Retrograde TrkAIII transport from ERGIC to ER: a re-localisation mechanism for oncogenic activity.

Antonietta Rosella Farina,Lucia Cappabianca,Luciana Gneo,Rita Maccarone,Pierdomenico Ruggeri,Andrew Reay Mackay

doi:10.18632/oncotarget.5802

Abstract

In human SH-SY5Y neuroblastoma (NB) cells, nascent immature N-glycosylated 110kDa TrkA moves rapidly from the endoplasmic reticulum (ER) to the Golgi Network (GN), where it matures into the 140kDa receptor prior to being transported to the cell surface, creating GN and cell surface pools of inactive receptor maintained below the spontaneous activation threshold by a full compliment of inhibitory domains and endogenous PTPases. In contrast, the oncogenic alternative TrkAIII splice variant is not expressed at the cell surface but re-localises to intracellular membranes, within which it exhibits spontaneous ERGIC/COPI-associated activation and oncogenic Akt signalling. In this study, we characterise the mechanism responsible for TrkAIII re-localisation. Spontaneous TrkAIII activation, facilitated by D4 IG-like domain and N-glycosylation site omission, increases spontaneous activation potential by altering intracellular trafficking, inhibiting cell surface expression and eliminating an important inhibitory domain. TrkAIII, spontaneously activated within the permissive ERGIC/COPI compartment, rather than moving in an anterograde direction to the GN exhibits retrograde transport back to the ER, where it is inactivated. This sets-up self-perpetuating TrkAIII re-cycling between the ERGIC and ER, that ensures continual accumulation above the spontaneous activation threshold of the ERGIC/COPI compartment. This is reversed by TrkA tyrosine kinase inhibitors, which promote anterograde transport of inactivated TrkAIII to the GN, resulting in GN-associated TrkAIII maturation to a 120kDa species that is degraded at the proteasome.

Highlights

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor tropomyosinrelated kinase, TrkA, is expressed by advanced stage human neuroblastomas (NBs), associates with poor prognosis and post therapeutic relapse in unfavourable high TrkA expressing tumours and exhibits oncogenic activity in NB models [1,2,3,4,5,6]
The Trk-T3 oncogene, involved in thyroid cancer, re-localises to endoplasmic reticulum (ER) exit sites (ERES), exhibits spontaneous activation within ERES-associated COPII vesicles and induces oncogenic signalling from this alternative location through Akt but not Ras, similar to that reported for the TrkAIII oncoprotein [1, 13]
We have previously reported [3] and confirm here (Figure 1A) that TrkA trafficking from ER to Golgi Network (GN) and to the cell surface in SH-SY5Y NB cells occurs without spontaneous activation and results in two distinct steadystate pools of inactive TrkA receptors at the cell surface and within the GN, the latter overlapping closely with the GN marker GM130 [3]

Summary

Introduction

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor tropomyosinrelated kinase, TrkA, is expressed by advanced stage human neuroblastomas (NBs), associates with poor prognosis and post therapeutic relapse in unfavourable high TrkA expressing tumours and exhibits oncogenic activity in NB models [1,2,3,4,5,6]. As a consequence and in contrast to TrkA, TrkAIII is not expressed at the cell surface but re-localises to intracellular membranes, within which it exhibits spontaneous ligand-independent activation, leading to oncogenic signalling through Akt but not Ras from this alternative location. Signalling from TrkAIII differs to classical signalling from cell surface ligand-activated TrkA through Ras/MAPK, suggesting that the re-localisation of TrkAIII is fundamental for it’s oncogenic activity [1]. This hypothesis is supported by recent reports that the oncogenic activity of several receptor tyrosine kinase (RTK) oncogenes depends www.impactjournals.com/oncotarget upon their re-localisation [10]. The Trk-T3 oncogene, involved in thyroid cancer, re-localises to ER exit sites (ERES), exhibits spontaneous activation within ERES-associated COPII vesicles and induces oncogenic signalling from this alternative location through Akt but not Ras, similar to that reported for the TrkAIII oncoprotein [1, 13]

Methods

Results

Conclusion