[Cancer Research 62, 5668-5671, October 15, 2002]
© 2002 American Association for Cancer Research
A Novel Mechanism of Nuclear Factor 
B Activation through the Binding between Inhibitor of Nuclear Factor-B
and the Processed NH2-terminal Region of Mig-61
Toshiyuki Tsunoda,
Junichi Inokuchi,
Iwai Baba,
Koji Okumura,
Seiji Naito,
Takehiko Sasazuki and
Senji Shirasawa2
Department of Pathology [T. T., J. I., I. B., S. S.] and Director-General [T. S.], Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan; and Department of Urology, Graduate School of Medical Sciences, Kyushu University [T. T., J. I., K. O., S. N.], Fukuoka 812-8582, Japan
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ABSTRACT
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Mitogene-inducible gene-6 (Mig-6), an adaptor molecule containing the Cdc42/Rac interaction and binding (CRIB) domain, is rapidly induced by mitogenic and stressful stimuli, and sustained mig-6 expression is observed in chronic pathological conditions. The function of this molecule has remained elusive. We find that mig-6 is constitutively expressed in many human cancer cell lines, and Mig-6 is cleaved into the NH2-terminal region containing the CRIB domain and the remainder of the COOH-terminal region by limited proteolytic processing. We report here that full-length Mig-6, but not CRIB domain-deleted Mig-6 (
Mig-6) or uncleavable mutant of Mig-6 (Mig-6-S38A), induces transcriptional activation of nuclear factor of 
B (NFB), which is inhibited by inhibitor of B
(IB), and that the processed NH2-terminal region of Mig-6 but not the full length is bound with IB through its NFB binding region. These findings suggest that the processed CRIB domain of Mig-6 will compete with NFB for IB and result in NFB activation. This novel NFB activation pathway provides new insights regarding tumorigenesis, and the specific inhibition of the cleavage of Mig-6 may be a target for clinical treatment.
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Introduction
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Mig-63
is an adaptor molecule containing CRIB (3)
domain, src homology 3 binding domain, and 14-3-3 binding domain (1, 2, 3)
, and deregulated mig-6 expression is observed in chronic pathological conditions (3)
. However, the function of Mig-6 has remained elusive. Gene 33, rat homologue of mig-6, expression is rapidly induced by insulin, cortisol, cyclic AMP, calcium ionophores, vasoactive peptide, and mechanical strain (3, 4, 5)
. Although mig-6/Gene 33 is one of the immediate early response genes, it is characterized by its sustained expression among them (3)
. This sustained mig-6 expression is thought to trigger cells to initiate hypertrophy in chronic pathological conditions such as diabetes and hypertension (3
, 6
, 7)
, through stress-activated protein kinase/c-Jun NH2-terminal kinase activation by binding between its CRIB domain and Cdc42 (3)
. Here, we have found that mig-6 expression is induced by activated Ki-ras in human colon cancer cells, and mig-6 is constitutively expressed in many human cancer cells. Furthermore, we found that Mig-6 induces transcriptional activation of NFB. NFB is a transcription factor that is related to the immune responses, cell proliferation, apoptosis, and cell migration, and it plays critical roles in human diseases, including inflammatory diseases and cancer. NFB resides in the cytoplasm as an inactive complex with IB in unstimulated cells, whereas the canonical NFB activation pathway is dependent on IB phosphorylation, resulting in ubiquitin-dependent degradation of IB by 26S proteasome and subsequent NFB activation (8, 9, 10, 11)
. Here, we obtained evidence that Mig-6 induces transcriptional activation of NFB through a novel mechanism of the binding between IB and the processed NH2-terminal region of Mig-6.
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Materials and Methods
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Plasmids and Antibodies.
COOH-terminal HA-tagged Mig-6 cDNA was subcloned into pSI vector (Promega, Madison, WI), and Mig-6 and Mig-6 (67–462a.a.) cDNA were subcloned into pCMV-HA expression vector (Clontech, Palo Alto, CA). pCMV-HAMig-6-S38A was mutated at the 38th amino acid residue from serine to alanine using a mutagenesis kit (Stratagene, La Jolla, CA). pNFB-luc, pAP1-luc, and p53-luc were from Stratagene. phRL-CMV, expressing Renilla luciferase, was from Promega. pCMV-IB and pCMV-IBM were from Clontech. IB1–3 sequences were subcloned into a pCMV-Myc expression vector (Clontech). Anti-HA antibody (3F10) was from Roche (Mannheim, Germany). Anti-Myc antibody (9E10) and anti-IB antibody (sc371) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti phospho-IB (5A5) antibody was from Cell Signaling Technology (Beverly, MA).
Transfections, Coimmunoprecipitation, and Immunoblottings.
293T cells were transiently transfected using polyfect (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. Total cell lysates were extracted 24 h after transfection then subjected to immunoblot analysis using Western blots (12)
. Proteins were also extracted using a radioimmunoprecipitation assay buffer then immunoprecipitated with anti-HA, anti-myc, or anti-IB antibodies. The immunoprecipitates were subjected to immunoblot analysis.
Determination of Amino Acid Sequences.
293T cells were transiently transfected with pSI-Mig-6HA (8 µg), and the cell extracts immunoprecipitated with anti-HA antibody were fractionated on an 8% polyacrylamide gel and stained with Coomassie Brilliant Blue. The processed COOH-terminal fragment of Mig-6 was cut out, and its NH2-terminal sequence was determined using the G1005A Protein Sequencing System (Hewlett Packard, Palo Alto, CA).
Dual Luciferase Assays.
NIH3T3 cells were transiently transfected in a 6-cm diameter dish and harvested 24 h after transfection. Luciferase assays were carried out using the Dual Luciferase kit (Promega). Firefly luciferase reporter constructs (500 ng of pNF-B-luc) were transfected together with 2 ng of the Renilla luciferase reporter plasmid phRL-CMV as an internal control. Cells were lysed in 750 µl of passive lysis buffer, and 20 µl of lysate was assayed for firefly and Renilla activity according to the manufacturer’s instructions. Transfection efficiencies were corrected through normalization of the firefly activity to that obtained from the Renilla. All of the experiments done in triplicate were carried out at least three times, and the results were averaged. Results are shown as fold activation considering activity of the transfection with empty vector as 1. For statistical analysis, we used Student’s t test (13)
.
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Results and Discussion
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We earlier identified the mig-6 gene to be one of the activated Ki-ras-induced transcripts, determined using a cDNA subtraction library between human colon cancer HCT116 cells and its activated Ki-ras-disrupted HKe3 cells (14)
. mig-6 expression was strongly observed in HKe3 transfectants expressing activated Ki-ras (Fig. 1a)
, thereby indicating that mig-6 expression is induced by activated Ki-ras-mediated signals in HCT116 cells. Northern blots revealed that mig-6 expression was rarely evident in the normal colon epithelium, whereas it was strongly expressed in several kinds of human colon cancer cells (Fig. 1b)
. Constitutive mig-6 expression was also observed in other cancer cells including urinary bladder, prostate, kidney, lung, breast, pancreas, and liver (Fig. 1c)
. Sustained mig-6 expression is thought to trigger cells to initiate hypertrophy in chronic pathological conditions such as diabetes and hypertension (3
, 6
, 7)
. Taken together, the findings suggested that mig-6 expression is probably associated with chronic diseases and also with cancer.
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Fig. 1. Constitutive mig-6 expression in human cancer cell lines. a, 20 µg of total RNA of HCT116, HKe3, and HKe3-derived transfectants expressing activated Ki-Ras (e3-MKRas#9 and e3-MKRas#14) at exponential-growth (Ex) and serum-starved (St) conditions were run in an agarose-formaldehyde gel, then Northern blots were prepared using mig-6 (top panel) and ß-actin (bottom panel) as probes as described (20)
. b and c, 20 µg of total RNA of each cancer cell line were run in an agarose-formaldehyde gel, and Northern blots were done using mig-6 (top panel) and ß-actin (bottom panel) as probes.
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Mig-6HA, HA-tagged Mig-6 in its COOH terminus, was detected as two bands of Mr 
57,000 and 50,000, respectively, with use of an anti-HA antibody (Fig. 2a)
. To determine whether these two translational products were produced by limited proteolytic processing of Mig-6, HAMig-6, HA-tagged Mig-6 in its NH2 terminus, was transiently transfected in 293T cells. HAMig-6 was detected in the form of two bands of Mr 57,000 and 7,000, respectively (Fig. 2b)
. Thus, the full length of Mig-6 was cleaved into the NH2-terminal region containing the CRIB domain and the remainder of the COOH-terminal region. To examine the cleavage site of Mig-6, we determined the NH2-terminal amino acid sequence of the processed COOH-terminal fragment of Mig-6. We found that Mig-6 is processed at the position between Ser 38 and Glu 39. As a mutant form of Mig-6, a single amino acid substitution of Ala for Ser at the 38th position of Mig-6 (Mig-6-S38A) was not processed in 293T cells (Fig. 2b)
. Specific mechanisms are probably involved in the limited proteolytic processing of Mig-6.
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Fig. 2. Limited proteolytic processing of Mig-6. a, 293T cells were transfected with pSI-Mig-6HA, then whole cell lysates were fractionated on an 8% polyacrylamide gel. b, 293T cells were transfected with pCMV-HAMig-6 and HAMig-6-S38A, then whole cell lysates were fractionated on a 420% polyacrylamide gel. For Western blotting we used an anti-HA antibody. Arrow, Mig-6 and Mig-6-S38A; white arrow, the processed COOH-terminal fragment of Mig-6; and arrowhead, the processed NH2-terminal fragment of Mig-6.
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To elucidate functional roles of Mig-6, luciferase-reporter assays for activation of several transcriptional factors, including AP1, NFB, and p53, were done. In NIH3T3 cells, Mig-6 specifically induced transcriptional activity of NFB by 18-fold compared with that of empty vector, whereas Mig-6 induced neither activator protein-1 nor p53 transcriptional activation (Fig. 3a)
. This Mig-6-induced NFB activation was in a dose-dependent fashion (Fig. 3b)
, and the Mig-6-induced NFB activation was similarly observed in 293T, COS7, HeLa, HepG2, and HKe3 cells (data not shown). Mig-6 (CRIB domain-deleted Mig-6) or Mig-6-S38A induced no NFB activation in NIH3T3 cells (Fig. 3b)
, whereas Mig-6-induced NFB activation in NIH3T3 cells was partially and completely blocked by IB and super-repressor IBM (15)
, respectively (Fig. 3c)
. Cumulatively, these findings suggest that: (a) the NH2-terminal region containing the CRIB domain of Mig-6 is required for NFB activation; (b) ser residue at the 38th amino acid of Mig-6 or the processed NH2-terminal region of Mig-6 will be critical for NFB activation; and (c) Mig-6-induced NFB activation is mediated by the IB-dependent pathway. However, phosphorylation of IB was not induced in cells transfected with Mig-6, compared with that of the cells treated with tumor necrosis factor (Fig. 3d)
, indicating that Mig-6-induced NFB activation may be independent of phosphorylation of IB and the subsequent degradation of IB (8
, 11
, 15)
.
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Fig. 3. NFB activation through the processed NH2-terminal region of Mig-6. a, NIH3T3 cells were transfected with pNF-B-luc, p53-luc, and pAP-1-luc plasmid (500 ng) together with an empty-vector or pCMV-HAMig-6 (2 µg). Firefly luciferase activity was normalized to Renilla luciferase activity as an internal control. The relative luciferase activity with pCMV-HAMig-6, as compared with the one with the empty vector, is taken as fold activation. * showed significant differences for empty vector established as P < 0.05. Expression of HAMig-6 was visualized in Western blots using an anti-HA antibody (arrow). b, NIH-3T3 cells were transfected with pNF-B-luc plasmid (500 ng) together with an empty vector, pCMV-HAMig-6, HAMig-6, or HAMig-6-S38A. The relative luciferase activity was determined as described in a. Expression of HAMig-6 (black arrow), HAMig-6 (white arrow), and HAMig-6-S38A (black arrow) was visualized in Western blots using an anti-HA antibody. * showed significant differences for empty vector established as P < 0.05. c, NIH3T3 cells were transfected with the pNFB-luc plasmid (500 ng) together with an empty vector or with pCMVHA-Mig-6 (1.75 µg), with or without pCMV-IB and IBM (0.25 µg). * and ** showed significant differences for Mig-6 and Mig6 with IB, Mig-6 with IB, and Mig-6 with IBM, respectively, established as P < 0.05. d, NIH3T3 cells were transfected with an empty vector or with pCMV-HAMig-6 (4 µg) and human recombinant tumor necrosis factor (20 ng/ml; Genzyme-Techne, Minneapolis, MN) was added for 5 min after 24 h after transfection, then whole cell lysates were fractionated on a 10% polyacrylamide gel. Western blotting was done using anti phospho-IB antibody; bars, ±SD.
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Of considerable interest is the similarity of amino acid sequences between the CRIB domain binding region of Cdc42 (residues 166–186; Ref. 16
) and the NFB binding region of IB (residues 123–143; Ref. 17
; Fig. 4a
). This binding site forms an -helical secondary structure, and Glu138 of IB is one of the amino acids interacting with NFB (17)
. On the basis of these similarities, we hypothesized that Mig-6, which is located in the cytoplasm (18)
, may interact with IB through its NFB binding region, the result being translocation of NFB into nucleus. To examine the binding between Mig-6 and IB, 293T cells were transiently transfected with a single construct or with various combinations of HAMig-6, HAMig-6, and IB. Equal amounts of each total cell lysate were immunoprecipitated with an anti-HA antibody and an anti-IB antibody, then blotted with an anti-HA antibody. Surprisingly, not the full length but the Mr 7,000-processed NH2-terminal region of Mig-6 was coimmunoprecipitated with IB (Fig. 4b)
. This means that only the processed NH2-terminal fragment containing the CRIB domain of Mig-6 can be associated with IB. Next, to confirm the binding site of IB with Mig-6, three kinds of IB deletion constructs (IB1–3) were used (Fig. 4c)
. 293T cells were cotransfected with HAMig-6 and MycIB1, MycIB2, or MycIB3, then total cell lysates was coimmunoprecipitated with an anti-myc antibody then immunoblotted with an anti-HA antibody, showing that only MycIB1 can interact with Mig-6 (Fig. 4d)
. Furthermore, Mig-6 can interact with the super-repressor of IBM (data not shown), indicating that the phosphorylation of IB at Ser32 and Ser36 is not required for this interaction. These results, taken together, suggest that the binding site of IB with Mig-6 will be the NFB binding region, and that the processed NH2-terminal region containing the CRIB domain of Mig-6 will compete with NFB for IB and consequently allow NFB to translocate into the nucleus, and to culminate in NFB activation.
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Fig. 4. Mig-6-induced NFB activation through binding between the processed NH2-terminal region of Mig-6 and the NFB binding region of IB. a, amino acid homology between the CRIB domain binding region of Cdc42 and the NFB binding region of IB. Gray and white box shows identities and conservative changes, respectively. Cylinders represent helices. The arrowhead shows amino acid residues that make contact with the p50 dimerization domain (16)
. b, 293T cells were cotransfected with pCMV-HA, pCMV-HAMig-6, or pCMV-HAMig-6 (7 µg), and pCMV-IB (1 µg) with 8 µg of the total amount of plasmids. Cells were harvested 24 h after transfection, and cell extracts were immunoprecipitated with an anti-HA or an anti-IB antibody then fractionated on a 420% polyacrylamide gel. Western blotting was done using an anti-HA antibody. c, deletion construct of IB. d, 293T cells were cotransfected with pCMV-HAMig-6 (7 µg) and pCMV-MycIB1, 2, or 3 (1 µg) with 8 µg of the total amount of plasmids. Cells were harvested 24 h after transfection, and cell extracts were immunoprecipitated with anti-Myc antibody and then fractionated on a 420% polyacrylamide gel. Western blotting was done using anti-Myc and anti-HA antibodies.
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Here we demonstrate that constitutive mig-6 expression, which is thought to be associated with chronic pathological conditions (3)
, will be involved in NFB activation through a novel mechanism of competition for IB with NFB. NFB plays critical roles in human diseases, including arthritis, asthma, autoimmunity (9
, 10) , and tumors (11
, 19)
, and modulation of NFB activity may provide effective therapeutic strategies. Our findings indicate that one of the most critical roles of Mig-6 will be NFB activation, in which limited proteolytic processing is additionally needed for NFB activation. Although the precise mechanism of the limited processing of Mig-6 has yet to be determined, specific inhibitors of the cleavage of Mig-6 may provide selective and potent drugs to treat subjects with chronic inflammatory diseases and tumors with constitutive expression of mig-6 through the inhibition of the NFB activity.
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ACKNOWLEDGMENTS
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We thank Mariko Ohara (Fukuoka) for language assistance.
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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.
1 Supported in part by a Grant-in-Aid for Scientific Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Phone: 81-3-3202-7181, extension 2833; Fax: 81-3-3202-7364; E-mail: sshirasawa{at}ri.imcj.go.jp
3 The abbreviations used are: Mig-6, mitogene-inducible gene-6; NFB, nuclear factor of B; CRIB, Cdc42/Rac interaction and binding; IB, inhibitor of B; HA, hemagglutinin A; CMV, cytomegalovirus.
Received 6/24/02.
Accepted 9/ 3/02.
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REFERENCES
|
|---|
- Burbelo P. D., Drechsel D., Hall A. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J. Biol. Chem., 270: 29071-29074, 1995.[Abstract/Free Full Text]
- Pirone D. M., Carter D. E., Burbelo P. D. Evolutionary expansion of CRIB-containing Cdc42 effector proteins. Trends Genet., 17: 370-373, 2001.[Medline]
- Makkinje A., Quinn D. A., Chen A., Cadilla C. L., Force T., Bonventre J. V., Kyriakis J. M. Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. J. Biol. Chem., 275: 17838-17847, 2000.[Abstract/Free Full Text]
- Lee K-L., Isham K. R., Stringfellow L., Rothrock R., Kenney F. T. Molecular cloning of cDNAs cognate to genes sensitive to hormonal control in rat liver. J. Biol. Chem., 260: 16433-16438, 1985.[Abstract/Free Full Text]
- Weinstock R. S., Messina J. L. Vanadate and insulin stimulate gene 33 expression. Biochem. Biophys. Res. Commun., 189: 931-937, 1992.[Medline]
- Wolf G., Ziyadeh F. N. Molecular mechanisms of diabetic renal hypertrophy. Kidney Int., 56: 393-405, 1999.[Medline]
- Mahgoub M. A., Abd-Elfattah A. S. Diabetes mellitus and cardiac function. Mol. Cell. Biochem., 180: 59-64, 1998.[Medline]
- Baldwin A. S. The NF-B and IB proteins: new discoveries and insights. Annu. Rev. Immunol., 14: 649-683, 1996.[Medline]
- Barnes P. J., Karin M. Nuclear factor- B: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med., 336: 1066-1071, 1997.[Free Full Text]
- Makarov S. S. NF-B as a therapeutic target in chronic inflammation: recent advances. Mol. Med. Today, 6: 441-448, 2000.[Medline]
- Karin M., Cao Y., Greten F. R., Li Z. W. NF-B in cancer: from innocent bystander to major culprit. Nat. Rev. Cancer, 2: 301-310, 2002.[Medline]
- Okumura K., Shirasawa S., Nishioka M., Sasazuki T. Activated Ki-Ras suppresses 12-O-tetradecanoylphorbol-13-acetate-induced activation of the c-Jun NH2-terminal kinase pathway in human colon cancer cells. Cancer Res., 59: 2445-2450, 1999.[Abstract/Free Full Text]
- Cammarano M. S., Minden A. Dbl and the Rho GTPases activate NFB by IKK-dependent and IKK-independent pathways. J. Biol. Chem., 276: 25876-25882, 2001.[Abstract/Free Full Text]
- Shirasawa S., Furuse M., Yokoyama N., Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science (Wash. DC), 260: 85-88, 1993.[Abstract/Free Full Text]
- Brown K., Gerstberger S., Carlson L., Franzoso G., Siebenlist U. Control of IB- proteolysis by site-specific, signal-induced phosphorylation. Science (Wash. DC), 267: 1485-1491, 1995.[Abstract/Free Full Text]
- Mott H. R., Owen D., Nietlispach D., Lowe P. N., Manser E., Lim L., Laue E. D. Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK. Nature (Lond.), 399: 384-388, 1999.[Medline]
- Huxford T., Huang D. B., Malek S., Ghosh G. The crystal structure of the IB/NF-B complex reveals mechanisms of NF-B inactivation. Cell, 95: 759-770, 1998.[Medline]
- Wick M., Bürger C., Funk M., Müller R. Identification of a novel mitogen-inducible gene (mig-6): Regulation during G1 progression and differentiation. Exp. Cell Res., 219: 527-535, 1995.[Medline]
- Rayet B., Gelinas C. Aberrant rel/nfkb genes and activity in human cancer. Oncogene, 18: 6938-6947, 1999.[Medline]
- Baba I., Shirasawa S., Iwamoto R., Okumura K., Tsunoda T., Nishioka M., Fukuyama K., Yamamoto K., Mekada E., Sasazuki T. Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells. Cancer Res., 60: 6886-6889, 2000.[Abstract/Free Full Text]
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ABSTRACT
Introduction
