Identification and Characterization of a Nuclear Interacting Partner of Anaplastic Lymphoma Kinase (NIPA)

Silvia Klumpen,Christian Peschel,Justus Duyster,Cornelius Miething,Stephan W Morris,Florian Bassermann,Tao Ouyang,Ren-Yuan Bai,Christine Von Klitzing

doi:10.1074/jbc.m300883200

Abstract

Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.

Highlights

A LexATPR-anaplastic lymphoma kinase (ALK) fusion protein was shown to be efficiently autophosphorylated in yeast, and this protein was used as bait to screen a library prepared from K562, a human chronic myeloid leukemia (CML) cell line
The expression of nuclear interacting partner of ALK (NIPA) in Anaplastic large-cell lymphoma (ALCL) cells was demonstrated by identification of the gene in a cDNA library prepared from the cell line Karpas 299, which was derived from an ALK-positive ALCL patient
We describe the isolation of NIPA as an interacting partner for kinase-active ALK fusion proteins only

Summary

Introduction

We and others demonstrated phospholipase C-␥ and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. Truncation, or recombination, receptor tyrosine kinases may escape their strict ligand-mediated control and exhibit unregulated kinase activity, leading to constitutive activation of intracellular downstream signaling molecules and causing cell transformation and aberrant proliferation. Since the initial discovery of the chromosomal translocation t(2;5)(p23;q35) that fuses nucleophosmin (NPM) on chromosome 5 to anaplastic lymphoma kinase (ALK) on chromosome 2, a number of oncogenic fusions involving ALK in ALCL have been identified, such as TPM3-ALK, TFG-ALK, ATIC-ALK, and CLTC-ALK among others [5,6,7,8,9,10,11,12]. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events

Methods

Results

Conclusion