Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Paper
  • Published:

Identification of NPM-ALK interacting proteins by tandem mass spectrometry

Abstract

Constitutive overexpression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a key oncogenic event in anaplastic large-cell lymphomas with the characteristic chromosomal aberration t(2;5)(p23;q35). Proteins that interact with ALK tyrosine kinase play important roles in mediating downstream cellular signals, and are potential targets for novel therapies. Using a functional proteomic approach, we determined the identity of proteins that interact with the ALK tyrosine kinase by co-immunoprecipitation with anti-ALK antibody, followed by electrospray ionization and tandem mass spectrometry (MS/MS). A total of 46 proteins were identified as unique to the ALK immunocomplex using monoclonal and polyclonal antibodies, while 11 proteins were identified in the NPM immunocomplex. Previously reported proteins in the ALK signal pathway were identified including PI3-K, Jak2, Jak3, Stat3, Grb2, IRS, and PLCĪ³1. More importantly, many proteins previously not recognized to be associated with NPM-ALK, but with potential NPM-ALK interacting protein domains, were identified. These include adaptor molecules (SOCS, Rho-GTPase activating protein, RAB35), kinases (MEK kinase 1 and 4, PKC, MLCK, cyclin G-associated kinase, EphA1, JNK kinase, MAP kinase 1), phosphatases (meprin, PTPK, protein phosphatase 2 subunit), and heat shock proteins (Hsp60 precursor). Proteins identified by MS were confirmed by Western blotting and reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by tandem mass spectrometry for the elucidation of ALK-binding proteins, and its potential signal transduction pathways.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  • Amin HM, Medeiros LJ, Ma Y, Feretzaki M, Das P, Leventaki V, Rassidakis GZ, O'Connor SL, McDonnell TJ and Lai R . (2003). Oncogene, 22, 5399ā€“5407.

  • Baba T, Fusaki N, Shinya N, Iwamatsu A and Hozumi N . (2003). Biochem. Biophys. Res. Commun., 304, 67ā€“72.

  • Bai RY, Dieter P, Peschel C, Morris SW and Duyster J . (1998). Mol. Cell. Biol., 18, 6951ā€“6961.

  • Bai RY, Ouyang T, Miething C, Morris SW, Peschel C and Duyster J . (2000). Blood, 96, 4319ā€“4327.

  • Bernhard OK, Burgess JA, Hochgrebe T, Sheil MM and Cunningham AL . (2003). Proteomics, 3, 139ā€“146.

  • Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ and Mann M . (2003). Nat. Biotechnol., 21, 315ā€“318.

  • Bonvini P, Gastaldi T, Falini B and Rosolen A . (2002). Cancer Res., 62, 1559ā€“1566.

  • Brazil DP, Park J and Hemmings BA . (2002). Cell, 111, 293ā€“303.

  • Davis TH, Morton CC, Miller-Cassman R, Balk SP and Kadin ME . (1992). N. Engl. J. Med., 326, 1115ā€“1122.

  • Edmondson RD, Vondriska TM, Biederman KJ, Zhang J, Jones RC, Zheng Y, Allen DL, Xiu JX, Cardwell EM, Pisano MR and Ping P . (2002). Mol. Cell. Proteomics, 1, 421ā€“433.

  • Epstein AL and Kaplan HS . (1974). Cancer, 34, 1851ā€“1872.

  • Figeys D, McBroom LD and Moran MF . (2001). Methods, 24, 230ā€“239.

  • Fischer P, Nacheva E, Mason DY, Sherrington PD, Hoyle C, Hayhoe FG and Karpas A . (1988). Blood, 72, 234ā€“240.

  • Fujimoto J, Shiota M, Iwahara T, Seki N, Satoh H, Mori S and Yamamoto T . (1996). Proc. Natl. Acad. Sci. USA, 93, 4181ā€“4186.

  • Griffis ER, Xu S and Powers MA . (2003). Mol. Biol. Cell, 14, 600ā€“610.

  • Hubinger G, Scheffrahn I, Muller E, Bai R, Duyster J, Morris SW, Schrezenmeier H and Bergmann L . (1999). Exp. Hematol., 27, 1796ā€“1805.

  • Hubinger G, Wehnes E, Xue L, Morris SW and Maurer U . (2003). Exp. Hematol., 31, 226ā€“233.

  • Kai M, Tanaka H and Wang TS . (2001). Mol. Cell. Biol., 21, 3289ā€“3301.

  • Kaiser BK, Zimmerman ZA, Charbonneau H and Jackson PK . (2002). Mol. Biol. Cell, 13, 2289ā€“2300.

  • Kelkar N, Gupta S, Dickens M and Davis RJ . (2000). Mol. Cell. Biol., 20, 1030ā€“1043.

  • Khoury JD, Medeiros LJ, Rassidakis GZ, Yared MA, Tsioli P, Leventaki V, Schmitt-Graeff A, Herling M, Amin HM and Lai R . (2003). Clin. Cancer Res., 9, 3692ā€“3699.

  • Knaus PI, Lindemann D, DeCoteau JF, Perlman R, Yankelev H, Hille M, Kadin ME and Lodish HF . (1996). Mol. Cell. Biol., 16, 3480ā€“3489.

  • Kuefer MU, Look AT, Pulford K, Behm FG, Pattengale PK, Mason DY and Morris SW . (1997). Blood, 90, 2901ā€“2910.

  • Luderus ME, de Graaf A, Mattia E, den Blaauwen JL, Grande MA, de Jong L and van Driel R . (1992). Cell, 70, 949ā€“959.

  • Lux A, Attisano L and Marchuk DA . (1999). J. Biol. Chem., 274, 9984ā€“9992.

  • Mason DY, Bastard C, Rimokh R, Dastugue N, Huret JL, Kristoffersson U, Magaud JP, Nezelof C, Tilly H and Vannier JP . (1990). Br. J. Haematol., 74, 161ā€“168.

  • Miyake I, Hakomori Y, Shinohara A, Gamou T, Saito M, Iwamatsu A and Sakai R . (2002). Oncogene, 21, 5823ā€“5834.

  • Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL and Look AT . (1994). Science, 263, 1281ā€“1284.

  • Morris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X and Witte DP . (1997). Oncogene, 14, 2175ā€“2188.

  • Nieborowska-Skorska M, Slupianek A, Xue L, Zhang Q, Raghunath PN, Hoser G, Wasik MA, Morris SW and Skorski T . (2001). Cancer Res., 61, 6517ā€“6523.

  • Ohta S, Shiomi Y, Sugimoto K, Obuse C and Tsurimoto T . (2002). J. Biol. Chem., 277, 40362ā€“40367.

  • Pandey A, Lazar DF, Saltiel AR and Dixit VM . (1994). J. Biol. Chem., 269, 30154ā€“30157.

  • Payen E, Verkerk T, Michalovich D, Dreyer SD, Winterpacht A, Lee B, De Zeeuw CI, Grosveld F and Galjart N . (1998). J. Biol. Chem., 273, 9099ā€“9109.

  • Peng J, Elias JE, Thoreen CC, Licklider LJ and Gygi SP . (2003). J. Proteome Res., 2, 43ā€“50.

  • Pratt RL and Kinch MS . (2002). Oncogene, 21, 7690ā€“7699.

  • Raetz EA, Perkins SL, Carlson MA, Schooler KP, Carroll WL and Virshup DM . (2002). Am. J. Pathol., 161, 875ā€“883.

  • Ranish JA, Yi EC, Leslie DM, Purvine SO, Goodlett DR, Eng J and Aebersold R . (2003). Nat. Genet., 33, 349ā€“355.

  • Rashid KA, Hevi S, Chen Y, Le Caherec F and Chuck SL . (2002). J. Biol. Chem., 277, 22010ā€“22017.

  • Rey M, Vicente-Manzanares M, Viedma F, Yanez-Mo M, Urzainqui A, Barreiro O, Vazquez J and Sanchez-Madrid F . (2002). J. Immunol., 169, 5410ā€“5414.

  • Rossi L, Deri P, Andreoli I, Gremigni V, Salvetti A and Batistoni R . (2003). Int. J. Dev. Biol., 47, 293ā€“298.

  • Ruchatz H, Coluccia AM, Stano P, Marchesi E and Gambacorti-Passerini C . (2003). Exp. Hematol., 31, 309ā€“315.

  • Sharfe N, Freywald A, Toro A and Roifman CM . (2003). J. Immunol., 170, 6024ā€“6032.

  • Shiio Y, Donohoe S, Yi EC, Goodlett DR, Aebersold R and Eisenman RN . (2002). EMBO J., 21, 5088ā€“5096.

  • Shiota M, Fujimoto J, Semba T, Satoh H, Yamamoto T and Mori S . (1994). Oncogene, 9, 1567ā€“1574.

  • Shiota M and Mori S . (1997). Leukemia, 11 (Suppl 3), 538ā€“540.

  • Shipp MA, Ross KN, Tamayo P, Weng AP, Kutok JL, Aguiar RC, Gaasenbeek M, Angelo M, Reich M, Pinkus GS, Ray TS, Koval MA, Last KW, Norton A, Lister TA, Mesirov J, Neuberg DS, Lander ES, Aster JC and Golub TR . (2002). Nat. Med., 8, 68ā€“74.

  • Slupianek A, Nieborowska-Skorska M, Hoser G, Morrione A, Majewski M, Xue L, Morris SW, Wasik MA and Skorski T . (2001). Cancer Res., 61, 2194ā€“2199.

  • Souttou B, Carvalho NB, Raulais D and Vigny M . (2001). J. Biol. Chem., 276, 9526ā€“9531.

  • Stein H, Mason DY, Gerdes J, O'Connor N, Wainscoat J, Pallesen G, Gatter K, Falini B, Delsol G, Lemke H, Schwarting R and Lennert K . (1985). Blood, 66, 848ā€“858.

  • Taimen P, Viljamaa M and Kallajoki M . (2000). Exp. Cell. Res., 256, 140ā€“149.

  • Tessier-Lavigne M . (1995). Cell, 82, 345ā€“348.

  • Walker-Daniels J, Coffman K, Azimi M, Rhim JS, Bostwick DG, Snyder P, Kerns BJ, Waters DJ and Kinch MS . (1999). Prostate, 41, 275ā€“280.

  • Zamo A, Chiarle R, Piva R, Howes J, Fan Y, Chilosi M, Levy DE and Inghirami G . (2002). Oncogene, 21, 1038ā€“1047.

  • Zelinski DP, Zantek ND, Stewart JC, Irizarry AR and Kinch MS . (2001). Cancer Res., 61, 2301ā€“2306.

  • Zhang Q, Raghunath PN, Xue L, Majewski M, Carpentieri DF, Odum N, Morris S, Skorski T and Wasik MA . (2002). J. Immunol., 168, 466ā€“474.

Download references

Acknowledgements

We thank Dr G Chris Fillmore for assistance with cell culture. This work was supported by the ARUP Institute for Clinical and Experimental Pathology.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Kojo SJ Elenitoba-Johnson or Megan S Lim.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crockett, D., Lin, Z., Elenitoba-Johnson, K. et al. Identification of NPM-ALK interacting proteins by tandem mass spectrometry. Oncogene 23, 2617ā€“2629 (2004). https://doi.org/10.1038/sj.onc.1207398

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1207398

Keywords

This article is cited by

Search

Quick links