TrkAIII Promotes Microtubule Nucleation and Assembly at the Centrosome in SH-SY5Y Neuroblastoma Cells, Contributing to an Undifferentiated Anaplastic Phenotype

Antonietta R Farina,Pierdomenico Ruggeri,Natalia Di Ianni,Marzia Ragone,Giulia Ianni,Alberto Gulino,Andrew R Mackay,Lucia Cappabianca

doi:10.1155/2013/740187

Antonietta R Farina, Pierdomenico Ruggeri + Show 6 more

Open Access

https://doi.org/10.1155/2013/740187

Copy DOI

Abstract

The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs) and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α-tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC.

Highlights

TrkAIII is a developmentally regulated alternative splice variant of the NGF receptor tropomyosin-related kinase TrkA that is expressed by advanced stage human neuroblastomas (NBs), characterised by exon 6-7 skipping and exon 9 omission, and exhibits oncogenic activity in NB models [1,2,3,4,5]
Cell phenotype, which is in stark contrast to ligand-activated cell surface TrkA, which signals through IP3k/Akt and Ras/MAPK and promotes a less aggressive phenotype characterised by neuronal differentiation associated with the inhibition of proliferation [1, 5, 8,9,10]
This possibility is supported by the observations that (a) MT assembly and nucleation at the centrosome in TrkAIII transfectants were reduced by CEP-701 at TrkAIII inhibitory concentrations; (b) kd-TrkAIII SH-SY5Y transfectants did not exhibit this pattern of MT nucleation and assembly; and (c) TrkAIII SHSY5Y transfectants exhibited significantly more rapid MT regrowth from the centrosome, when compared to control, TrkAI, and CEP-701-treated TrkAIII transfectants

Summary

Introduction

TrkAIII is a developmentally regulated alternative splice variant of the NGF receptor tropomyosin-related kinase TrkA that is expressed by advanced stage human neuroblastomas (NBs), characterised by exon 6-7 skipping and exon 9 omission, and exhibits oncogenic activity in NB models [1,2,3,4,5]. As a consequence and in contrast to fully spliced cell surface TrkA, TrkAIII exhibits intracellular expression and spontaneous, ligand-independent activation that is restricted to interphase within the intracellular membrane compartment. This results in chronic signalling through IP3k/Akt but not Ras/MAPK and promotes a more aggressive proliferating, undifferentiated stressresistant, angiogenic, and tumourigenic stem cell-like NB cell phenotype, which is in stark contrast to ligand-activated cell surface TrkA, which signals through IP3k/Akt and Ras/MAPK and promotes a less aggressive phenotype characterised by neuronal differentiation associated with the inhibition of proliferation [1, 5, 8,9,10]. MTs differ in MTOC usage and nucleate at cytoplasmic and/or perinuclear/Golgi-associated extracentrosomal MTOCs, resulting in the formation of more diffuse cytoplasmic MT mats and coils, and during neuronal differentiation, the formation of long MTs organised at the cell periphery is required for neuritogenesis and axon genesis [11,12,13,14,15,16,17,18,19,20,21]

Methods

Results

Conclusion