).

The present studies demonstrate that MUC1 is imported into the nucleus by a mechanism involving binding to Nup62. We also demonstrate that MUC1 forms oligomers through a CQC motif in the MUC1 cytoplasmic domain and that MUC1 oligomerization is necessary for nuclear import.


Figure 1
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 1.
MUC1 associates with importin β and Nup62. A, lysates from ZR-75-1/vector and ZR-75-1/MUC1 small interfering RNA cells were subjected to immunoprecipitation (IP) with anti-importin {alpha} (left) or anti-importin β (right). The immunoprecipitates were analyzed by immunoblotting (IB) with anti-MUC1-C, anti-c-Abl, anti-importin {alpha} (left), or anti-importin β (right). Lysates not subjected to immunoprecipitation were used as controls. B, anti-Nup62 or control IgG immunoprecipitates prepared from ZR-75-1/vector and ZR-75-1/MUC1 small interfering RNA cells were subjected to immunoblotting with anti-MUC1-C and anti-Nup62. C, 293 cells were transfected to express MUC1 and Myc or Myc-Nup62. Anti-Nup62 or control IgG immunoprecipitates were immunoblotted with anti-MUC1-C and anti-Myc. D, lysates from ZR-75-1 cells were incubated with GST or GST-Nup62. The adsorbates were analyzed by immunoblotting with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie Blue staining.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human ZR-75-1/vector and ZR-75-1/MUC1 small interfering RNA breast cancer cells (13) were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 µg/ml streptomycin, 100 units/ml penicillin, and 2 mM L-glutamine. Human HCT116 colon carcinoma and 293 kidney cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 µg/ml streptomycin and 100 units/ml penicillin.

Immunoprecipitation and Immunoblot Analysis—Cell lysates were prepared as described (26). Soluble proteins were subjected to immunoprecipitation as described (26) with anti-importin {alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-importin β (Santa Cruz Biotechnology), anti-Nup62 (Santa Cruz Biotechnology), or anti-Myc (Santa Cruz Biotechnology). Immunoprecipitates and soluble proteins were analyzed by immunoblotting with anti-MUC1-C (Ab1; NeoMarkers), anti-c-Abl (Santa Cruz Biotechnology), anti-importin {alpha}, anti-importin β, anti-Nup62, anti-green fluorescence protein (GFP; Clontech), anti-His (Invitrogen), anti-I{kappa}B{alpha} (Santa Cruz Biotechnology), anti-proliferating cell nuclear antigen (PCNA) (Upstate Biotechnology), anti-MUC1-N (monoclonal antibody DF3) (1), or anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated second antibodies and chemiluminescence (PerkinElmer Life Sciences).

Vector Generation and Cell Transfection—Myc-tagged MUC1-CD (20) and Myc-tagged Nup62 were expressed by cloning into the pCMV-Myc vector (Clontech). Mutants were generated by site-directed mutagenesis and confirmed by sequencing. GFP-tagged MUC1-CD and its mutants were expressed by cloning into the pEGFP-C1 vector (Clontech). 293 cells were transfected with the expression vectors in the presence of Lipofectamine. Glutathione S-transferase (GST) fusion proteins were prepared by expression of pGEX4T1-based vectors in Escherichia coli BL21 (DE3). His-tagged MUC1-CD proteins were prepared as described (26). The EYFP-Nuc vector was obtained from Clontech. The pIRESpuro2-MUC1(CQC-AQA) mutant was generated as described (20) and transfected into HCT116 cells with Lipofectamine. Stable clones were selected in puromycin (Calbiochem).


Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 2.
MUC1-CD binds to Nup62. A, lysates from 293 cells expressing Myc-MUC1-CD were incubated with GST or GST-Nup62 (left). Lysates from 293 cells expressing Myc-Nup62 were incubated with GST or GST-MUC1-CD (right). The adsorbates were analyzed by immunoblotting (IB) with anti-Myc (top). Input of the GST proteins was assessed by Coomassie Blue staining (bottom). B, schemes depicting the MUC1-CD and Nup62 proteins. MUC1-CD is highlighted at the CQC motif, phosphorylation sites, and the β-catenin binding domain. NTD, N-terminal domain. CTD, C-terminal domain. C, lysates from 293 cells expressing Myc-Nup62 or the indicated deletion mutants were incubated with GST or GST-MUC1-CD. The adsorbates were immunoblotted with anti-Myc. Lysates not subjected to the pull-downs were immunoblotted as controls. D, anti-Myc or IgG immunoprecipitates (IP) prepared from 293 cells expressing the indicated vectors were immunoblotted with anti-MUC1-C or anti-Myc. Lysates not subjected to immunoprecipitation were used as controls. aa, amino acids.

 
Binding Assays—Cell lysates were incubated with 5 µgof GST or GST fusion proteins bound to glutathione beads. After incubation for 2 h at 4 °C, the adsorbates were washed and then subjected to immunoblot analysis. An aliquot of total lysate (2%, v/v) was included as a control. For direct binding assays in vitro, purified GST proteins were incubated with purified His-tagged proteins in binding buffer (250 mM NaCl, 20 mM HEPES, pH 7.0, 5 mM EDTA, 0.5 mM dithiothreitol, and 0.1% Tween 20) for 1 h at room temperature. The adsorbates were analyzed by immunoblotting.

Cellular Fractionation—Nuclear and cytosolic fractions were prepared as described (25).

Luciferase Assays—Cells were transfected with pcycD1(161)-luc (34) and β-galactosidase in the presence of Lipofectamine. Luciferase assays (Luciferase Assay System; Promega, Madison, WI) were performed at 48 h after transfection. Transfection efficiency was normalized by β-galactosidase expression.

Anchorage-independent Growth—Cells (1 x 105) were suspended in 0.33% Noble agar (Difco) in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. The cell suspension was layered over 0.5% agar in medium in 100-mm dishes. Colonies of >10 cells were counted at 2 weeks.

Tumorigenicity Assays—Cells (1 x 106) were injected subcutaneously into the flanks of 4–6-week old nude (nu/nu) mice. Tumor volumes were calculated from bidimensional measurements.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MUC1-C Associates with Nup62—To investigate mechanisms responsible for nuclear import of MUC1-C, we first asked if MUC1-C associates with importins in MUC1-positive and, as a control, MUC1-negative ZR-75-1 cells. Immunoblot analysis of anti-importin {alpha} immunoprecipitates with an antibody against the C terminus of MUC1 showed no detectable reactivity (Fig. 1A, left). As a control, the c-Abl protein, which contains an NLS, was detectable in complexes with importin {alpha} (Fig. 1A, left). When a similar analysis was performed on anti-importin β immunoprecipitates, reactivity with the anti-MUC1-C antibody was detectable with 15–25-kDa proteins (Fig. 1A, right). The anti-MUC1-C signals detected in the anti-importin β immunoprecipitates corresponded to those obtained with lysates not subjected to immunoprecipitation, indicating that MUC1-C associates with importin β and not importin {alpha}. Importin β binds to the C-terminal domain of the nucleoporin p62 (Nup62) (32). Consequently, we asked if MUC1-C associates with Nup62. The results demonstrate that MUC1-C is detectable in anti-Nup62 immunoprecipitates (Fig. 1B). Using 293 cells that express MUC1 and Myc-tagged Nup62 (Myc-Nup62), immunoblot analysis of anti-Nup62 immunoprecipitates with anti-MUC1-C confirmed the association of MUC1-C and Nup62 (Fig. 1C). In other studies, lysates from ZR-75-1 cells were incubated with GST or GST-Nup62. Analysis of the adsorbates with anti-MUC1-C demonstrated binding of MUC1-C to GST-Nup62 and not to GST (Fig. 1D). These findings indicate that MUC1-C associates with Nup62.


Figure 3
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 3.
MUC1 forms oligomers. A, purified His-MUC1-CD was incubated for 1 h at room temperature and then separated under reducing and nonreducing conditions. Proteins were detected by Coomassie Blue staining. B, purified His-MUC1-CD was incubated with GST-MUC1-CD or GST-MUC1-CD(CQC-AQA) for 1 h at room temperature. The adsorbates and input were immunoblotted (IB) with anti-His. C, His-MUC1-CD was immobilized on a Ni2+-nitrilotriacetic acid chip. GST-MUC1-CD was injected over the chip at the indicated concentrations. Raw binding data were analyzed by BIAevaluation software version 3.0 and fit to a 1.1 Languir binding model. D, anti-Myc immunoprecipitates (IP) prepared from 293 cells expressing the indicated vectors were immunoblotted with the indicated antibodies. Lysates not subjected to immunoprecipitation were used as controls.

 
MUC1-CD Associates with the Nup62 T-rich and C-terminal Coiled-coil Regions—To localize the region of MUC1-C that associates with Nup62, lysates from 293 cells expressing a Myc-tagged MUC1-CD were incubated with GST or GST-Nup62. The results demonstrate that Nup62 binds to MUC1-CD (Fig. 2A, left). In the reciprocal experiment, lysates from 293 cells expressing Myc-Nup62 were incubated with GST or GST-MUC1-CD. Immunoblot analysis of the adsorbates with anti-Myc confirmed binding of Nup62 to MUC1-CD (Fig. 2A, right). To define the region of Nup62 involved in the association with MUC1-CD (Fig. 2B), 293 cells were transfected with vectors expressing the Nup62 N-terminal FXFG repeats, the T-rich linker region, and the C-terminal {alpha}-helical coiled-coil domain (Fig. 2B). Probing the lysates with GST-MUC1-CD demonstrated that MUC1-CD associates with Nup62-(178–327) and Nup62-(328–522) but not with Nup62-(1–177) (Fig. 2C). To confirm these observations, 293 cells expressing MUC1 and Myc-tagged full-length Nup62 or the deletion mutants were immunoprecipitated with anti-Myc or IgG. Immunoblot analysis of the precipitates with anti-MUC1-C confirmed binding of MUC1 to Nup62-(178–327) and Nup62-(328–522) and not to Nup62-(1–177) (Fig. 2D). These findings demonstrate that MUC1-CD associates with the Nup62 T-rich linker and C-terminal coiled-coil regions.

MUC1-CD Forms Oligomers—To assess binding of MUC1-CD to Nup62 in vitro, we purified a His-tagged MUC1-CD. Immunoblot analysis under reducing conditions showed that the ~10-kDa His-MUC1-CD is also detectable as ~20-kDa and higher molecular mass forms (Fig. 3A). Moreover, most of the His-MUC1-CD was found at ~20 kDa or higher under nonreducing conditions, indicating that MUC1-CD forms oligomers in vitro (Fig. 3A). A CQC motif is present at amino acids 1–3 of MUC1-CD (Fig. 2B, scheme). To determine if the cysteines are involved in the formation of MUC1-CD oligomers, we generated MUC1-CD with C-A mutations. Incubation of His-MUC1-CD with GST-MUC1-CD or GST-MUC1-CD(CQC-AQA) and immunoblot analysis of the adsorbates with anti-His demonstrated binding of His-MUC1-CD to GST-MUC1-CD and not GST-MUC1-CD(CQC-AQA) (Fig. 3B). To define the kinetics of oligomer formation, the parameters of His-MUC1-CD binding were determined using His-MUC1-CD immobilized to the sensor chip in a BIAcore. GST-MUC1-CD bound to His-MUC1-CD with a dissociation constant (Kd) of 33 nM (Fig. 3C). Substitution of His-MUC1-CD with His-MUC1-CD(CQC-AQA) showed little if any binding (data not shown). To investigate whether MUC1-CD forms oligomers in cells, we coexpressed Myc-MUC1-CD and GFP-MUC1-CD. Immunoblot analysis of anti-Myc immunoprecipitates with anti-GFP showed that Myc-MUC1-CD associates with GFP-MUC1-CD (Fig. 3D). By contrast, there was no detectable association when Myc-MUC1-CD was coexpressed with a GFP-MUC1-CD-(7–72) mutant deleted at the CQCRRK motif (Fig. 3D). There was also no detectable binding of Myc-MUC1-CD with GFP-MUC1-CD(CQC-AQA), indicating that the cysteines are essential for the interaction (Fig. 3D). These findings demonstrate that MUC1-CD forms oligomers in vitro and in cells by a mechanism dependent on the cysteine residues.


Figure 4
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 4.
Direct binding of MUC1 oligomers and Nup62. A, purified His-MUC1-CD was incubated with GST or GST-Nup62 for 1 h at room temperature (left). Purified His-MUC1-CD or His-MUC1-CD(CQC-AQA) was incubated with GST-Nup62 (right). The adsorbates were immunoblotted (IB) with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie Blue staining. B, purified His-MUC1-CD was incubated with GST or the indicated GST-Nup62 deletion mutants for 1 h at room temperature. The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie Blue staining. C, lysates from 293 cells expressing GFP-MUC1-CD or GFP-MUC1-CD(CQC-AQA) were incubated with GST-Nup62 (left). Lysates from 293 cells expressing Myc-Nup62 were incubated with GST, GST-MUC1-CD, or GST-MUC1-CD(CQC-AQA) (right). The adsorbates and lysates were immunoblotted with the indicated antibodies. Input of the GST proteins was determined by Coomassie Blue staining. D, anti-Myc or IgG immunoprecipitates (IP) from 293 cells expressing the indicated vectors were immunoblotted with anti-GFP and anti-Nup62. Lysates not subjected to immunoprecipitation were used as controls.

 
MUC1-CD Binds Directly to Nup62—Incubation of purified GST-Nup62 and purified His-MUC1-CD demonstrated direct binding of MUC1-CD to Nup62 (Fig. 4A, left). Notably, oligomeric MUC1-CD was detectable in the GST-Nup62 adsorbates (Fig. 4A, left). Moreover, there was no detectable binding of MUC1-CD(CQC-AQA) to Nup62 in vitro (Fig. 4A, right). When His-MUC1-CD was incubated with the GST-Nup62 deletion mutants, binding of oligomeric MUC1-CD was detectable with Nup62-(178–327) and not Nup62-(1–177) or Nup62-(328–522) (Fig. 4B). These results indicate that, in contrast to the association of MUC1-CD with both Nup62-(178–327) and Nup62-(328–522) in cells, direct binding of MUC1-CD is selective for Nup62-(178–327) in vitro. In concert with the results obtained with purified proteins, binding of GST-Nup62 to GFP-MUC1-CD in cell lysates was abrogated by mutating CQC to AQA (Fig. 4C, left). Binding of GST-MUC1-CD to Myc-Nup62 in cell lysates was also abrogated by the CQC-AQA mutations (Fig. 4C, right). In addition, coprecipitation of Nup62 and MUC1-CD was abrogated by expressing MUC1-CD-(7–72) or MUC1-CD(CQC-AQA) (Fig. 4D). These findings indicate that MUC1-CD binds directly to Nup62 and that this interaction is dependent on the cysteines in the CQC motif.

Nuclear Import of MUC1 Involves Binding to Nup62—To determine if the interaction with Nup62 contributes to nuclear import of MUC1, immunoblot analysis was performed on nuclear lysates. The results demonstrate the presence of GFP-MUC1-CD and little if any GFP-MUC1-CD-(CQC-AQA) (Fig. 5A). Equal loading and purity of the nuclear fractions were documented by immunoblotting for levels of nuclear PCNA and cytoplasmic I{kappa}B{alpha} (Fig. 5A). To determine if MUC1 is imported into the nucleus by binding to Nup62, Myc-Nup62-(178–328) was expressed as a decoy to interfere with the interaction between GFP-MUC1-CD and endogenous Nup62. Immunoblot analysis of nuclear lysates demonstrated that Nup62-(178–328) blocks nuclear localization of MUC1-CD (Fig. 5B). By contrast, nuclear localization of EYFP-Nuc, which contains an NLS, was unaffected by Nup62-(178–328) expression (Fig. 5C). These findings indicate that MUC1-CD is imported into the nucleus by interacting with Nup62.


Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 5.
Nuclear import of MUC1 involves the CQC motif and Nup62. A, nuclear and cytosolic fractions from 293 cells expressing GFP-MUC1-CD or GFP-MUC1-CD(CQC-AQA) were immunoblotted (IB) with the indicated antibodies. B and C, nuclear and cytosolic lysates from 293 cells expressing the indicated vectors were immunoblotted with anti-GFP, anti-I{kappa}B{alpha}, or anti-PCNA.

 
Stable Expression of Full-length MUC1(CQC-AQA) Attenuates Binding to Nup62 and Nuclear Localization—To further assess involvement of the MUC1 CQC motif in subcellular localization, MUC1-negative HCT116 carcinoma cells were stably transfected to express the empty vector, wild-type MUC1, or the MUC1(CQC-AQA) mutant. As shown previously (9, 13), expression of MUC1-C was detectable in HCT116/MUC1, but not HCT116/vector, cells (Fig. 6A, left). MUC1-C expression was also detectable in two independently selected clones (A and B) of HCT116/MUC1(CQC-AQA) cells (Fig. 6A, left). Analysis of the transfectants by flow cytometry with anti-MUC1-N further demonstrated that, as found with wild-type MUC1 (13), MUC1(CQC-AQA) is detectable on the surface of HCT116 cells (Fig. 6A, right). To determine if MUC1(CQC-AQA) interacts with Nup62, anti-Nup62 immunoprecipitates were analyzed by immunoblotting with anti-MUC1-C. The results show that binding of MUC1 to Nup62 is abrogated in the HCT116 cells expressing MUC1(CQC-AQA) (Fig. 6B). To extend this analysis, nuclear and cytosolic lysates were subjected to immunoblot analysis with anti-MUC1-C. The results show that MUC1-C is detectable to a greater extent in nuclear lysates from HCT116/MUC1, as compared with HCT116/MUC1(CQC-AQA), cells (Fig. 6C). By contrast, cytosolic MUC1-C levels were similar in the HCT116/MUC1 and HCT116/MUC1 (CQC-AQA) cells (Fig. 6C). MUC1 functions as a coactivator of Tcf4-mediated transcription of the cyclin D1 promoter (9, 11). To analyze the effects of MUC1(CQC-AQA), we used the pcycD1(–161)-Luc reporter, which contains three Tcf-binding sites (34). As shown previously (9) and as compared with HCT116/vector cells, activation of pcycD1(–161)-Luc was increased by MUC1 expression (Fig. 6D). By contrast, MUC1-induced coactivation of pcycD1(–161)-Luc was attenuated in the HCT116/MUC1(CQC-AQA) cells (Fig. 6D). These findings indicate that, like MUC1-CD, mutation of the CQC motif in full-length MUC1 attenuates binding to Nup62 and nuclear localization.

Nuclear Import of MUC1 Contributes to Transformation—To determine if attenuation of nuclear localization affects the MUC1 transforming function, the HCT116 cell transfectants were assessed for anchorage-dependent and -independent growth. Expression of MUC1 or MUC1(CQC-AQA) had little if any effect on anchorage-dependent growth in tissue culture flasks (data not shown). As shown previously for growth in soft agar (9, 26), the MUC1 transfectants formed colonies that were substantially larger than those obtained with HCT116/vector cells (Fig. 7A). By contrast, expression of MUC1(CQC-AQA) resulted in the formation of colonies that were similar to those found with HCT116/vector cells (Fig. 7A). Quantification of the number of colonies demonstrated plating efficiencies of ~6% for the HCT116/MUC1 cells and less than 0.1% for the HCT116/vector and HCT116/MUC1(CQC-AQA) cells (Fig. 7B). To determine if expression of MUC1(CQC-AQA) affects tumorigenicity, 1 x 106 HCT116/vector, HCT116/MUC1, or HCT116/MUC1(CQC-AQA) cells were injected subcutaneously into nude mice. As shown previously (9, 13), HCT116/MUC1 cells formed tumors that were somewhat larger than those obtained with HCT116/vector cells (Fig. 7C). Significantly, however, tumors were substantially smaller in mice injected with the HCT116/MUC1(CQC-AQA) cells as compared with those formed with both HCT116/vector and HCT116/MUC1 cells (Fig. 7C). These findings indicate that the function of wild-type MUC1 in supporting anchorage-independent growth and tumorgenicity is attenuated by the MUC1(CQC-AQA) mutant.


Figure 6
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 6.
Stable expression of MUC1(CQC-AQA) in HCT116 cells. HCT116 cells were stably transfected to express the empty vector, MUC1, or MUC1(CQC-AQA). Clones (A and B) were selected from two independent transfections. A, lysates were subjected to immunoblot (IB) analysis with anti-MUC1-C and anti-β-actin (left). Cells were incubated with anti-MUC1-N (closed symbols) or control mouse IgG (open symbols) and analyzed for immunofluorescence by flow cytometry (right). B, anti-Nup62 or IgG immunoprecipitates (IP) from the indicated cells were analyzed by immunoblotting with anti-MUC1-C and anti-Nup62. C, nuclear and cytoplasmic lysates from the indicated cells were immunoblotted with anti-MUC1-C, anti-I{kappa}B{alpha}, and anti-PCNA. D, the indicated cells were transfected with pcycD1(–161)-Luc and β-galactosidase. The results are expressed as -fold activation (mean ± S.D. of three separate experiments) compared with that obtained with pcycD1(–161)-Luc in HCT116/vector cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nuclear Import of MUC1-C—The MUC1 protein is expressed as a transmembrane heterodimer that consists of N-terminal and C-terminal subunits. Previous work has shown that the MUC1-C, and not MUC1-N, localizes to the nucleus in diverse cell types (8, 11, 21, 35–39). The mechanisms responsible for nuclear import of MUC1-C, which is devoid of a classical NLS, have been unclear. Mutation of the RRK motif in the MUC1 cytoplasmic tail attenuated nuclear targeting of MUC1-C in the response to heregulin stimulation (21), indicating that this region of MUC1 is of importance to its nuclear import. In concert with the lack of a classical NLS, the present results show that there is no detectable binding of MUC1-C to importin {alpha}. Moreover, the association of MUC1-C with importin β suggested that MUC1-C may be imported by a nonclassical pathway. Indeed, certain proteins, including parathyroid hormone-related protein (40), Smad-3 (41), cAMP-response element-binding protein, Jun, and Fos (42), contain nonclassical NLSs that are transported through the NPC by direct binding to importin β. In this regard, importin β forms a complex with Nup62 (32). Other proteins, such as β-catenin, enter the nucleus by an importin {alpha}/β- and nucleoporin-independent mechanism (33). The present studies demonstrate that MUC1-C associates with importin β and with Nup62. These results indicated that MUC1-C could enter the nucleus by forming a complex with Nup62 (Fig. 7D). Little, however, is known about binding of nuclear proteins to the nucleoporins.


Figure 7
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 7.
MUC1(CQC-AQA) attenuates the MUC1 transforming function. A, photomicrographs of the indicated cells (1 x 105) after suspension in soft agar and incubation for 2 weeks. B, colonies of >10 cells were counted from three plates of the indicated cells. The results are expressed as the number of colonies (mean ± S.E.) per plate. C, the indicated cells (1 x 106) were injected subcutaneously into the flanks of nude mice. Tumor volumes were calculated by bidimensional measurements on day 25. The results are expressed as the tumor volume (mean ± S.D.) obtained from 6 mice/group. D, scheme depicting the proposed interactions of MUC1 oligomers with importin β and Nup62. TRL, T-rich linker region. CTD, C-terminal domain.

 
MUC1-CD Binds Directly to the Nup62 Central Domain—The Nup62 protein consists of an N-terminal domain (amino acids 1–177) that contains FXFG repeats, a central threonine-rich linker (amino acids 178–327), and a C-terminal domain (amino acids 328–522) that contains a heptad repeat characteristic of an {alpha}-helical coiled-coil (43–45). The Nup62 C-terminal {alpha}-helical coiled-coil domain functions as a binding site for importin β (32). From pull-down and coimmunoprecipitation studies, the present results demonstrate that MUC1-C binds to the Nup62 central (amino acids 178–327) and C-terminal (amino acids 328–522) regions. There was no detectable binding of MUC1-C to the Nup62 N-terminal region, indicating that the association with Nup62 is mediated by a mechanism independent of the FXFG repeats. Direct binding studies using purified proteins in vitro confirmed the interaction between MUC1-C and the Nup62 T-rich linker region. However, there was no detectable binding of MUC1-C and the Nup62 C-terminal domain. These results indicate that the association between MUC1-C and the Nup62 C-terminal domain observed in pull-down and coimmunoprecipitation experiments is indirect. These findings are also consistent with a model in which importin β delivers MUC1 to the Nup62 C terminus and thereby promotes direct binding of MUC1 to the Nup62 central domain (Fig. 7D).

MUC1 Oligomers Interact with Nup62—Our studies on binding of purified MUC1-CD and Nup62 indicated that MUC1 forms oligomers. Incubation of purified MUC1-CD in vitro and immunoblot analysis showed the presence of dimers and higher order structures. Pull-down and coimmunoprecipitation studies further demonstrated that Nup62 associates predominantly, if not exclusively, with MUC1 oligomers. These results suggested that the interaction between MUC1 and Nup62 is dependent on the formation of MUC1 oligomers. Previous work had shown that MUC1/Y forms heterodimeric complexes with a 15–20-kDa protein by a mechanism involving the CQC motif (46). However, to our knowledge, there is no previous evidence indicating that MUC1 forms homodimers. To investigate the role of MUC1 oligomers, we mutated the two cysteines in the MUC1-CD CQC motif. MUC1-CD(CQC-AQA) failed to form oligomers in vitro and in cells. MUC1-CD(CQC-AQA) also failed to bind to Nup62. In addition, we found that, in contrast to MUC1-CD, there was no detectable binding of MUC1-CD(CQC-AQA) to importin β (data not shown). These results indicate that the CQC motif and oligomerization are necessary for the formation of MUC1 complexes with importin β and Nup62. In concert with binding of the MUC1 oligomers, and not MUC1-CD(CQC-AQA), to Nup62, nuclear targeting of full-length MUC1 was disrupted by the CQC -> AQA mutant. These findings provide the first evidence that MUC1 forms oligomers and that oligomerization is involved in targeting MUC1 to the nucleus.

MUC1 Is Imported into the Nucleus by Interacting with Nup62—The finding that MUC1 oligomers bind directly to the T-rich linker region of Nup62 suggested that this interaction may be responsible for transport of MUC1 through the nuclear pore. To address this possibility, the Nup62-(178–328) T-rich linker region was expressed to block binding of MUC1 to endogenous Nup62. The results showed that Nup62-(178–328) blocks nuclear localization of MUC1, indicating that binding of MUC1 to the T-rich linker region of endogenous Nup62 is necessary for nuclear import. To our knowledge, there are no previous reports that provide evidence for nuclear import of a cargo by direct binding to Nup62. Nuclear import of the Ran exchange factor, RCC1, does not require the importins or other soluble factors (47). The E7 transforming protein of the type 16 human papillomavirus also enters the nucleus in the absence of soluble factors (48). However, it is not known if RCC1 or E7 is imported into the nucleus by direct binding to a nucleoporin. Nuclear import of the HIV-1 genome is mediated by the small virally encoded Vpr protein, which binds directly to the nucleoporin CG1 (49). Otherwise, little is known about nuclear import that is directly mediated by nucleoporins.

Nuclear Localization of MUC1 May Contribute to Transformation—MUC1-C forms cytosolic and nuclear complexes with β-catenin, {gamma}-catenin, and p120ctn (8, 11, 21, 25, 35, 36, 50). The available evidence indicates that MUC1 coactivates β-catenin/Tcf-mediated transcription of the cyclin D1 promoter-reporter (9, 11). Expression of MUC1 with a Tyr46 -> Phe mutation in the cytoplasmic domain attenuates coactivation of the cyclin D1 promoter, fails to confer anchorage-independent growth, and blocks the formation of HCT116 tumors (9). Previous work demonstrated that mutation of the MUC1 CQC motif to AQA blocks localization of MUC1 to the surface of MDCK cells (51). However, the present studies clearly show that the MUC1(CQC-AQA) mutant is expressed on the surface of HCT116 cells. The basis for this discrepancy in findings may be related to cell context. Expression of Tac-MUC1 fusion proteins, as used in other work (52), has shown that the CQC motif is necessary for palmitoylation and cell surface recycling, indicating that the MUC1(CQC-AQA) mutant could accumulate in endosomes. The present studies further demonstrate that binding of full-length MUC1 to Nup62 and nuclear import is attenuated by the CQC -> AQA mutations. In concert with these results, expression of the MUC1(CQC-AQA) mutant attenuated MUC1-mediated coactivation of the cyclin D1 promoter. Moreover, like MUC1(Y46F), expression of MUC1(CQC-AQA) was ineffective in supporting anchorage-independent growth and attenuated the formation of HCT116 tumors. These findings collectively indicate that MUC1 may contribute to transformation through nuclear localization. Therefore, strategies designed to disrupt the formation of MUC1 oligomers could attenuate localization of MUC1 to the nucleus and thereby its transforming function.


   FOOTNOTES
 
* This work was supported by NCI, National Institutes of Health Grant CA97098 and United States Army Grant DAMD17-03-1-0672. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Beijing Institute of Biotechnology, Beijing 100850, China. Back

2 To whom correspondence should be addressed: Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: donald_kufe{at}dfci.harvard.edu.

3 The abbreviations used are: MUC1-N, MUC1 N-terminal subunit; MUC1-C, MUC1 C-terminal subunit; CD, cytoplasmic domain; NLS, nuclear localization signal; GFP, green fluorescence protein; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen. Back


   ACKNOWLEDGMENTS
 
We acknowledge Kamal Chauhan for excellent technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kufe, D., Inghirami, G., Abe, M., Hayes, D., Justi-Wheeler, H., and Schlom, J. (1984) Hybridoma 3, 223–232[Medline] [Order article via Infotrieve]
  2. Ligtenberg, M. J., Kruijshaar, L., Buijs, F., van Meijer, M., Litvinov, S. V., and Hilkens, J. (1992) J. Biol. Chem. 267, 6171–6177[Abstract/Free Full Text]
  3. Macao, B., Johansson, D. G., Hansson, G. C., and Hard, T. (2006) Nat. Struct. Mol. Biol. 13, 71–76[CrossRef][Medline] [Order article via Infotrieve]
  4. Levitin, F., Stern, O., Weiss, M., Gil-Henn, C., Ziv, R., Prokocimer, Z., Smorodinsky, N. I., Rubinstein, D. B., and Wreschner, D. H. (2005) J. Biol. Chem. 280, 33374–33386[Abstract/Free Full Text]
  5. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. A. (1988) J. Biol. Chem. 263, 12820–12823[Abstract/Free Full Text]
  6. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and Kufe, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2320–2323[Abstract/Free Full Text]
  7. Merlo, G., Siddiqui, J., Cropp, C., Liscia, D. S., Lidereau, R., Callahan, R., and Kufe, D. (1989) Cancer Res. 49, 6966–6971[Abstract/Free Full Text]
  8. Li, Y., Liu, D., Chen, D., Kharbanda, S., and Kufe, D. (2003) Oncogene 22, 6107–6110[CrossRef][Medline] [Order article via Infotrieve]
  9. Huang, L., Ren, J., Chen, D., Li, Y., Kharbanda, S., and Kufe, D. (2003) Cancer Biol. Ther. 2, 702–706[Medline] [Order article via Infotrieve]
  10. Schroeder, J. A., Masri, A. A., Adriance, M. C., Tessier, J. C., Kotlarczyk, K. L., Thompson, M. C., and Gendler, S. J. (2004) Oncogene 23, 5739–5747[CrossRef][Medline] [Order article via Infotrieve]
  11. Huang, L., Chen, D., Liu, D., Yin, L., Kharbanda, S., and Kufe, D. (2005) Cancer Res. 65, 10413–10422[Abstract/Free Full Text]
  12. Yin, L., and Kufe, D. (2003) J. Biol. Chem. 278, 35458–35464[Abstract/Free Full Text]
  13. Ren, J., Agata, N., Chen, D., Li, Y., Yu, W.-H., Huang, L., Raina, D., Chen, W., Kharbanda, S., and Kufe, D. (2004) Cancer Cell 5, 163–175[CrossRef][Medline] [Order article via Infotrieve]
  14. Raina, D., Kharbanda, S., and Kufe, D. (2004) J. Biol. Chem. 279, 20607–20612[Abstract/Free Full Text]
  15. Yin, L., Huang, L., and Kufe, D. (2004) J. Biol. Chem. 279, 45721–45727[Abstract/Free Full Text]
  16. Raina, D., Ahmad, R., Kumar, S., Ren, J., Yoshida, K., Kharbanda, S., and Kufe, D. (2006) EMBO J. 25, 3774–3783[CrossRef][Medline] [Order article via Infotrieve]
  17. Yin, L., Kharbanda, S., and Kufe, D. (2007) J. Biol. Chem. 282, 257–266[Abstract/Free Full Text]
  18. Vermeer, P. D., Einwalter, L. A., Moninger, T. O., Rokhlina, T., Kern, J. A., Zabner, J., and Welsh, M. J. (2003) Nature 422, 322–326[CrossRef][Medline] [Order article via Infotrieve]
  19. Abe, M., and Kufe, D. (1989) Cancer Res. 49, 2834–2839[Abstract/Free Full Text]
  20. Li, Y., Ren, J., Yu, W.-H., Li, G., Kuwahara, H., Yin, L., Carraway, K. L., and Kufe, D. (2001) J. Biol. Chem. 276, 35239–35242[Abstract/Free Full Text]
  21. Li, Y., Yu, W.-H., Ren, J., Huang, L., Kharbanda, S., Loda, M., and Kufe, D. (2003) Mol. Cancer Res. 1, 765–775[Abstract/Free Full Text]
  22. Yamamoto, M., Bharti, A., Li, Y., and Kufe, D. (1997) J. Biol. Chem. 272, 12492–12494[Abstract/Free Full Text]
  23. Li, Y., Bharti, A., Chen, D., Gong, J., and Kufe, D. (1998) Mol. Cell. Biol. 18, 7216–7224[Abstract/Free Full Text]
  24. Li, Y., Kuwahara, H., Ren, J., Wen, G., and Kufe, D. (2001) J. Biol. Chem. 276, 6061–6064[Abstract/Free Full Text]
  25. Li, Y., and Kufe, D. (2001) Biochem. Biophys. Res. Commun. 281, 440–443[CrossRef][Medline] [Order article via Infotrieve]
  26. Ren, J., Li, Y., and Kufe, D. (2002) J. Biol. Chem. 277, 17616–17622[Abstract/Free Full Text]
  27. Weis, K. (2003) Cell 112, 441–451[CrossRef][Medline] [Order article via Infotrieve]
  28. Stewart, M. (2007) Nat. Rev. Mol. Cell. Biol. 8, 195–208[CrossRef][Medline] [Order article via Infotrieve]
  29. Hodel, M. R., Corbett, A. H., and Hodel, A. E. (2001) J. Biol. Chem. 276, 1317–1325[Abstract/Free Full Text]
  30. Kau, T. R., Way, J. C., and Silver, P. A. (2004) Nat. Rev. Cancer 4, 106–117[Medline] [Order article via Infotrieve]
  31. Ryan, K. J., and Wente, S. R. (2000) Curr. Opin. Cell Biol. 12, 361–371[CrossRef][Medline] [Order article via Infotrieve]
  32. Percipalle, P., Clarkson, W. D., Kent, H. M., Rhodes, D., and Stewart, M. (1997) J. Mol. Biol. 266, 722–732[CrossRef][Medline] [Order article via Infotrieve]
  33. Suh, E. K., and Gumbiner, B. M. (2003) Exp. Cell Res. 290, 447–456[CrossRef][Medline] [Order article via Infotrieve]
  34. Tetsu, O., and McCormick, F. (1999) Nature 398, 422–426[CrossRef][Medline] [Order article via Infotrieve]
  35. Li, Y., Chen, W., Ren, J., Yu, W., Li, Q., Yoshida, K., and Kufe, D. (2003) Cancer Biol. Ther. 2, 187–193[Medline] [Order article via Infotrieve]
  36. Wen, Y., Caffrey, T., Wheelock, M., Johnson, K., and Hollingsworth, M. (2003) J. Biol. Chem. 278, 38029–38039[Abstract/Free Full Text]
  37. Wei, X., Xu, H., and Kufe, D. (2005) Cancer Cell 7, 167–178[CrossRef][Medline] [Order article via Infotrieve]
  38. Wei, X., Xu, H., and Kufe, D. (2006) Mol. Cell 21, 295–305[CrossRef][Medline] [Order article via Infotrieve]
  39. Wei, X., Xu, H., and Kufe, D. (2007) Cancer Res. 67, 1853–1858[Abstract/Free Full Text]
  40. Lam, M. H., Briggs, L. J., Hu, W., Martin, T. J., Gillespie, M. T., and Jans, D. A. (1999) J. Biol. Chem. 274, 7391–7398[Abstract/Free Full Text]
  41. Xiao, Z., Liu, X., and Lodish, H. F. (2000) J. Biol. Chem. 275, 23425–23428[Abstract/Free Full Text]
  42. Forwood, J. K., Lam, M. H., and Jans, D. A. (2001) Biochemistry 40, 5208–5217[Medline] [Order article via Infotrieve]
  43. Carmo-Fonseca, M., Kern, H., and Hurt, E. C. (1991) Eur. J. Cell Biol. 55, 17–30[Medline] [Order article via Infotrieve]
  44. Cordes, V., Waizenegger, I., and Krohne, G. (1991) Eur. J. Cell Biol. 55, 31–47[Medline] [Order article via Infotrieve]
  45. Starr, C. M., and Hanover, J. A. (1991) BioEssays 13, 145–146[CrossRef][Medline] [Order article via Infotrieve]
  46. Zrihan-Licht, S., Vos, H. L., Baruch, A., Elroy-Stein, O., Sagiv, D., Keydar, I., Hilkens, J., and Wreschner, D. H. (1994) Eur. J. Biochem. 224, 787–795[Medline] [Order article via Infotrieve]
  47. Nemergut, M. E., and Macara, I. G. (2000) J. Cell Biol. 149, 835–850[Abstract/Free Full Text]
  48. Angeline, M., Merle, E., and Moroianu, J. (2003) Virology 317, 13–23[CrossRef][Medline] [Order article via Infotrieve]
  49. Le Rouzic, E., Mousnier, A., Rustum, C., Stutz, F., Hallberg, E., Dargemont, C., and Benichou, S. (2002) J. Biol. Chem. 277, 45091–45098[Abstract/Free Full Text]
  50. Schroeder, J. A., Adriance, M. C., Thompson, M. C., Camenisch, T. D., and Gendler, S. J. (2003) Oncogene 22, 1324–1332[CrossRef][Medline] [Order article via Infotrieve]
  51. Pemberton, L. F., Rughetti, A., Taylor-Papadimitriou, J., and Gendler, S. J. (1996) J. Biol. Chem. 271, 2332–2340[Abstract/Free Full Text]
  52. Kinlough, C. L., McMahan, R. J., Poland, P. A., Bruns, J. B., Harkleroad, K. L., Stremple, R. J., Kashlan, O. B., Weixel, K. M., Weisz, O. A., and Hughey, R. P. (2006) J. Biol. Chem. 281, 12112–12122[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
D. Raina, R. Ahmad, M. D. Joshi, L. Yin, Z. Wu, T. Kawano, B. Vasir, D. Avigan, S. Kharbanda, and D. Kufe
Direct Targeting of the Mucin 1 Oncoprotein Blocks Survival and Tumorigenicity of Human Breast Carcinoma Cells
Cancer Res., June 15, 2009; 69(12): 5133 - 5141.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. P. Pitroda, N. N. Khodarev, M. A. Beckett, D. W. Kufe, and R. R. Weichselbaum
From the Cover: MUC1-induced alterations in a lipid metabolic gene network predict response of human breast cancers to tamoxifen treatment
PNAS, April 7, 2009; 106(14): 5837 - 5841.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. K. Singh, M. E. Behrens, J. P. Eggers, R. L. Cerny, J. M. Bailey, K. Shanmugam, S. J. Gendler, E. P. Bennett, and M. A. Hollingsworth
Phosphorylation of MUC1 by Met Modulates Interaction with p53 and MMP1 Expression
J. Biol. Chem., October 3, 2008; 283(40): 26985 - 26995.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
D. D. Carson
The Cytoplasmic Tail of MUC1: A Very Busy Place
Sci. Signal., July 8, 2008; 1(27): pe35 - pe35.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/27/19321    most recent
M703222200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leng, Y.
Right arrow Articles by Kufe, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leng, Y.
Right arrow Articles by Kufe, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement