Upon neurotrophin binding, Trk receptor signaling promotes two distinct outcomes; neuronal survival and the extension of processes or ‘neurites’. The recruitment of specific subsets of intracellular effectors to the Trk receptors governs the type of cellular response initiated upon neurotrophin binding (Lee et al. 2001; Huang and Reichardt 2003) and the subcellular localization of the activated Trk receptors influences the type of signal that is propagated (Zhang et al. 2000; Delcroix et al. 2003).
The suppressors of cytokine signaling (SOCS) proteins are a family of intracellular proteins implicated in the negative regulation of a variety of cytokine, growth factor, and hormone signals (O'Sullivan et al. 2007; Croker et al. 2008; Piessevaux et al. 2008). Each member of the SOCS family is characterized by a N-terminus of variable length, a central SH2 domain, and a highly conserved C-terminal SOCS box motif (Hilton et al. 1998). The SH2 domain binds to phosphorylated tyrosine residues within proteins. The SOCS box mediates association with elongin BC which recruits the E3 ubiquitin ligase scaffold protein Cullin5 (Babon et al. 2009). By incorporating these two separate functional domains, the SOCS proteins integrate specific substrate recognition via the SH2 domain and ubiquitination of SOCS-associated proteins via the SOCS box.
SOCS proteins have pleiotropic effects in the nervous system (Wang and Campbell 2002; Campbell 2005), including regulation of neurodevelopment (Turnley et al. 2001; Feng et al. 2007), adult neurogenesis (Ransome and Turnley 2007), neuroinflammation (Campbell et al. 2010; Baker et al. 2009; Gilli et al. 2010, 2011; Turnley et al. 2002b), and neurotrauma (Girolami et al. 2010; Qin et al. 2008; Hellstrom et al. 2011; Choi et al. 2009; Stark and Cross 2006). SOCS2 in particular has been shown to have neuron-specific effects. Cortical neurons from transgenic mice over-expressing SOCS2 (SOCS2TG) displayed enhanced neurite outgrowth and differentiated neurospheres from SOCS2TG mice contained more neurons with more numerous and complex processes (Goldshmit et al. 2004a; Scott et al. 2006). Conversely, cortical neurons and differentiated neurospheres from mice lacking SOCS2 (SOCS2KO) showed reduced numbers of neurites and reduced neurite length (Goldshmit et al. 2004a). Thus the level of SOCS2 expression is proportional to the amount of neurite outgrowth in cortical neurons and differentiated neurospheres. In addition, over-expression of SOCS2 promoted neurite outgrowth in PC12 cells, augmented by nerve growth factor (NGF) (Goldshmit et al. 2004b). This highlighted a potential interplay between Trk receptor signaling and SOCS2 activity.
Approximately 80% of dorsal root ganglion (DRG) neurons in newborn mice are sensory neurons that require NGF for their survival and neurite outgrowth in culture (Levi-Montalcini and Angeletti 1963; Skaper et al. 1982; Eichler and Rich 1989) and thus provide a useful in vitro system for the study of NGF-dependent survival and neurite outgrowth and hence SOCS2 and TrkA interactions. In order to further elucidate the mechanism by which SOCS2 regulates neurite outgrowth and to determine whether regulation of TrkA signaling is involved, we have now used a range of cellular and molecular analyses of primary sensory neurons derived from SOCS2 over-expressing and SOCS2 null mice, and transfection of TrkA and SOCS2 mutants into 239T cells and PC12 Tet-On cells. We report that SOCS2 interacts with and is a novel regulator of signal transduction by TrkA.
- Top of page
- Materials and methods
- Acknowledgments and conflict of interest disclosure
- Supporting Information
This paper aimed to better define the role of SOCS2 in neurons with particular attention to responses mediated by NGF. The extent and complexity of neurite outgrowth in DRG neurons cultured with NGF was found to be proportional to the amount of SOCS2 expression, consistent with the findings of previous studies of primary cortical neurons and differentiated neurospheres (Goldshmit et al. 2004a; Scott et al. 2006). SOCS2 has a credible role in regulation of DRG neuron development as it is highly expressed in embryonic day 14 mouse DRG and more generally the onset of SOCS2 expression in the nervous system appears to coincide with that of neuronal differentiation in the developing mouse (Polizzotto et al. 2000). As no alteration in DRG survival in the presence of limiting amounts of NGF was noted, it suggests that its developmental role may be limited to regulation of neurite outgrowth rather than survival, although an early role in DRG neurogenesis, as shown for cortical neurons (Turnley et al. 2002a), cannot be excluded at this stage. Given that the DRG neurons were taken from postnatal pups, where neurites have already been extended, it is possible that the effects of SOCS2 on NGF-mediated neurite outgrowth relate more to regeneration of severed neurites and we cannot exclude an effect on neuron survival at earlier embryonic time-points at this stage. The effects of SOCS2 on neurite length were relatively modest, with an apparently greater effect on neurite branching. NGF can differentially regulate neurite length versus branching in DRG neurons, with an enhancement of length at neonatal ages and promotion of branching in adult neurons (Yasuda et al. 1990). Further, the promotion of neurite branching induced by NGF is mediated by the AKT signaling pathway (Markus et al. 2002), which correlates with the SOCS2-induced enhancement of the AKT pathway we observed in PC12 cells. The capacity for SOCS2 to discriminate between neurite outgrowth and survival signals downstream of NGF stimulation draws interesting parallels with the function of the TrkA adapter molecule Fibroblast growth factor receptor substrate 2 (FRS2) (Peng et al. 1995) and suggests that SOCS2 may interact with FRS2 or be part of the same regulatory pathway.
TrkA expressed on the cell surface is required for binding the NGF ligand, thus the observed increase in TrkA at the surface of the cell soma of DRG neurons over-expressing SOCS2 may be a key to the altered responsiveness of these cells to NGF stimulation. The amount of the TrkA receptor presented at the cell surface and the rate at which it is internalized, recycled, or transported to different intracellular compartments can influence sensitivity of a cell to NGF and the type of signaling output (Zhang et al. 2000). In both DRG neurons and PC12 Tet-On cells, increased levels of TrkA were expressed on the cell surface. In DRG neurons, this increased surface expression was most notable in the absence of NGF, indicating that the internalization of TrkA in response to NGF was apparently normal in SOCS2-over-expressing cells. TrkA was internalized within 10 min in both wildtype and SOCS2 over-expressing cells, although at this stage we could not exclude a difference in the rate of internalization. In the PC12 Tet-On cells the increase in surface expression was difficult to distinguish from a total increase in TrkA expression levels. In contrast to control cells, PC12 Tet-On cells expressing SOCS2 showed increased surface TrkA phosphorylation upon NGF stimulation, suggesting an alteration in the kinetics of NGF-induced TrkA internalization or recycling. TrkA is known to undergo glycosylation during maturation and this post-translational modification ensures that it is properly trafficked to the cell surface and is only activated upon ligand binding (Watson et al. 1999b). TrkA is characterized by an immature ‘under-glycosylated’ species of 110 kDa that is the primary translated product that undergoes further glycosylation to become the mature fully glycosylated species of 140 kDa that is inserted into the plasma membrane (Clary and Reichardt 1994; Martin-Zanca et al. 1989). There is some evidence that the 110 kDa TrkA species is less phosphorylated than the 140 kDa species (Mardy et al. 2001) and this is the case in the TrkA expressed in PC12 cells (Fig. 4b).
As total levels of SOCS2 protein were not altered in SOCS2 over-expressing DRG neurons, a shift in the subcellular distribution of TrkA under basal conditions indicates a role for SOCS2 in the intracellular trafficking of the TrkA receptor. Constitutive TrkA receptor recycling between the plasma membrane and intracellular endosomes in the absence of ligand has been observed in sympathetic neurons, and appears to occur primarily within the cell body (Ascano et al. 2009). It is not known if sensory DRG neurons display similar TrkA recycling kinetics. The reason for the normal levels of TrkA in SOCS2-over-expressing DRG neurons, compared with increased levels in transiently transfected PC12 and 293T cells is not clear at this stage. Given that the effect of SOCS2 on TrkA levels does not require direct interaction with SOCS2, it suggests that increased levels of SOCS2 sequester ubiquitination complexes, protecting TrkA from degradation. In DRG neurons the lack of increased TrkA levels may reflect compensatory mechanisms and homeostasis, given the long term expression of both SOCS2 and TrkA in these cells from early developmental stages. It may also reflect the comparative level of expression of SOCS2 in the different cell types: in DRG neurons from SOCS2TG mice, the level of SOCS2 expression was approximately 2 fold higher than wildtype; in SOCS2 over-expressing PC12 cells expression levels ranged from 10 to 20 fold higher than controls (data not shown), whereas 293T cells do not normally express detectable levels of SOCS2.
Different signaling intermediates are recruited to the activated receptors in different parts of the cell, thus the regulated endocytosis and trafficking of activated NGF-TrkA complexes permits fine-tuning of downstream signaling events. Upon NGF binding to TrkA at the cell surface, the activated-receptor complex is rapidly internalized in PC12 cells (Hosang and Shooter 1987; Zhou et al. 1995; Grimes et al. 1996). These internalized membrane vesicles mature to become early endosomes, and the fate of these endosomes dictates the duration and type of signal transmitted. As some of these vesicles return to the plasma membrane either directly or via recycling endosomes and restore sensitivity to further stimuli, others fuse with lysosomes that enclose the cytoplasmic receptor tail and shut down the signal by degrading the internalized protein cargo (Chen et al. 2005; Georgieva et al. 2011). Importantly, a subset of early endosomes evades degradation by the lysosomes to become ‘signaling endosomes’ that contain activated TrkA receptors that are trafficked toward the cell soma to propagate cell survival and differentiation signals (Grimes et al. 1996; Bhattacharyya et al. 1997; Ehlers et al. 1995; Tsui-Pierchala and Ginty 1999; Watson et al. 1999a; Jullien et al. 2002, 2003; Delcroix et al. 2003). Ubiquitination is a post-translational modification that has an important role in both secretory and endocytic pathways [reviewed by (Acconcia et al. 2009; MacGurn et al. 2012)]. Ubiquitin modifiers such as TNF receptor associated factor-6 (TRAF6), Nedd4-2, and c-Cbl regulate the trafficking, sorting and stability of TrkA (Geetha et al. 2005; Arevalo et al. 2006; Georgieva et al. 2011; Yu et al. 2011; Takahashi et al. 2011). Differential expression of one or more of these signaling intermediates in DRG neurons versus PC12 Tet-On cells probably explains the different effects of SOCS2 over-expression on total levels of TrkA. In DRG neurons TrkA turnover appears normal, whereas in PC12 Tet-On cells it appears to be impaired.
SOCS2 is an ubiquitin ligase that can promote the degradation of a variety of cytokine and growth factor signaling intermediates (Pezet et al. 1999; Bullock et al. 2006; Rico-Bautista et al. 2006; Piessevaux et al. 2008; Babon et al. 2009; Lee et al. 2010; Kazi and Ronnstrand 2013) and directly ubiquitinates the growth hormone receptor, regulating its half-life (Vesterlund et al. 2011). Interestingly, SOCS2 is implicated in the ubiquitination and proteasomal degradation of the E3 ubiquitin ligase TRAF6 (McBerry et al. 2012), and altered regulation of TRAF6 has been shown to influence the accumulation of Lys-63 ubiquitinated TrkA in vivo (Wooten et al. 2008). Thus, ubiquitin modification may be a mechanism by which SOCS2 influences signaling via the TrkA receptor.
To better define the biochemical relationship between SOCS2 and neurotrophin signaling, protein complexes were immunoprecipitated from lysates of 293T cells over-expressing neurotrophin receptors. SOCS2 bound to TrkA, TrkB, and TrkC, but not p75NTR. It is not known if the site of interaction is a single motif that is highly conserved amongst these proteins, or whether SOCS2 has a unique interface with each of these receptors. For this study, only TrkA receptor association with SOCS2 was examined in detail. Immunoprecipitation of truncated forms of the TrkA receptor with SOCS2 revealed that the juxtamembrane region (residues 452-493) of TrkA is at least partially required for interaction with SOCS2. This region of TrkA does not contain any tyrosine motifs that might directly bind the SH2 domain of SOCS2, thus suggesting that SOCS2 may interact with TrkA via an adapter protein. A conserved motif adjacent to this region (449–452 KFG) has been implicated in the association of TrkA with the adapter molecule FRS2 (Peng et al. 1995), however, this region was shown to be dispensable for association with SOCS2. The TrkA juxtamembrane region (472–493) has previously been shown to associate with the 14 kDa dynein light chain and 74 kDa dynein intermediate chain (Yano et al. 2001), p62/ZIP/Sequestosome 1 (Geetha and Wooten 2003), and the PDZ domain protein GAIP interacting protein (GIPC) (Lou et al. 2001). Furthermore, this region contains residue K845 that is the site of TRAF6-mediated ubiquitination (Geetha et al. 2005). Thus it is possible that SOCS2 is associating with TrkA via association with any number of adapter molecules. Systematic mutation of individual residues within 452–493 may address the precise molecular basis for interaction between SOCS2 and TrkA and mass spectrometry of immunoprecipitated protein complexes is required to determine the breadth of SOCS2 interacting proteins.
Neurite outgrowth of PC12 Tet-On cells transfected with SOCS2 or SOCS2 mutants was examined under basal conditions and with NGF. As shown in previous studies (Goldshmit et al. 2004b), SOCS2 over-expression increased neurite outgrowth under basal conditions and with NGF. Mutation of the SH2 domain prevented this enhanced neurite outgrowth and deletion of the SOCS box blocked NGF-induced neurite outgrowth. Over-expression of SOCS2 in these cells increased the level and duration of phosphorylation of several signal transduction pathways in response to NGF stimulation, in particular pAKT and pErk1/2, both of which are well known to play roles in regulation of neurite outgrowth (Segal 2003). Interestingly, given that SOCS2 is an ubiquitin ligase, these same pathways were potentiated in PC12 cells exposed to the proteasome inhibitor MG132, which resulted in increased neurite outgrowth and stabilization of the TrkA receptor but did not require internalization of the TrkA receptor (Song et al. 2009; Song and Yoo 2011).
This study has focused on the role of SOCS2 in TrkA receptor biology in neurons. There is also scope for broader investigation into non-neuronal cell types, such as glial and hematopoietic lineages, that engage Trk receptors (Althaus et al. 2008; Koch et al. 2008) and SOCS2 (Rico-Bautista et al. 2006). Further, elevated levels of NGF have been implicated in chronic pain conditions and NGF and the TrkA receptor have been the targets of various therapeutic strategies to address this condition (Watson et al. 2008; Eibl et al. 2012; McKelvey et al. 2012). A better understanding of molecules that regulate the signaling downstream of NGF stimulation may inform the development of future therapeutic interventions.
We have demonstrated a novel role for SOCS2 in the regulation of the biology of the NGF receptor TrkA. The central hypothesis was that SOCS2 modulated the responsiveness of the TrkA receptor to an NGF stimulus and thus altered downstream signaling events that culminated in changes to neurite outgrowth. SOCS2 was shown to regulate neurite complexity and localization of TrkA in cultured dorsal root ganglia neurons from mice with altered SOCS2 expression. Mechanisms of interaction and effects on TrkA-mediated neurite outgrowth correlated with alterations in AKT and Erk1/2 signaling. This work provides compelling evidence for a novel role for SOCS2 in the regulation of neurotrophin signaling.