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

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.

TrkAIII signals endoplasmic reticulum stress to the mitochondria in neuroblastoma cells, resulting in glycolytic metabolic adaptation

“…In addition to density gradient ultracentrifugation-purified mitochondria, TrkAIII was also detected in purified ER membranes, confirming previous reports [ 3 , 11 ], and in density gradient ultracentrifugation-purified MAMs but was not detected in membrane-free 100,000 x g ultracentrifugation cytosol fractions (Figure 2C ). TrkAIII positive MAMs were positive for TrkAIII, calnexin and TOM20 but not α-tubulin, whereas ER membranes were positive for TrkAIII and calnexin but not TOM20 and α-tubulin, confirming MAM purification as previously reported [ 44 , 45 ].…”

“…As a consequence, TrkAIII is not expressed at the cell surface but accumulates within pre-Golgi membranes and at the centrosome, where it exhibits spontaneous ligand-independent activation. Spontaneous intracellular TrkAIII activation leads to chronic signaling through the IP3K/Akt but not RAS/MAPK pathway and promotes a more stem cell-like, anaplastic, pro-angiogenic, stress-resistant, genetically unstable, tumourigenic and metastatic phenotype [ 1 – 3 , 6 , 7 , 11 – 13 ]. In NB cell lines, alternative TrkAIII splicing is promoted by a hypoxia mimic, suggesting that it represents a mechanism through which tumour suppressing signals from fully spliced TrkA receptors can switch to tumor promoting signals from TrkAIII within the hypoxic tumour microenvironment [ 1 , 2 , 6 ].…”

“…In NB cell lines, alternative TrkAIII splicing is promoted by a hypoxia mimic, suggesting that it represents a mechanism through which tumour suppressing signals from fully spliced TrkA receptors can switch to tumor promoting signals from TrkAIII within the hypoxic tumour microenvironment [ 1 , 2 , 6 ]. Furthermore, spontaneous activation of TrkAIII within the ERGIC-COP1 compartment and at the centrosome provides novel alternatives to “classical” cell surface oncogenic receptor tyrosine kinase (RTK) signaling and fuels the growing hypothesis that the RTK oncoprotein mislocalization underpins oncogenic activity [ 11 , 14 , 15 ].…”

Regulation of cargo export and sorting at the trans‐Golgi network

“…The role of AREX is to quickly handle and respond to fluctuations in protein synthesis that might occur under a variety of conditions . Such fluctuations, if not controlled, might lead to system failure or to aberrant cargo processing and sorting, and hence to disease . AREX acts through a surprisingly complex set of molecular components at the ER exit sites which include the signalling protein Gα12, AKAP scaffolds, adenylate cyclase and phosphorylation cascade that results in activation of the export machinery towards the Golgi as well as in attenuation of protein synthesis, thereby reducing the amount of folded cargo in the ER lumen .…”

TrkA is a binding partner of NPM‐ALK that promotes the survival of ALK+ T‐cell lymphoma

“…For instance, NGF/TrkA induces differentiation, apoptosis, and growth inhibition in neuroblastoma and medulloblastoma (Chou et al ., 2000; Lavenius et al ., 1995; Matsushima and Bogenmann, 1993), and high expression of TrkA is considered a favorable prognostic indicator in neuroblastoma (Nakagawara et al ., 1993). Whereas TrkAI splice variant was found to induce these effects, the TrkAIII splice variant was shown to possess significant oncogenic effects in neuroblastoma (Farina et al ., 2015; Tacconelli et al ., 2004). Moreover, the association of TrkA with its ligand NGF leads to the phosphorylation and activation of cascades involving PI3K/AKT and MAPK, which contributes to the survival of pheochromocytoma (Yao and Cooper, 1995).…”