Activation of Anaplastic Lymphoma Kinase Receptor Tyrosine Kinase Induces Neuronal Differentiation through the Mitogen-activated Protein Kinase Pathway

Abstract

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.The common structural features of a receptor tyrosine kinase (RTK) 1 include an extracellular ligand binding region, a hydrophobic membrane-spanning segment, and a cytoplasmic domain that carries the catalytic function. Following ligand binding, the RTK dimerizes and autophosphorylates (1). The activated RTK initiates signal transduction cascades through binding of SH2 domain-containing proteins to specific receptor phosphotyrosine residues (2). RTKs can regulate a wide variety of cellular processes involved in cell division, differentiation, survival, and motility. A number of RTKs play essential roles during the development of the nervous system by contributing to neuronal differentiation, survival, and function (reviewed in Ref.3). Most of these receptors have specific or shared ligands called neurotrophic factors that have been identified (reviewed in Ref. 3). However, for some of them, named orphan receptors, their ligands are still unknown (4 -6).Anaplastic lymphoma kinase (ALK), a novel orphan neuronal receptor, was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused in the t(2;5) chromosomal rearrangement associated with the non-Hodgkin lymphoma (7). This translocation produces a fusion gene that encodes a soluble chimeric transforming protein comprised of the N-terminal portion of the phosphoprotein nucleophosmin (NPM), a highly conserved RNA-binding nucleolar protein, linked to the cytoplasmic portion of ALK (7). The NPM-ALK fusion protein was localized within both the cytoplasm and the nucleoplasm and also within the nucleoli of t(2;5)-translocation-positive lymphoma cells (8). However, whereas the NPM sequence is essential for the transforming activity (9), the nuclear localization, occurring via the shuttling activity of NPM (10...

Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway

“…It may be relevant in this context that the structural relationship of ALK to the insulin receptor family (Morris et al, 1994;Shiota et al, 1994) might explain the ability of NPM/ALK to activate mTOR. In agreement with this hypothesis is the identification of MEK/ERK activation in neural cells transfected with ALK (Souttou et al, 2001) or stimulated through endogenous ALK (Motegi et al, 2004). Furthermore, these observations suggest that, in addition to NPM/ALK, the full-length ALK and possibly other ALK hybrid proteins might activate mTOR.…”

What have we learnt from mouse models of NPM-ALK-induced lymphomagenesis?

“…50 Consistent with hyperactivation of the Ras pathway, phosphorylated ERK (pErk, the activated form of the kinase) has been detected in tumour cells derived from NPM-ALK transgenic mice, 22,23 confirming data generated in Drosophila and cell lines. 41,44,[51][52][53] The stress-activated JNK MAP Kinase pathway is also active in the tumour tissues of NPM-ALK transgenic mice. 23 Whereas in some cellular contexts stress-induced JNK activation appears to mediate apoptosis, in tumour cells JNK can signal cell survival.…”

“…Investigations based largely on cell line data suggest that the NPM-ALK signalosome stimulates pathways regulating mitogenesis and survival, involving the phosphoinositide 3-kinase (PI 3-kinase) and Ras/MAP kinase pathways, and the STAT family of transcription factors. [36][37][38][39][40][41][42][43] Here we focus on the contribution of mouse models to our understanding of these signals in situ.…”

Dominant Negative Effectors of Heparin Affin Regulatory Peptide (HARP) Angiogenic and Transforming Activities

“…Construction and Production of ALK Extracellular Domain (RECA)-A construct coding for the entire extracellular domain of human ALK (that we named RECA) linked to a His 8 tag was generated using PCR experiments from the pCDNA3-ALK (26). The resulting RECA-His cDNA was further subcloned into the pCEP4 vector 6, respectively) was separated on a 10% SDS-PAGE.…”