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 PLCc1. 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 ALKbinding proteins, and its potential signal transduction pathways.

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas

“…Our group and others have described modifications of the expression of regulators of the Rho GTPases (Crockett et al, 2004;Cussac et al, 2006). We observed the extinction of the GTPase inhibitor RhoGDI2 in the proteome of an NPM-ALK( þ ) cell line, an effect shown to correlate with higher metastatic activity and poor prognosis in bladder cancers (Theodorescu et al, 2004;Cussac et al, 2006).…”

“…New partners have been found that account for different functions of NPM-ALK, such as the regulation of mRNA turnover (Fawal et al, 2006). Among others, proteins regulating cell shape and cytoskeleton plasticity, two important features altered in transformed cells, were identified, leading to investigations of molecules classified as 'cytoskeleton and motility regulators' in the context of ALCLs (Crockett et al, 2004;Cussac et al, 2006). Ambrogio et al (2005) reported the association of NPM-ALK with p130Crk associated substrate (p130Cas) and described its role in actin depolymerization and transformation.…”

“…These findings suggested that upregulation of Rho GTPases might be of importance during the progression of the disease. Recently, about 45 proteins were reported to interact with NPM-ALK, including various Rho GTPase activating proteins whose pattern of expression was also altered in a study of the transcriptome of NPM-ALK( þ ) cells (Crockett et al, 2004;Lamant et al, 2006). Rho GTPases mediate many aspects of cell biology including proliferation, regulation of the cell survival, polarity, adhesion, membrane trafficking and motility (Hall, 2005).…”

Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

“…First, the lack of any effect of the pan-Jak inhibitor on the ALK þ TCL cells (Figures 1-4) rules out the involvement of any member of this family that, beside Jak3, includes Jak1, Jak2 and Tyk2. 34 Furthermore, two SRC family inhibitors, SU6656 and PP2, used at the doses of up to 1 and 2 mM, respectively, also had no effect on the Stat3 phosphorylation and cell growth (MTT conversion assay) of the ALK þ TCL Karpas 299 cells (data not shown). This observation corroborates the previous finding that mouse embryonal fibrobast cell line devoid of three members of src family known to activate STAT3 were able to display STAT3 phosphorylation upon transfection with NPM/ALK.…”

Proteomics in pathology research

“…Proteins identified by MS were confirmed by Western blotting and reciprocal immunoprecipitation. 83 Proteomic-based methods for the identification and quantitation of proteins associated with the chromatin 84 or specific transcription factor complexes have been developed. Using the ICAT technique of quantitative mass spectrometry, proteins associated with a transcription factor NFE2p18/Mafk, essential for b-globin expression in murine erythroleukemia cells were determined 85 during two differentiation states.…”