Oncogene (2004) 23, 2617–2629 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Identification of NPM-ALK interacting proteins by tandem mass spectrometry David K Crockett1, Zhaosheng Lin1, Kojo SJ Elenitoba-Johnson*,1,2 and Megan S Lim*,1,2 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA; 2Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132, USA Constitutive overexpression of nucleophosmin-anaplastic Introduction lymphoma kinase (NPM-ALK) is a key oncogenic event in anaplastic large-cell lymphomas with the characteristic Anaplastic large-cell lymphoma (ALCL) is a distinct chromosomal aberration t(2;5)(p23;q35). Proteins that subtype of peripheral T-cell lymphomas harboring interact with ALK tyrosine kinase play important roles in chromosomal translocations involving the ALK tyro- mediating downstream cellular signals, and are potential sine kinase (Stein et al., 1985; Mason et al., 1990). The targets for novel therapies. Using a functional proteomic t(2;5)(p23;q35) chromosomal aberration resulting in approach, we determined the identity of proteins that overexpression of a chimeric oncogene, nucleophos- interact with the ALK tyrosine kinase by co-immunopre- min-anaplastic lymphoma kinase (NPM-ALK) (Morris cipitation with anti-ALK antibody, followed by electro- et al., 1994; Shiota et al., 1994), is the most common spray ionization and tandem mass spectrometry (MS/ translocation found in these tumors. The resulting MS). A total of 46 proteins were identified as unique to NPM-ALK fusion protein has been well documented the ALK immunocomplex using monoclonal and poly- as a constitutively active tyrosine kinase and the clonal antibodies, while 11 proteins were identified in the causative oncogene in t(2;5)-positive ALCLs (Morris NPM immunocomplex. Previously reported proteins in et al., 1997; Shiota and Mori, 1997). The NPM-ALK the ALK signal pathway were identified including PI3-K, protein plays a key role in ALCL lymphomagenesis, and Jak2, Jak3, Stat3, Grb2, IRS, and PLCc1. More has been shown to cause lymphoid malignancy in vitro importantly, many proteins previously not recognized to and in vivo (Kuefer et al., 1997). be associated with NPM-ALK, but with potential NPM- The NPM-ALK fusion protein transduces its trans- ALK interacting protein domains, were identified. These forming capacity via several key signaling pathways include adaptor molecules (SOCS, Rho-GTPase activat- including the PI3-K pathway leading to Akt activation ing protein, RAB35), kinases (MEK kinase 1 and 4, PKC, (Slupianek et al., 2001), as well as activation of the JAK/ MLCK, cyclin G-associated kinase, EphA1, JNK kinase, STAT pathway (Nieborowska-Skorska et al., 2001; MAP kinase 1), phosphatases (meprin, PTPK, protein Zamo et al., 2002; Ruchatz et al., 2003). The src phosphatase 2 subunit), and heat shockproteins (Hsp60 homology SH2 and phosphotyrosine-binding (PTB) precursor). Proteins identified by MS were confirmed by protein motifs within the cytoplasmic domain of ALK Western blotting and reciprocal immunoprecipitation. are known to mediate its interaction with other signaling This study demonstrates the utility of antibody immuno- molecules (Morris et al., 1997). Co-immunoprecipitation precipitation and subsequent peptide identification by studies have demonstrated the interaction of ALK with tandem mass spectrometry for the elucidation of ALK- PLCg (Bai et al., 1998), IRS-1 (Fujimoto et al., 1996), binding proteins, and its potential signal transduction Hsp90 (Bonvini et al., 2002), Grb, ShcC (Miyake et al., pathways. 2002), Jak3 (Zamo et al., 2002), Jak2 (Ruchatz et al., Oncogene (2004) 23, 2617–2629. doi:10.1038/sj.onc.1207398 2003), Stat5 (Nieborowska-Skorska et al., 2001), and Published online 16 February 2004 Stat3 (Zamo et al., 2002; Zhang et al., 2002). In many instances, it has been well established that Keywords: NPM-ALK; anaplastic lymphoma kinase; proteins do not act alone but form multimeric com- functional proteomics; mass spectrometry plexes with potential substrates and regulatory proteins that influence its biologic activity (Edmondson et al., 2002). Since ALK plays a critical role in the activation of multiple signaling pathways, determination of its interacting proteins and those that formthe complexof proteins within its ‘interactome’ may provide novel *Correspondence: MS Limor KSJ Elenitoba-Johnson, Departmentof insights as to its function, as well as reveal potential Pathology, University of Utah School of Medicine, 50 North Medical targets for novel therapies. Drive, RoomA565, Salt Lake City, UT 84132, USA; E-mail: [email protected], [email protected] The emerging field of proteomics provides a powerful Received 8 May 2003; revised 13 October 2003; accepted 25 November avenue to carry out functional studies of protein– 2003 protein interactions and characterization of signal Proteomic analysis of NPM-ALK interacting proteins DK Crockett et al 2618 transduction pathways (Figeys et al., 2001; Shiio et al., 2002). This is in large part due to advances in protein sample preparation, analytical sensitivity of mass spectrometers (Blagoev et al., 2003; Peng et al., 2003), and improvements in instrument software and protein databases (Edmondson et al., 2002; Ranish et al., 2003). Until recently, assessment of interacting proteins has only been feasible via laborious and time-consuming molecular biologic techniques such as the yeast- two-hybrid system. Using a variety of approaches including protein complex purification, immunoprecipi- tation, affinity chromatography followed by high- performance liquid chromatography (HPLC), and electrospray ionization and tandem mass spectrometry (ESI-MS/MS), interacting proteins of CD4 receptor complex (Bernhard et al., 2003), nonmuscle myosin heavy chain-IIA (Rey et al., 2002), proliferating cell nuclear antigen (Ohta et al., 2002), protein kinase Ce signaling complex (Edmondson et al., 2002), and apolipoprotein B (Rashid et al., 2002) have been identified, to list a few. Although the specific nature of the protein interactions needs to be functionally validated, these approaches represent a high-throughput method for the identification of proteins within a complex, and provide insights into the fundamental basis of their biologic activities. In this study, we sought to determine the identity of proteins that interact with NPM-ALK tyrosine kinase using immunoprecipitation, followed by liquid chroma- tography (LC) and ESI-MS/MS (Figure 1). In addition to confirming the identity of previously reported proteins in the ALK signal pathway including phos- pholipase C-gamma (PLCg1) (Bai et al., 1998), phos- phoinositide-3-kinase (PI3-K) (Bai et al., 2000; Slupianek et al., 2001), Jak2 (Ruchatz et al., 2003), Jak3 (Zamo et al., 2002), and IRS (Morris et al., 1997), we have identified a comprehensive list of proteins that may represent novel mediators of ALK signaling. This Figure 1 Overview of sample enrichment by antibody immuno- study demonstrates the utility of antibody immunopre- precipitation, separation of proteins by SDS–PAGE, trypsin cipitation and peptide identification by nanoflow ESI- digestion of unique bands, and analysis by ion trap tandemmass spectrometry LC/MS/MS for the high-throughput identification of proteins within the ALK signaling complex and potential definition of its signaling pathways. Results Expression of NPM-ALK in t(2;5)-positive cell lines To determine the basal level of expression of the NPM- ALK fusion protein in a t(2;5)-positive cell line Karpas 299 and the t(2;5)-negative cell line Mac 2A, we Figure 2 Confirmation of NPM-ALK expression in Karpas 299, performed Western blot analysis of total cell lysates but not in Mac 2A. Total cell lysates were analysed by SDS–PAGE and expression of NPM-ALK protein was detected using an anti- and probed with an anti-ALK antibody (Figure 2). The ALK antibody. b-actin served as the loading control 80 kDa NPM-ALK fusion protein is expressed in the Karpas 299 cell line, but not in Mac 2A. 299 cells using both monoclonal and polyclonal anti- Immunoprecipitation with anti-ALK antibody ALK antibodies. Figure 3a demonstrates separation of the ALK immunocomplex by 1-dimensional- To determine the complexes of proteins associated with SDS–PAGE in the Karpas 299 cells, with subsequent the NPM-ALK tyrosine kinase, we performed immu- silver staining of the separated proteins. Immunopreci- noprecipitation of total cell lysate obtained fromKarpas pitation using a control IgG antibody was also Oncogene Proteomic analysis of NPM-ALK interacting proteins DK Crockett et al 2619 anti-ALK immunoprecipitate revealed unique bands at approximately 175, 80, 43, and 28 kDa. Each lane was divided into 12 equal pieces (regardless of staining) and analysed by MS/MS. The proteins identified in the IgG control lane were subtracted fromthose identified in the ALK immunocomplex of the Karpas 299 cell line. For reproducibility, anti-ALK immunoprecipitation experi- ments using monoclonal antibodies were performed in duplicate. Experiments using polyclonal anti-ALK antibodies were performed in triplicate. Identical band- ing patterns seen in the SDS–PAGE separation, with only slight differences in intensity between monoclonal and polyclonal lanes, demonstrate the reproducibility of immunoprecipitation experiments. Immunoprecipitation with anti-NPM antibody To determine the protein interactions specifically associated with the NPM component of the NPM- ALK