Department of Anatomy and Neuroscience, Centre for Neuroscience Research, The University of Melbourne, Parkville, Victoria, Australia
Address correspondence and reprint requests to Ann Turnley, Department of Anatomy and Neuroscience, Kenneth Myer Building, 30 Royal Parade, The University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: firstname.lastname@example.org
Suppressor of cytokine signaling-2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over-expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet-On cells. Over-expression of SOCS2 in PC12 Tet-On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over-expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2-induced neurite outgrowth in PC12 Tet-On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.
Neurotrophins such as Nerve growth factor (NGF) are important for the neurite outgrowth and survival of sensory neurons. Increased expression of SOCS2 enhanced NGF-dependent neurite outgrowth and increased TrkA receptor surface localization in dorsal root ganglion neurons and PC12 cells. This correlated with increased and prolonged activation of pAKT and pErk1/2. In 293T cells SOCS2 was shown to interact with the TrkA receptor in the juxtamembrane region. We thus propose that SOCS2 is a novel regulator of neurotrophin signaling.
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.
Materials and methods
Mice were maintained on the C57Bl/6 background. SOCS2 transgenic (SOCS2TG) mice constitutively over-express SOCS2 in all tissues under the control of the human ubiquitin C promoter (Greenhalgh et al. 2002). SOCS2 knockout (SOCS2KO) mice lack the entire SOCS2 coding region and thus do not express any SOCS2 transcript or protein (Metcalf et al. 2000). Animal experimentation was approved by the University of Melbourne Animal Ethics Committee and performed in accordance with the NH & MRC of Australia code for the care and use of animals for scientific purposes.
Mammalian expression constructs for rat TrkA, TrkB, TrkC, and HA tagged p75NTR were in pCDNA3, rat TrkA and mutants in pCMV5 and mouse SOCS2 and mutants in pFLAG-CMV2.
PC12 Tet-On cell culture and transfection
The PC12 Tet-On cell line (ATCC) was propagated essentially as previously described (Goldshmit et al. 2004b). The Amaxa Cell line Nucleofector Kit V (Lonza, Cologne, Germany) was employed for PC12 Tet-On transfections, according to manufacturer's instructions, using program U-029 with approximately 3 million cells per reaction and 2 μg pFLAG-CMV2 vector or pFLAG-CMV2- SOCS2 FL, pFLAG-CMV2-SOCS2 SH2 3M or pFLAG-CMV2 SOCS2ΔBox (Greenhalgh et al. 2005) and 1 μg pMAX green fluorescent protein (GFP) control vector (Lonza). Cells were plated at 500 000 cells per well in a 6-well plate for protein analyses or 2500 cells per well in a 24-well plate for morphological analyses. The day following nucleofection, the media was replaced with proliferation medium or differentiation medium. Green fluorescence was observed within 24 h post-nucleofection and cells were incubated for 72 h before assay. For morphological analyses, the proportion of cells with at least one neurite greater than one cell body in length was recorded and maximum neurite length was measured as previously described (Goldshmit et al. 2004b). In some experiments, the K252a Trk inhibitor (Sigma-Aldrich, St Louis, MO, USA) or dimethylsulfoxide control was added to the culture media at least 30 min prior to addition of NGF.
293T cell culture
293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% v/v heat inactivated calf serum and 100 U/mL penicillin and 100 μg/mL streptomycin. For transfection, approximately 2 million 293T cells were cultured per well in a 6-well plastic plate in 2 mL of media with no antibiotics. For each well, cells were transfected with 2 μg of plasmid DNA and 10 μL Lipofectamine 2000 (Invitrogen, Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions.
Dorsal root ganglion dissection and neuronal culture
DRG neuron cultures were prepared from individual neonatal mice, essentially as previously described (Turnley et al. 2002b). Cells were plated in culture media Neurobasal A (Gibco, Life Technologies) with 2% v/v B27 (Gibco), 2 mM GlutaMax I (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0-50 ng/mL NGF or 10 ng/mL LIF. For DRG survival assays, cells were plated on fibronectin-coated 96-well plastic plates, in triplicate. For DRG neurite outgrowth assays, cells were plated on 24-well plastic plate or 8-well glass chamber slides coated with P-orn and mouse laminin (Invitrogen) in 50 ng/mL NGF. DRG neurite outgrowth cultures were fixed in 4% w/v paraformaldehyde, permeabilized with methanol, and then examined by immunocytochemistry for the neuronal marker βIII tubulin, using rabbit anti-βIII tubulin (Promega, Madison, WI, USA) and standard protocols.
Protein extraction, immunoprecipitation, and analysis
Protein lysates were prepared from tissues or cultured cells by washing in phosphate-buffered saline ( PBS) followed by incubation with lysis buffer [20 mM Tris-HCl, pH 7.6, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% v/v Triton X-100, 10% v/v glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, Complete PI protease inhibitor cocktail (Roche, Basel, Switzerland), and 0.5 mM PefaBloc SC (Roche)] for 30 min on ice. Lysates were clarified by centrifugation at 18 000 g for 10 min at 4°C. Protein lysates were pre-cleared with Protein G agarose (Sigma) for 1 h at 4°C before immunoprecipitation (IP). A 50 μg input sample was retained and 450 μg lysate was incubated with 1 μg of FLAG M2 (Sigma) or 5 μL pan-Trk C-14 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. For each IP, 30 μL Protein G agarose beads were incubated with 450 μg lysate at 4°C with gentle rotation for 2 h. Beads were washed four times with lysis buffer and immunoprecipitated proteins eluted by incubation with reducing Laemmli sample buffer for 10 min at 95°C. The IP eluates and input samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. Antibodies to detect pan-Trk C14 (Santa Cruz Biotechnology), TrkA, TrkB, TrkC, HA, and ubiquitin (clone P4D1) (Cell Signaling Technology, Danvers, Beverly, MA, USA), TrkA extracellular domain (Millipore AB1577), FLAG M2 (Sigma), and SOCS2 (Abcam, Cambridge, UK) were employed.
Cell surface proteins on PC12 Tet-On cells transfected with SOCS2 FL, SOCS2ΔBox or vector control were biotinylated using the NHS-Ezy Biotinylation kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Biotinylated cell surface proteins were immunoprecipitated using streptavidin-conjugated beads then total and surface levels of TrkA were analyzed by SDS–PAGE and western blot analysis.
Signaling pathway analyses
PC12 Tet-On cells transfected with SOCS2 FL and mutant constructs were starved for 2 to 4 h, NGF (50 ng/mL) was added for 10-20 min, then washed out, with lysates taken at indicated time-points before and after washout. SDS–PAGE and western blots were used to detect levels of total and phosphorylated Extracellular signal-regulated kinase 1/2 (Erk1/2) and Protein Kinase B (AKT).
TrkA Antibody feeding assay
DRG neuron cultures were washed twice to remove residual NGF 24 h post-plating. Starve media (500 μL DRG culture media with 1% v/v fetal calf serum (FCS) was incubated with cells for 2 h to achieve steady-state levels of TrkA surface expression in the absence of NGF. Half of the media was removed from each well and starve media applied to ‘no NGF’ and negative control wells or starve media with 100 ng/mL NGF applied to ‘NGF’ treated wells, and incubated for 10 min at 37°C. Media was then removed from each well and replaced with starve media (negative control wells only) or anti-TrkA antibody that recognized the extracellular domain of TrkA (1 : 100; Biosensis, Thebarton, Australia) and incubated for 10 min at 37°C. Cells were fixed in 4% paraformaldehyde for 20 min at 23°C, washed twice with PBS, incubated in blocking solution [PBS, 2% v/v heat inactivated calf serum and 2% v/v normal goat serum (Invitrogen)] for 20 min at 23°C, followed by goat anti-rabbit Cy3 conjugated secondary antibody (1 : 1000; Invitrogen), and 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. Images of immunostained neurons were captured using the same exposure time for all samples. TrkA staining on the surface of the cell soma of DRG neurons was assessed using ImageJ software (version 1.44o, National Institutes of Health, USA, http://imagej.nih.gov/ij) to quantify the signal intensity. The image was inverted and the measurement criteria set to record the area, integrated density and limit the measurement to the area covered by the chosen threshold. The threshold was manually adjusted to match the TrkA signal. A box was drawn to surround the cell body of interest. The integrated density of pixels within the box that were covered by the threshold was measured. TrkA labeling was performed on two separate cohorts of animals. Both SOCS2TG and wildtype animals (n = 4 of each genotype per experiment) were represented in both cohorts. In order to effectively pool signal intensity data from both cohorts, the data from each experiment were transformed by subtracting the global mean of the experiment from each of the observations in that experiment. This resulted in the distribution of observations from all experiments being centred at zero. The data were presented as relative frequency histograms and a mean and SEM from the transformed values from each animal (n = 8 per genotype) were calculated to enable statistical testing between genotypes and conditions.
DRG neuron survival assays
Live cells growing in 96-well plates were imaged at low (4×) magnification. Viable DRG neurons were identified by their large, rounded cell bodies, and the presence of neurites. The same wells were assessed at 1, 24, 48, and 72 h and cell numbers were normalized within each sample by expressing the ‘relative survival’ as the number of viable cells at 24, 48 or 72 h post-plating as a percentage of the number of viable cells at 1 h post-plating.
DRG neurite outgrowth and morphology
A short time-point of 4 h was chosen for measurement of neurite length, as neurite outgrowth was so robust that cells incubated for longer time-points, such as 18 h had neurites that were too long, too branched, and overlapping to be accurately measured. ImageJ was employed for image analysis. Neurite complexity was qualitatively described and categorized as no neurites greater than one cell body in length, short neurites between one and three cell bodies in length, and long neurites greater than three cell bodies in length. Neurite complexity was quantified for non-overlapping neurons using the ImageJ Sholl Analysis Plugin, obtained from the Ghosh Lab (http://www-biology.ucsd.edu/labs/ ghosh/software/). In the plugin dialog box, the starting radius was 0.1, the ending radius was as per the length of the current line selection (that is the length of the longest neurite), the radius step size was 0.05, the radius span was 0.001, and the radius span type defined as median. The number of crossings at each radius for each measured cell was exported for analysis.
All experiments were performed at least three independent times for cell lines or from at least three independent mice of each genotype (exact numbers provided in figure legends). Results were expressed as mean ± SEM and significance of effects assessed by use of unpaired t-test, with p <0.05 considered significant.
Gain or loss of SOCS2 does not alter NGF-dependent survival of DRG neurons
To assess whether altered levels of SOCS2 could influence the survival of cultured DRG neurons, DRG neurons were dissected from wildtype, SOCS2TG, SOCS2KO, and SOCS2KO heterozygous (SOCS2HET) neonatal mice and cultured in 0-50 ng/mL NGF for 3 days. To determine whether any altered survival that may be because of altered SOCS2 expression was specific to NGF, a separate treatment of 10 ng/mL LIF, another factor known to support DRG neuron survival (Murphy et al. 1993, 1991), was included. The number of viable cells was assessed at 1, 24, 48, and 72 h post-plating. As expected, DRG neurons showed enhanced viability in higher concentrations of NGF (50 ng/mL), confirming that a proportion of the cells cultured were dependent upon NGF for survival. The genotype of the animals had no influence on the relative survival of DRG neurons at each concentration of NGF suggesting that neither gain nor loss of SOCS2 altered the survival in response to NGF (Fig. 1a). Similarly, there was no difference in survival observed in the presence of LIF within each genotype (Fig. 1b).
Neurite outgrowth in DRG neuron cultures is regulated by SOCS2 expression
To assess whether altered levels of SOCS2 could influence the neurite outgrowth of cultured DRG neurons, DRG neurons were dissected from SOCS2TG, SOCS2KO, and SOCS2HET neonatal mice and cultured in NGF. After 4 h, neurons were fixed and immunostained for the neuronal marker βIII tubulin. Neurite morphology was initially broadly classified using a qualitative measure on the basis of neurite length as either bearing no neurites, short neurites, or long neurites (Fig. 2a). Over-expression of SOCS2 promoted an increased proportion of neurons with longer, generally more complex neurites, whereas loss of SOCS2 resulted in shorter and simpler neurites (Fig. 2b). DRG neurons with half the normal expression of SOCS2 (SOCS2HET) exhibited neurite morphology similar to that of the SOCS2KO DRG neurons, suggesting that loss of a single copy of SOCS2 can elicit differences in neurite outgrowth responses to NGF.
The number of intersecting processes at intervals from the cell soma was also measured using the quantitative Sholl analysis method (Fig. 2c). Sholl analysis revealed that DRG neurons from SOCS2TG animals exhibited a significant increase in the extent of neurite branching (Fig. 2d). Loss of SOCS2 (SOCS2KO and SOCSHET) had no impact on the extent of neurite branching (Fig. 2e).
SOCS2 associates with TrkA, TrkB, and TrkC, but not p75NTR
To explore the molecular basis of the SOCS2 action on NGF-mediated neurite outgrowth, 293T cells were transfected with expression constructs for SOCS2 and each of the neurotrophin Trk receptors; the Trk receptors TrkA, TrkB, and TrkC and the tumor necrosis receptor family member p75NTR. Cell lysates were harvested 24–48 h post-transfection and immunoprecipitated with a FLAG antibody to isolate the FLAG-tagged SOCS2 protein and any interacting proteins. Western blots of total lysates and immunoprecipitates were probed for TrkA, TrkB, TrkC or HA to detect HA-tagged p75NTR and showed that SOCS2 interacted with TrkA, TrkB, and TrkC but not p75NTR (Fig. 3a). In addition, the reciprocal immunoprecipitation, using an anti-pan Trk antibody, pulled down SOCS2 (Fig. 3b).
SOCS2 associates with the juxtamembrane region of TrkA
To determine the region of the TrkA receptor required for interaction with SOCS2, TrkA with various deletions or point mutations in the cytoplasmic tail of the receptor (Fig. 3c) were co-expressed with FLAG-SOCS2 in 293T cells and protein associations tested by co-immunoprecipitation (Fig. 3d). Progressive truncations of the cytoplasmic tail of TrkA showed a diminished capacity to interact with SOCS2. Once TrkA was truncated to amino acids 1-493, it only very weakly immunoprecipitated with SOCS2. Almost all binding of TrkA was lost upon deletion to residues 1-472 and binding was completely ablated with deletion to 1-452. These data suggest that the region of critical binding between SOCS2 and TrkA lies somewhere within residues 453–493 of TrkA. To specifically interrogate the TrkA juxtamembrane region for binding with SOCS2, TrkA receptor mutants with discrete portions of the juxtamembrane region deleted or mutated were examined (Fig. 3d). Deletion of the five residues surrounding the Src binding site Y499 (residues 495–500) did not alter expression levels or capacity to bind SOCS2. A highly conserved motif (K450, F451, and G452) immediately adjacent to the proposed 453–493 binding region was also investigated. These three residues were examined because they are highly conserved amongst the Trk receptors and have published significance with respect to NGF-dependent neurite outgrowth (Peng et al. 1995). Deletion of residues 450–452 had no effect on the ability of TrkA to immunoprecipitate with SOCS2. Deletion of a majority of the juxtamembrane region (residues 453–517) resulted in a partial loss of association between SOCS2 and TrkA, depending on the antibody used to detect TrkA – use of the anti-TrkA from Cell Signaling Technology indicated no interaction (Fig. 3d) whereas an anti-TrkA extracellular domain from Millipore indicated loss of interaction with the higher MW (glycosylated) form of TrkA (Fig. 3e). Interestingly, the TrkA Y499A substitution resulted in TrkA with enhanced expression levels and this mutant exhibited stronger binding to SOCS2 than wildtype TrkA. Similarly, more subtle and somewhat variable increases in TrkA levels were observed with the different TrkA constructs upon co-expression with SOCS2 (Fig. 3d,e). This SOCS2-induced increase in TrkA expression was not influenced by the extent to which each TrkA construct could immunoprecipitate with SOCS2 in 293T cells (Fig. 3e), indicating that the regulation of total TrkA levels by SOCS2 is independent of direct binding.
SOCS2 interacts with endogenous TrkA in PC12 Tet-On cells and increases TrkA protein expression levels
To determine whether SOCS2 could interact with endogenous, rather than over-expressed TrkA, FLAG-SOCS2 was transfected into PC12 Tet-On cells, which endogenously expressed TrkA, as well as SOCS2. IP and TrkA western blots confirmed that SOCS2 interacted with endogenously expressed TrkA (Fig. 4a). SOCS2 over-expression in these cells also increased total TrkA levels independent of NGF, whereas NGF enhanced the amount of TrkA that interacted with SOCS2 (Fig. 4a).
The effect of SOCS2 on surface expression and phosphorylation of endogenous TrkA was then examined. Full length SOCS2 (SOCS2FL), a mutant of SOCS2 lacking the SOCS box domain (SOCS2ΔBox; Fig. 6a) and empty vector control were transfected into PC12 Tet-On cells and surface proteins were biotinylated. Analysis of total levels of TrkA again showed an increased expression induced by SOCS2 over-expression, with no effect on these levels by SOCS2ΔBox, NGF addition or inhibition of TrkA signaling by the inhibitor K252a. IP of biotinylated proteins followed by western blot for TrkA indicated that there were increased levels of surface TrkA when SOCS2 was over-expressed, which correlated with the increased total levels, and decreased levels of surface TrkA when the SOCS2ΔBox mutant was expressed (Fig. 4b). Addition of NGF induced a reduction of surface TrkA, particularly for control and SOCS2ΔBox. There was little phosphorylated TrkA at the cell surface with the control vector, reflecting internalization of NGF-bound TrkA, whereas phospho-TrkA levels in total lysates correlated with total TrkA levels. Surface phospho-TrkA was detected in the presence of SOCS2FL and SOCS2ΔBox constructs, which was ablated by addition of the TrkA inhibitor K252a (Fig. 4b).
SOCS2 over-expression does not significantly alter total TrkA protein expression in DRG neurons but increases TrkA cell surface expression
Given that SOCS2 regulated TrkA levels and surface localization in PC12 Tet-On cells, we next determined if over-expression of SOCS2 altered the total amount of TrkA protein in primary DRG neurons. Lysates were prepared from whole DRGs dissected from wildtype or SOCS2TG neonatal mice and probed by western blot. In contrast to findings from SOCS2 transfected PC12 Tet-On cell lines, no significant differences were detected (Fig. 5a).
To determine whether SOCS2 regulated the location of TrkA, surface expression of the TrkA receptor with and without NGF stimulation was examined in cultured DRG neurons from SOCS2 transgenic and wildtype animals. Cells were cultured in NGF for 24 h, then starved for 2 h. Media with or without NGF was then added to the culture for 10 min, followed by TrkA antibody. TrkA staining on the surface of the cell soma was imaged (Fig. 5b and Fig. S2) and the intensity of TrkA labeling on the surface of the cell soma was quantified. Data were transformed by subtraction of the ‘global mean’ intensity from all observations within each experiment, to allow comparison of neurons from different cohorts of mice and presented as a TrkA intensity distribution (Fig. 5c) and the mean transformed TrkA intensity (Fig. 5d). TrkA receptors were internalized upon NGF stimulation and less surface labeling of TrkA was detected after addition of NGF in cells of both genotypes compared with no NGF. SOCS2TG DRG neurons showed significantly more surface TrkA without NGF than with NGF, however, the difference was not significant in wildtype DRG neurons. When comparing TrkA staining in DRG neurons of different genotypes, there was no significant difference in surface TrkA staining between SOCS2TG and wildtype DRG neurons upon the addition of NGF but SOCS2TG DRG neurons displayed an increased intensity of surface labeled TrkA in the absence of NGF, which was also visible in neurites (Fig. 5b).
SOCS2 SH2 and SOCS box domains are required for SOCS2-induced neurite outgrowth in PC12 Tet-On cells
To explore the domains of SOCS2 required for the regulation of neurite outgrowth, PC12 Tet-On cells were transiently transfected with vectors encoding SOCS2FL, SOCS2 SH2 3M, SOCS2ΔBox (Fig. 6a) or a GFP vector control and neurite outgrowth in the presence or absence of NGF was analyzed. Cells were co-transfected with a GFP expression construct to help visualize cell morphology. Cells were cultured for 3 days post-transfection in either proliferative media or differentiation media containing NGF. GFP expressing cells were scored for the presence of neurites. Under proliferative conditions (−NGF) over-expression of SOCS2FL induced a significant increase in the amount of neurite outgrowth in PC12 Tet-On cells relative to the other expression constructs, as previously shown (Goldshmit et al. 2004b) (Fig. 6b). Under differentiation conditions (+NGF) over-expression of SOCS2FL enhanced the amount of NGF-induced neurite outgrowth. Under all circumstances, mutation of the SOCS2 SH2 domain yielded similar neurite outgrowth as the GFP-only control, whereas the SOCS2ΔBox protein attenuated NGF-induced neurite outgrowth both in terms of the percentage of cells producing neurites and the length of the neurites (Fig. 6b,c). Collectively, these data suggested that the SH2 domain and the SOCS box were both required for SOCS2-induced neurite extension.
To determine whether the altered neurite outgrowth induced by SOCS2 correlated with alterations in signal transduction pathways, expression of a range of TrkA signaling pathway proteins was examined in an NGF pulse-chase experiment in PC12 Tet-On cells. Of the pathways examined, pAKT (Ser473) (Fig. 6d) and pErk1/2 (Fig. 6e) showed the most robust differences, with SOCS2FL inducing higher levels and sustained activation of pAKT and pErk1/2.
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.
Acknowledgments and conflict of interest disclosure
This work was supported by an NHMRC Fellowship (grant number #628344) to A.M.T, an Australian Postgraduate Award to R.T.U. and a Melbourne Faculty Research Grant. The authors declare no conflicts of interest.
All experiments were conducted in compliance with the ARRIVE guidelines.