The Alternative TrkAIII Splice Variant Targets the Centrosome and Promotes Genetic Instability

Abstract

The hypoxia-regulated alternative TrkAIII splice variant expressed by human neuroblastomas exhibits oncogenic potential, driven by in-frame exon 6 and 7 alternative splicing, leading to omission of the receptor extracellular immunoglobulin C 1 domain and several N-glycosylation sites. Here, we show that the TrkAIII oncogene promotes genetic instability by interacting with and exhibiting catalytic activity at the centrosome. This function depends upon intracellular TrkAIII accumulation and spontaneous interphase-restricted activation, in cytoplasmic tyrosine kinase (tk) domain orientation, predominantly within structures that closely associate with the fully assembled endoplasmic reticulum intermediate compartment and Golgi network. This facilitates TrkAIII tk-mediated binding of ␥-tubulin, which is regulated by endogenous protein tyrosine phosphatases and geldanamycin-sensitive interaction with Hsp90, paving the way for TrkAIII recruitment to the centrosome. At the centrosome, TrkAIII differentially phosphorylates several centrosome-associated components, increases centrosome interaction with polo kinase 4, and decreases centrosome interaction with separase, the net results of which are centrosome amplification and increased genetic instability. The data characterize TrkAIII as a novel internal membrane-associated centrosome kinase, unveiling an important alternative mechanism to "classical" cell surface oncogenic receptor tk signaling through which stressregulated alternative TrkAIII splicing influences the oncogenic process.Alternative splicing is fundamental for differential protein expression from the same gene and not only increases the proteomic complexity of higher organisms (29) but is also involved in cancer pathogenesis, activating several oncogenes and inactivating several oncosuppressors (17).The neurotrophin receptor tropomyosin-related kinase A (TrkA) is among the proto-oncogenes activated by alternative splicing, with a novel hypoxia-regulated oncogenic alternative TrkAIII splice variant recently identified in advanced-stage human neuroblastomas (NB) and primary glioblastomas (44,45). In contrast to wild-type TrkAI/TrkAII, the expression of which is associated with better prognosis for NB, induces NB cell differentiation, exhibits a tumor suppressor function in NB models in vivo (9,19,22,30,44,45), and may regulate both spontaneous and therapy-induced NB regression (30), TrkAIII is expressed by more-advanced-stage NB and exhibits oncogenic activity in NB models (44,45). This has challenged the hypothesis of an exclusively tumor-suppressing function for TrkA in NB by providing a way through which tumor-suppressing signals from TrkA can be converted to oncogenic signals from TrkAIII during tumor progression.The oncogenic potential of TrkAIII, characterized by NIH 3T3 cell transforming and NB xenograft primary and metastatic tumor-promoting activity (44,45), is driven by in-frame alternative splicing of exons 6 and 7. This results in the omission of the receptor extracellular immunoglobulin C 1 (Ig C...

On the death Trk

“…This link has been confirmed in many studies over the past 15 years, establishing full length TrkA as one of most robust indicators of positive prognosis in neuroblastoma (reviewed in Brodeur, 2003;Shimada et al, 2004;Brodeur et al, 2009). It should however be noted that a less widely expressed alternatively spliced non-NGF responsive variant, TrkAIII, has negative effects on prognosis (Tacconelli et al, 2004;Farina et al, 2009).…”

Alternative TrkA Splicing and Neuroblastoma

“…This is supported by the inverse relationship exhibited by TrkA expression and NB stage (Nakagawara et al, 1992;Nakagawara & Koger, 2000;Nakagawara, 2001) and suggests that the reintroduction of adequate TrkA expression levels and/or the activation of post cell surface TrkA receptor signalling represents an important potential therapeutic goal in NB. Recently, however, a darker side to TrkA involvement in NB has been revealed by the discovery of an alternative TrkA splice variant "TrkAIII" expressed by advanced stage primary human NBs that exhibits oncogenic activity in NB models (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b.…”

“…The mature gp140 TrkAI receptor is expressed predominantly at the cell surface with GN accumulation of the immature gp110TrkAI receptor, whereas TrkAIII is not expressed at the cell surface but is retained within intracellular membranes, within which it exhibits relatively equal distribution between the endoplasmic reticulum (ER), endoplasmic reticulum and Golgi intermediate (ERGIC); Golgi network (GN) and associated vesicle compartments (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b; c) Differences in spontaneous versus liganddependent activation. TrkAI exhibits ligand-dependent but not spontaneous activity, whereas TrkAIII exhibits spontaneous ligand-independent activation and does not bind neurotrophins; d) Differences in post receptor signal transduction.…”

“…Spontaneous ligand-independent TrkAIII activation most likely depends upon the omission of the extracellular D4 Ig-like domain plus associated N-glycosylation sites, which prevent ligand-independent TrkA activation (Arevalo et al, 2000). Differences in adapter protein binding exhibited by ligand-activated TrkAI and spontaneously-active TrkAIII and subsequent differences in post receptor signalling most likely reflect differences in receptor localisation, with lack of post TrkAIII signalling through Ras/MAPK explained either by dislocation from caveolae-associated Ras/MAPK (Paratcha & Ibanez, 2002;Rajagopal et al, 2004), altered adapter protein binding (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b; TrkAIII activity below the Ras/MAPK activation threshold (Hallberg et al, 1998); and/or direct PI3K/Akt inhibition of Raf-MEK-ERK signalling (Moelling et al, 2002), the latter possibility supported by TrkAIII antagonism of NGF/TrkAI-induced ERK phosphorylation (Tacconelli et al, 2004). Interestingly, post TrkAIII receptor signalling through PI3K/Akt but not Ras/MAPK resembles post receptor signalling from GN-associated immature gp110TrkAI transactivated by G-protein associated A 2A adenosine receptors (Rajagopal et al, 2004), indicating that an intracellular location alters post TrkA receptor signalling.…”

“…In response to ligand, activated TrkA receptors exhibit receptor-associated tyrosine kinase and PI3K activity, are phosphorylated on Y490, Y674/675 and Y758 residues, bind Shc, Grb2 and FRS2 adapters and signal through PI3K/Akt and Ras/MAPK, whereas spontaneously active TrkAIII exhibits tyrosine kinase and PI3K activity, is constitutively phosphorylated on Y490, Y674/675 and Y758 residues, binds only low levels of non-phosphorylated Shc, does not bind FRS2 or GRB2 and signals through IP3K/Akt but not Ras/MAPK (Tacconelli et al, 2004); and e) differences in biological activity, with TrkAI exhibiting tumour-suppressing and TrkAIII oncogenic activity in NB models (Tacconelli et al, 2004). These differences depend upon the omission of sequences encoded within exons 6/7 and form the basis of the differential TrkAI/II tumour-suppressing and TrkAIII oncogenic activity observed (Tacconelli et al, 2004;Farina et al, 2009aFarina et al, , 2009b. Intracellular TrkAIII retention versus cell surface TrkAI expression may depend upon differences in extracellular domain N-glycosylation, which regulates cell surface TrkA expression (Watson et al, 1999).…”