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RING finger-dependent ubiquitination by PRAJA is dependent on TGF-β and potentially defines the functional status of the tumor suppressor ELF

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Abstract

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-β) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-β signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-β-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P<0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Δ-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Δ-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-β signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t1/2) and rate constant for degradation (kD) of ELF is 1.91 h and 21.72 min−1 when coupled with ectopic expression of PRAJA in cells stimulated by TGF-β, compared to PRAJA-transfected unstimulated cells (t1/2=4.33 h and kD=9.6 min−1). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.

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Abbreviations

RING:

Really Interesting New Gene

TGF-β:

transforming growth factor-beta

ELF:

embryonic liver fodrin

SARA:

Smad anchor for receptor activation

ORF:

open-reading frame

HECT:

homologous to E6-AP C-terminus

HEF1:

human enhancer of filamentation 1

APC:

anaphase-promoting complex

IκB:

inhibitor of nuclear factor κB

PML:

promyelocytic leukemia protein

RFP:

ret finger protein

IAP:

inhibitors of apoptosis

PH:

partial hepatectomy

PBS:

phosphate-buffered saline

BSA:

bovine serum albumin

ATCC:

American Type Culture Collection

HCC:

hepatocellular carcinoma

DMEM:

Dulbecco's modified Eagle's medium

FBS:

fetal bovine serum

IP:

immunoprecipitation

IB:

immunoblotting

FITC:

fluorescein isothiocyanate

SDS:

sodium dodecyl sulfate

ECL:

enhanced chemiluminescence

WB:

Western blotting

SDS–PAGE:

SDS–polyacrylamide gel electrophoresis

VA-1:

ELF-specific polyclonal antibody

HA:

hemagglutinin

R-Smad:

receptor-regulated Smad

PDB:

protein data bank

References

  • Alarcon-Vargas D, Tansey WP, Ronai Z . (2002). Oncogene 21: 4384–4391.

  • Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K . (2004). J Clin Invest 113: 253–264.

  • Bhanumathy CD, Tang Y, Monga SP, Katuri V, Cox JA, Mishra B et al. (2002). Dev Dyn 223: 59–69.

  • Bonni S, Wang HR, Causing CG, Kavsak P, Stroschein SL, Luo K et al. (2001). Nat Cell Biol 3: 587–595.

  • Braun L, Mead JE, Panzica M, Mikumo R, Bell GI, Fausto N . (1988). Proc Natl Acad Sci USA 85: 1539–1543.

  • Brzovic PS, Meza J, King MC, Klevit RE . (1998). J Biol Chem 273: 7795–7799.

  • Chatterjee-Kishore M, van Den Akker F, Stark GR . (2000). J Biol Chem 275: 20406–20411.

  • Chen D, Kon N, Li M, Zhang W, Qin J, Gu W . (2005). Cell 121: 1071–1083.

  • Derynck R, Zhang YE . (2003). Nature 425: 577–584.

  • Dinudom A, Harvey KF, Komwatana P, Young JA, Kumar S, Cook DI . (1998). Proc Natl Acad Sci USA 95: 7169–7173.

  • Dupont S, Zacchigna L, Cordenonsi M, Soligo S, Adorno M, Rugge M et al. (2005). Cell 121: 87–99.

  • Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T et al. (2001). J Biol Chem 276: 12477–12480.

  • Fukuchi M, Imamura T, Chiba T, Ebisawa T, Kawabata M, Tanaka K et al. (2001). Mol Biol Cell 12: 1431–1443.

  • Heldin C-H, Miyazono K, ten Dijke P . (1997). Nature 390: 465–471.

  • Hicke L, Dunn R . (2003). Annu Rev Cell Dev Biol 19: 141–172.

  • Honda R, Yasuda H . (1999). EMBO J 18: 22–27.

  • Itoh S, Thorikay M, Kowanetz M, Moustakas A, Itoh F, Heldin C-H et al. (2003). J Biol Chem 278: 3751–3761.

  • Izzi L, Attisano L . (2004). Oncogene 23: 2071–2078.

  • Janse DM, Crosas B, Finley D, Church GM . (2004). J Biol Chem 279: 21415–21420.

  • Joazeiro CA, Wing SS, Huang H, Leverson JD, Hunter T, Liu YC . (1999). Science 286: 309–312.

  • Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH et al. (2000). Mol, Cell 6: 1365–1375.

  • Koepp DM, Harper JW, Elledge SJ . (1999). Cell 97: 431–434.

  • Kretzschmar M, Massague J . (1998). Curr Opin Genet Dev 8: 103–111.

  • Laney JD, Hochstrasser M . (1999). Cell 97: 427–430.

  • Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W . (2003). Science 302: 1972–1975.

  • Liu X, Elia AE, Law SF, Golemis EA, Farley J, Wang T . (2000). EMBO J 19: 6759–6769.

  • Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM . (1999). Proc Natl Acad Sci USA 96: 11364–11369.

  • Markowitz SD, Roberts AB . (1996). Cytokine Growth Factor Rev 7: 93–102.

  • Mishra L, Cai T, Levine A, Weng D, Mezey E, Mishra B et al. (1998). Int J Dev Biol 42: 221–224.

  • Mishra L, Cai T, Yu P, Monga SP, Mishra B . (1999). Oncogene 18: 353–364.

  • Mishra L, Tully RE, Monga SP, Yu P, Cai T, Makalowski W et al. (1997). Oncogene 15: 2361–2368.

  • Miyazono K, Suzuki H, Imamura T . (2003). Cancer Sci 94: 230–244.

  • Moser MJ, Gong Y, Zhang MN, Johnston J, Lipschitz J, Minuk GY . (2001). Dig Dis Sci 46: 907–914.

  • Ohta T, Michel JJ, Schottelius AJ, Xiong Y . (1999). Mol Cell 3: 535–541.

  • Pan Y, Haines DS . (1999). Cancer Res 59: 2064–2067.

  • Roberts AB, Sporn MB . (1990). Peptide growth factors and their receptors. In: Handbook of Experimental Pathology, Sporn MB, Roberts AB (eds). Springer-Verlag: New York, pp. 419–472.

    Google Scholar 

  • Russell WE, Coffey Jr RJ, Ouellette AJ, Moses HL . (1988). Proc Natl Acad Sci USA 85: 5126–5130.

  • Sasaki A, Masuda Y, Iwai K, Ikeda K, Watanabe K . (2002). J Biol Chem 277: 22541–22546.

  • Saurin AJ, Borden KL, Boddy MN, Freemont PS . (1996). Trends Biochem Sci 21: 208–214.

  • Schwartz DC, Hochstrasser M . (2003). Trends Biochem Sci 28: 321–328.

  • Schwede T, Kopp J, Guex N, Peitsch MC . (2003). Nucleic Acids Res 31: 3381–3385.

  • Shi Y, Massague J . (2003). Cell 113: 685–700.

  • Siegel PM, Massague J . (2003). Nat Rev Cancer 3: 807–821.

  • Souchelnytskyi S, Moustakas A, Heldin C-H . (2002). Trends Cell Biol 12: 304–307.

  • Stroschein SL, Bonni S, Wrana JL, Luo K . (2001). Genes Dev 15: 2822–2836.

  • Sun Y . (2003). Cancer Biol Ther 2: 623–629.

  • Tang B, Vu M, Booker T, Santner SJ, Miller FR, Anver MR et al. (2003a). J Clin Invest 112: 1116–1124.

  • Tang Y, Katuri V, Dillner A, Mishra B, Deng CX, Mishra L . (2003b). Science 299: 574–577.

  • Tang Y, Katuri V, Iqbal S, Narayan T, Wang Z, Lu RS et al. (2002). Oncogene 21: 5255–5267.

  • Tang Y, Katuri V, Srinivasan R, Fogt F, Redman R, Anand G et al. (2005). Cancer Res 65: 9228–9237.

  • Wang T . (2003). Front Biosci 8: d1109–d1127.

  • Weinstein M, Monga SP, Liu Y, Brodie SG, Tang Y, Li C et al. (2001). Mol Cell Biol 21: 5122–5131.

  • Weinstein M, Yang X, Deng C . (2000). Cytokine Growth Factor Rev 11: 49–58.

  • Wieser R . (2001). Curr Opin Oncol 13: 70–77.

  • Xu J, Attisano L . (2000). Proc Natl Acad Sci USA 97: 4820–4825.

  • Xu X, Brodie SG, Yang X, Im YH, Parks WT, Chen L et al. (2000). Oncogene 19: 1868–1874.

  • Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H et al. (1999). EMBO J 18: 1280–1291.

  • Yin Y, Wang ZY, Mora-Garcia S, Li J, Yoshida S, Asami T et al. (2002). Cell 109: 181–191.

  • Zhang Y, Chang C, Gehling DJ, Hemmati-Brivanlou A, Derynck R . (2001). Proc Natl Acad Sci USA 98: 974–979.

  • Zhang Y, Musci T, Derynck R . (1997). Curr Biol 7: 270–276.

  • Zhong Q, Gao W, Du F, Wang X . (2005). Cell 121: 1085–1095.

  • Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH . (1999). Nature 400: 687–693.

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Acknowledgements

We thank Drs M Zasloff and S Evans for their critical review of the manuscript, and R Redman and A Rashid for their invaluable assistance with histopathology specimens and extensive confirmatory review of all slides. Grant Support: NIHR01 DK56111 (LM), NIHR01 CA106614-01A2 (LM), NIHR01 DK58637 (BM), VA Merit Award (LM), and R Robert and Sally D Funderburg Research Scholar (LM).

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Authors and Affiliations

  1. Departments of Surgical Sciences, Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA

    T Saha, D Vardhini, Y Tang, V Katuri, W Jogunoori, E A Volpe, A Sidawy, B Mishra & L Mishra

  2. Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA, USA

    D Haines

  3. Department of Cell Biology, Georgetown University, Washington, DC, USA

    X Zhou & I Gallicano

  4. Department of Pathology, Georgetown University, Washington, DC, USA

    R Schlegel

  5. Department of Veterans Affairs Medical Center, Washington, DC, USA

    A Sidawy & L Mishra

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  1. T Saha
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  2. D Vardhini
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Correspondence to B Mishra or L Mishra.

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The authors have no competing financial interests.

Supplementary Information accompanies the paper on Oncogene website (http://www.nature.com/onc)

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Saha, T., Vardhini, D., Tang, Y. et al. RING finger-dependent ubiquitination by PRAJA is dependent on TGF-β and potentially defines the functional status of the tumor suppressor ELF. Oncogene 25, 693–705 (2006). https://doi.org/10.1038/sj.onc.1209123

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