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Endocrinology |
1 Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania; and 2 Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Menin interacts with the transcription factors JunD and nuclear factor-
b and represses their activation of gene transcription (13, 14, 15)
. Additionally, menin interacts with RPA2, a component of single-stranded DNA–binding protein involved in DNA repair and replication (16)
. Recently, we found that menin interacts with FancD2, a nuclear protein that plays a critical role in maintaining genome stability in concert with breast cancer–associated gene (Brca1; ref. 17
). To better understand the role of menin in regulating cell proliferation, we used affinity chromatography coupled with mass spectrometry analysis to isolate additional menin-interacting proteins. We find that menin interacts with activator of S-phase kinase (ASK), an essential component of the Cdc7/ASK kinase complex (18
, 19)
, which plays a critical role in DNA replication in part by regulating the function of the MCM complex. ASK and cdc7 are essential for cell proliferation. Thus, regulation of ASK by menin may be a means of repression of cell proliferation.
Here, we show that targeted disruption of menin enhances cell proliferation, whereas complementation with menin inhibits cell proliferation. Moreover, we identify ASK as a novel menin-interacting protein and find that the COOH terminus of menin specifically interacts with ASK. We demonstrate that ASK induces a modest yet consistent increase in cell proliferation but that wild-type menin inhibits this increase. Importantly, two disease-related mutations in the COOH terminus of menin not only block the menin-ASK interaction, but also fail to inhibit ASK-induced cell proliferation. Collectively, these results link menin to ASK in the regulation of cell proliferation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of Menin-Interacting Proteins.
Procedures for isolating menin-interacting proteins are as described previously except for several modifications (17)
. Human embryonic kidney 293 cells were stably transfected with either vector or pcDNA3-F-menin. Cells were lysed in high-salt lysis buffer by Dounce homogenization, and samples were diluted with lysis buffer without NaCl, yielding a final concentration of 150 mmol/L NaCl. The diluted samples were centrifuged at 105 x g to remove insoluble debris, and the supernatant was incubated with equilibrated agarose beads coupled with an anti-Flag antibody (M2; Sigma, St. Louis, MO) at 4°C for 2 hours. The beads were thoroughly washed with lysis buffer containing 200 mmol/L NaCl and then incubated at 4°C for 20 minutes with elution buffer containing 0.5 mg/mL Flag peptide. The purified proteins from the control cells and the cells expressing F-menin were separated by SDS-PAGE, stained with a Colloidal Blue Staining kit (Invitrogen, Carlsbad, CA), and excised under a sterile hood. The peptide sequences from these proteins were identified using mass spectrometry at the Proteomics Core Facility at the University of Pennsylvania.
Cell Culture.
Human embryonic kidney 293 and 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, penicillin (100 units/mL), and streptomycin (100 µg/mL; ref. 23
). The cells were transfected using the calcium phosphate precipitation method as described previously (21)
. Menin-null and menin-complemented murine embryonic fibroblasts, isolated and immortalized as described previously (17)
, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1x nonessential amino acid mixtures, L-glutamine, and antibiotics (MEF medium).
Immunoprecipitation, Glutathione S-Transferase–Pulldown, and Immunoblotting Analysis.
Transfected 293 cells were lysed with cell lysis buffer [50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 100 mmol/L NaCl, 1 mmol/L EDTA and EGTA, 0.4% NP40, 20 mmol/L ß-glycerophosphate, and 2 x Protease Inhibitor Mixture Complete (Boehringer Mannheim)] as described previously (24)
. Lysed cells were sonicated (Sonic Dismembranator; Fisher Scientific, Pittsburgh, PA) for 20 s on ice and centrifuged at 1.3 x 104 rpm in a microcentrifuge (Eppendorf, Hamburg, Germany) for 15 minutes. The resulting lysates were either incubated with the M2 beads conjugated to an anti-Flag antibody (Sigma) or GammaBind Plus Sepharose beads (Amersham Biosciences) and the indicated primary antibodies, with addition of 10% (v/v) glycerol to the lysates, and rotated at 30 rpm at 4°C for 2 hours. The beads were collected by brief centrifugation and washed three times with washing buffer (the lysis buffer supplemented with 10% glycerol). To detect the interaction between GST-menin and ASK, cell lysates from 293T cells transfected with pF-ASK were incubated with glutathione beads coupled with either control GST or various GST-menin fusion proteins, in the presence of 10% glycerol (v/v) for 2 hours. The beads were then extensively washed with washing buffer, and proteins on the beads were separated by SDS-PAGE. Immunoblotting analysis using the M2 antibody was performed to detect the association with ASK.
To detect menin, menin mutant, and ASK protein expression in retrovirally transduced menin-null cells, whole-cell lysates were prepared with whole-cell lysis buffer [50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 0.4% Triton X-100, 0.1% NP40, 150 mmol/L NaCl, 10 mmol/L MgCl2, 0.5 mmol/L EDTA, 2.5 mmol/L EGTA, 0.2 mmol/L Na3VO4, 1 mmol/L NaF, 10 mmol/L ß-glycerophosphate, 1 mmol/L dithiothreitol, 0.2 mmol/L phenylmethylsulfonyl fluoride, 4 µg/mL leupeptin, aprotenin, and pepstatin A]. The primary antibody against a human menin peptide was raised in rabbits and affinity purified with the corresponding peptide-conjugated agarose beads (17) . Endogenous ASK was detected by an affinity purified rabbit antihuman ASK antibody (19) . An antiactin antibody was used as a loading control (Santa Cruz Biotechnology, Santa Cruz, CA). The enhanced chemiluminescence system (Amersham Biosciences) was used, as described previously (17) .
Production of Glutathione S-Transferase–Menin Proteins.
Exponentially growing DH5
Escherichia coli cells containing various GST-menin constructs were induced with 150 µmol/L isopropyl-1-thio-ß-D-galactopyranoside at room temperature. Bacteria were collected and lysed at 4°C in 1x PBS buffer with protease inhibitors and Triton X-100 (1%), followed by sonication. The supernatant from the lysates was incubated with Glutathione Sepharose 4B beads (Amersham Biosciences), and beads were washed extensively with 1x PBS buffer with protease inhibitors.
[3H]Thymidine Incorporation Assays.
Three thousand cells in 200 µL of MEF medium were seeded per well of a 96-well plate on day 0. On day 3, [3H]thymidine (ICN Biomedicals, Inc., Irvine, CA) was added to the medium. Cells were incubated with [3H]thymidine for 4 hours before being trypsinized and harvested. Incorporation of [3H]thymidine was determined using a model 1450 Microbeta Trillux Scintillation Counter (Wallac, Boston, MA) as described previously (25)
. All samples were analyzed in triplicate.
Fluorescence-Activated Cell Sorting Analysis.
On day 0, cells were seeded at a density of 2 x 105 cells/100-mm dish. On day 2, cells were pulsed for 45 minutes with 10 µmol/L 5'-bromo-2'-deoxyuridine-5'-triphosphate (BrDU; Sigma). After harvesting, 10 (6)
cells per sample were fixed in 70% ethanol while vortexing. Cell pellets were washed in washing buffer (PBS plus 0.5% bovine serum albumin), pelleted, and denatured in denaturing solution (3 N HCl and 0.5% Tween-20) for 10 minutes. After pelleting, cells were then incubated with anti-BrDU antibody (PharMingen, San Jose, CA) for 20 minutes, followed by washing and pelleting. Next, fluorescein isothiocyanate-conjugated goat antimouse IgG (PharMingen) antibody was added for 15 minutes. After another washing and pelleting, cells were resuspended in 0.5 mL of propidium iodide (10 µg/mL in PBS; Sigma) and incubated for 30 minutes. Samples were analyzed on a FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ).
In vitro Kinase Assays.
In vitro phosphorylation assays of human Cdc7-ASK kinase were conducted as described previously (24)
.
Statistical Analysis and Quantitation.
SEM bars are calculated using Microsoft Excel. The Student’s t test was used to determine significance of results.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, bottom panel). Fig. 1A
shows that the menin-null cells incorporate 2.5-fold more [3H]thymidine than do the menin-expressing cells (P < 0.0008). This provides genetic evidence for a crucial role of menin in regulating cell proliferation.
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, bottom panel). Fig. 1B
shows that complementation of menin-null cells with wild-type menin lowers the rate of cell proliferation by approximately 2-fold (top panel, P < 0.03). The kinetics of cell proliferation for menin-null cells and menin-complemented cells were also monitored (Fig. 1C)
. The number of menin-null cells is significantly higher than that of the menin-complemented cells, on days 3 and 4 of culture (P < 0.0007 and 0.02, respectively). Collectively, these results reinforce the crucial role of menin in repression of cell proliferation.
Menin Associates with Activator of S-Phase Kinase, the Regulatory Subunit of Cdc7 Kinase.
To explore the mechanism whereby menin inhibits cell proliferation, we attempted to identify menin-interacting proteins, which may shed light on menin-mediated repression of cell proliferation. Cell lysates from control 293 cells and 293 cells expressing Flag epitope-tagged-menin (F-menin) were passed through agarose beads conjugated with an anti-Flag antibody (M2 beads). Menin and menin-interacting proteins were retained on the beads, separated by SDS-PAGE, and stained with Colloid Coomassie Blue. A protein band immediately above menin was observed in F-menin-expressing cells but not in control cells. This band was excised from the gel (Fig. 2A
, Lane 2) and subjected to sequence analysis by mass spectrometry. Peptide sequences from the excised band matched the sequences from human ASK. ASK, a nuclear protein, is an obligatory component of the active Cdc7/ASK kinase, which is essential for cell proliferation in eukaryotic cells (18)
. Additionally, to identify novel menin-interacting proteins, we performed a yeast-two hybrid screen with an E17 murine embryonic library (BD Bioscience, San Jose, CA) and full-length menin. Two clones expressing ASK were identified during the screening, further supporting the interaction between menin and ASK (data not shown).3
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, top panel, Lane 3). As a control, immunoblotting confirmed the expression of ASK and menin (Fig. 2B
, bottom panels). To demonstrate that menin interacts with endogenous ASK, cell lysates from cells stably expressing the epitope-tagged menin were incubated with the M2 beads, and the immunoprecipitated proteins were subjected to immunoblotting analysis. Fig. 2C
shows that endogenous ASK is immunoprecipitated by menin, indicating that menin interacts with endogenous ASK (top panel, Lane 2). The expression of ASK and menin was confirmed by immunoblotting (Fig. 2C
, bottom panels).
Activator of S-Phase Kinase Associates with the COOH Terminus of Menin.
To determine which domain of menin interacts with ASK, we expressed and purified GST fusion proteins containing either the NH2 terminus (F1), the middle region (F2), or the COOH terminus (F3) of menin (Fig. 3A)
. Expression of the various fusion proteins was confirmed by Coomassie Blue staining (Fig. 3B)
. Control GST or various GST-menin fusion proteins were incubated with cell lysates containing the epitope tagged-ASK, and the interaction of ASK with the fusion proteins was detected by immunoblotting analysis. Fig. 3C
(Lane 4) shows that ASK specifically interacts with the COOH terminus (F3), spanning amino acid residues 396 to 610, but does not interact with the control GST or other regions of menin (Lanes 1–3).
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). Expression of the wild-type and mutant fusion proteins was confirmed by Coomassie Blue staining (Fig. 4B)
. The ability of menin and menin truncation mutants to bind ASK was examined as described above. Fig. 4C
shows that the wild-type COOH terminus of menin interacts with ASK (Lane 2); however, neither 527X nor 558X interacts with ASK (Lanes 3 and 4). Therefore, two independent disease-related mutations in menin compromise the interaction between menin and ASK.
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shows that MCM2 is vigorously phosphorylated by Cdc-7-ASK, as are MCM4 and MCM6 (top panel, Lane 2). However, addition of increasing amounts of menin does not affect the ability of Cdc-7-ASK to phosphorylate MCM2 (Fig. 4D
, top panel, Lanes 3–8). Menin is weakly phosphorylated by Cdc7-ASK, but the significance of this is unknown. Fig. 4D
(bottom panel) shows silver staining for proteins used for the in vitro kinase assay. It is possible that menin may affect the interaction of ASK with other important, but unknown, substrates. Alternatively, the interaction between menin and ASK may affect additional functions of ASK other than its Cdc7-associated activity.
Menin Suppresses Activator of S-Phase Kinase-Mediated Stimulation of Cell Proliferation.
Because menin and ASK interact specifically, we sought to determine whether menin interferes with the function of ASK in cell proliferation. We infected menin-null cells with control retroviruses or retroviruses expressing ASK and menin, separately or in various combinations. Fig. 5A
shows that expression of ASK leads to a modest yet consistent increase in the proliferation of menin-null cells, whereas expression of menin inhibits cell proliferation. Interestingly, coexpression of ASK and menin suppresses ASK-mediated stimulation of cell growth (Fig. 5A)
. The levels of expression for both menin and ASK are shown in Fig. 5B
. Collectively, these results indicate that menin can suppress ASK-mediated stimulation of cell proliferation.
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. Consistent with the observation in Fig. 5A
, ASK induces cell proliferation, and menin represses ASK-induced proliferation (Fig. 6A)
. Notably, the COOH-terminal menin mutants, 527X and 558X, fail to repress the ASK-mediated induction of cell proliferation. ASK and menin were all expressed as detected by Western blotting analysis (Fig. 6B)
. Together, these results indicate that loss of the ASK-interacting domain in the COOH terminus of menin deprives menin of the ability to repress ASK-induced cell proliferation. They suggest that menin may repress cell proliferation, at least in part, through interaction with ASK at its COOH terminus. Loss of function of the MEN1-derived menin mutants suggests a potential role for the menin-ASK interaction in regulating the development of MEN1.
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. Under these conditions, we failed to observe a significant difference among the various cell lines (Fig. 6C)
. It is possible that menin does decrease cell proliferation by slightly altering the cell cycle but that this assay is not sensitive enough to detect such differences. Alternatively, it is also possible that ASK and menin affect one or more phases of the cell cycle, which makes detection of an obvious difference in cell cycle more difficult. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These results provide genetic evidence for the critical role of menin in regulating cell proliferation. They suggest that one way in which menin suppresses tumorigenesis may be its inhibition of cell proliferation.
The current studies demonstrate a physical interaction between menin and ASK (Fig. 2)
, and suggest that menin and ASK cooperate in the regulation of cell proliferation. Whereas the NH2 terminus and middle region of menin mediate the interaction with proteins including JunD, nuclear factor-B, Pem, RPA2, FancD2, and mSin3A (13
, 14
, 16
, 17
, 26
, 27)
, we show that the COOH terminus of menin mediates the specific interaction with ASK (Fig. 3)
. The fact that various domains of menin interact with different proteins may partly explain how menin regulates diverse cellular functions, including gene transcription, genome stability, and cell proliferation.
Importantly, two disease-related COOH-terminal mutations, 527X and 558X, lose the ability to interact with ASK (Fig. 4)
, suggesting a potentially critical role for the interaction between ASK and menin in repressing MEN1 development. The specific interaction between menin and ASK provides a potential mechanistic link between menin and the regulation of cell proliferation. ASK is essential for cell proliferation, and a neutralized antibody against ASK blocks cell proliferation after microinjection into cells (19)
. Consistent with this observation, the expression of ASK alone leads to enhanced proliferation of menin-null cells, whereas coexpression of menin and ASK inhibits proliferation (Fig. 5)
. In other words, the role of ASK in inducing cell proliferation depends on the status of menin in cells; ASK induces cell proliferation in the absence of menin, whereas such a role is inactivated in the presence of menin. In this regard, it is interesting to note that JunD, a transcription factor that interacts with menin, inhibits cell proliferation in the presence of menin but promotes cell growth in the absence of menin (28)
. It is not yet clear whether JunD and ASK functionally interact. Collectively, our studies suggest that menin interacts with ASK and thus represses the role of ASK in stimulating cell proliferation.
If the interaction between menin and ASK is crucial for the ability of menin to repress ASK-mediated cell growth, then mutants of menin that do not bind ASK should fail to inhibit ASK-induced cell proliferation. In fact, two disease-related mutations that cannot bind ASK do fail to inhibit ASK-induced cell proliferation (Fig. 6)
. Thus, the COOH terminus of menin, through its interaction with ASK, may play a crucial role in regulating cell proliferation. In agreement with this notion, approximately 63% of reported germ line and sporadic mutations are truncations that eventually lead to removal of the COOH terminus (1)
. These results strongly suggest that the menin-ASK interaction is functionally important for the role of menin in regulating cell proliferation. However, these results do not rule out that additional pathways are also involved in menin-mediated regulation of cell proliferation.
The exact mechanism by which menin and ASK work together to modulate cell proliferation requires additional study. In vitro biochemical assays show that ASK is essential for Cdc7 protein kinase activity (24) . Furthermore, yeast Cdc7 and Dbf4, a homologue of ASK, are crucial for DNA replication (18) . These findings suggest that ASK and menin might influence proliferation via modulation of the cell cycle. Although infection of menin-null cells with ASK resulted in a moderate, but reproducible, increase in cell number, which was completely suppressed by menin, but not by the two COOH-terminal mutants, no obvious differences in cell cycle profiles were seen between the menin-null cells and the cells ectopically expressing ASK. It is possible that PI/BrDU staining is not sensitive enough to detect these differences in our system. Alternatively, it is also possible that ASK and menin affect one or more phases of the cell cycle, which makes detection of an obvious difference in cell cycle more difficult. Moreover, it remains unclear whether the major role of ASK in mammalian cells is the regulation of cell cycle progression, because overexpression of both Cdc7/ASK fails to alter the cell cycle profile (29) . Recent data show that loss of mammalian Cdc7 function leads to rapid arrest of replication forks, followed by G2-M arrest and eventual apoptosis (30) . Thus, in mammalian cells, the ASK/Cdc7 complex may not solely influence proliferation of cells by regulating DNA replication. No matter what the detailed mechanisms are, the current findings have provided a link between menin and ASK in the regulation of cell proliferation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Requests for reprints: Xianxin Hua, Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104-6160. Phone: 215-746-5565; Fax: 215-746-5525; E-mail: huax{at}mail.med.upenn.edu
Received 2/27/04. Revised 7/ 2/04. Accepted 7/12/04.
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