UBXN2A regulates nicotinic receptor degradation by modulating the E3 ligase activity of CHIP

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit are known for their prominent role in normal ganglionic transmission while their involvement in the mechanisms underlying nicotine addiction and smoking-related disease has been emerging only in recent years. The amount of information available on the maturation and trafficking of α3-containing nAChRs is limited. We previously showed that UBXN2A is a p97 adaptor protein that facilitates the maturation and trafficking of α3-containing nAChRs. Further investigation of the mechanisms of UBXN2A actions revealed that the protein interacts with CHIP (carboxyl terminus of Hsc70 interacting protein), whose ubiquitin E3 ligase activity regulates the degradation of several disease-related proteins. We show that CHIP displays E3 ligase activity toward the α3 nAChR subunit and contributes to its ubiquitination and subsequent degradation. UBXN2A interferes with CHIP-mediated ubiquitination of α3 and protects the nicotinic receptor subunit from endoplasmic reticulum associated degradation (ERAD). UBXN2A also cross-talks with VCP/p97 and HSC70/HSP70 proteins in a complex where α3 is likely to be targeted by CHIP. Overall,we identify CHIP as an E3 ligase for α3 and UBXN2A as a protein that may efficiently regulate the stability of CHIP's client substrates.

Keywords: CHIP; Proteasomal degradation; UBX domain; UBXN2A; p97; α3 Nicotinic subunit.

Fig. 1

UBXN2A interacts with the E3 ligase CHIP, and CHIP mediates α3 nAChR subunits ubiquitination. (a) A Y2H screen using full length mouse UBXN2A cDNA as bait revealed that UBXN2A interacts with the tetratricopeptide repeat (TPR) domain of CHIP. The TPR domain mediates CHIP–Hsc70–Hsp70 and Hsp90 interaction whereas two other domains of CHIP i.e., U-box and mixed charge with predicted coiled-coil region mediate the E3 ubiquitin ligase and dimerization activities of CHIP, respectively. A set of nutritional selection and β-GAL assays were performed to confirm the identified interactions. The cartoon shows the location of CHIP’s domains and where UBXN2A binds to CHIP according to the Y2H screen. (b) HA-UBXN2A can be detected in the (His)₆-CHIP samples pulled down from lysates of HEK293 cells co-transfected with His-CHIP and HA-UBXN2A, but not in the pull-down samples from lysates of HEK cells co-transfected with HA-UBXN2A and (His)₆-empty vector. (c) Differentiated PC12 cells were homogenized and subjected to immunoprecipitation with anti-α3 antibodies followed by SDS-PAGE and immunoblotting with anti-ubiquitin antibodies. These experiments showed that CHIP overexpression (right lane, left panel) significantly increases the levels of ubiquitinated α3 in this cell culture system (n = 3 separate cultures, *p <0.02). (d) We also examined how lack of CHIP affects α3 ubiquitination in mice null for the E3 ligase and their littermate controls. The levels of ubiquitinated α3 in brain homogenates from CHIP −/− mice were significantly lower than those from wild-type littermates (n = 5 mice/group, **p <0.001).

Fig. 2

CHIP decreases the stability of the α3 nAChR subunit. (a) The levels of α3 protein decreased in correspondence with increasing expression of CHIP in differentiated PC12 cells co-transfected with pcDNA-α3 expression plasmids and different amounts of pcDNA-(His)₆-CHIP plasmid (n = 3 separate cultures, *p < 0.02, **p < 0.005). (b) In differentiated PC12 cells, overexpression of the H260Q CHIP mutant, which is devoid of E3 ligase activity, had no significant effect on α3 stability compared to wild-type CHIP overexpression (n = 4 separate experiments, *p ≤ 0.02). (c) The levels of α3 are significantly higher in the brain homogenates of CHIP −/− mice than those of wild type mice (n = 4 mice/group, *p <0.02). (d and e) We also examined the levels of β2 nAChR subunits in brain homogenates of CHIP−/− and wild type mice and found that the levels of both ubiquitinated (panel d, n = 4 mice/group, p > 0.5) and total (panel e, n = 3 mice/group, p > 0.05) β2 protein were not different between the two mouse groups. (f) Finally, as an example of another ligand-gated ion channel, we measured the levels of GABA_A receptor proteins and found no significant changes in control vs. CHIP −/− brains (n = 4 mice/group, p > 0.5). ROD, relative optical density.

Fig. 3

UBXN2A affects CHIP/α3 interactions and interferes with CHIP-mediated α3 nAChR ubiquitination and degradation. (a and c). The interaction between CHIP and α3 was investigated in differentiated PC12 cells. PC12 cells were co-transfected with either CHIP+empty vector or CHIP + UBXN2A followed by immunoprecipitation experiments with anti-CHIP antibodies. UBXN2A overexpression weakened α3/CHIP interactionsin differentiated PC12 cells (n = 3 separate experiments, **p <0.01). (b and c). Similar immunoprecipitation experiments were conducted to examine the interaction between CHIP and α3 when UBXN2A levels were reduced using UBXN2A shRNA. The interaction between CHIP and α3 was significantly enhanced upon UBXN2A knock-down (n = 4 separate experiments, **p <0.01). (d and e) Levels of ubiquitinated α3 were measured in cell lysates of differentiated PC12 cells co-transfected with CHIP, CHIP + UBXN2A or corresponding empty vectors. The experiments revealed that UBXN2A overexpression inhibits CHIP-mediated ubiquitination of α3 (panel d, n = 4 separate experiments, *p <0.05 CHIP vs. CHIP + UBXN2A), resulting in higher levels of total α3 protein (panel e, n = 4 separate experiments, *p <0.05 CHIP vs. CHIP + UBXN2A.

Fig. 4

UBXN2A is found in a complex with CHIP/p97/α3 and facilitates the formation of p97/α3 complexes. (a) Immunoprecipitation experiments conducted on differentiated PC12 cell lysates using anti-CHIP antibodies revealed the presence of a complex containing CHIP, p97, α3 and UBXN2A. (b) UBXN2A, CHIP and α3 were also detected in p97 immunoprecipitates from differentiated PC12 cell lysates. (c) Differentiated PC12 cells transiently transfected with CHIP were treated with DBeQ, a potent and selective p97 inhibitor, followed by SDS-PAGE and probing with anti-α3 antibodies. The experiments showed that DBeQ treatment significantly blocked CHIP-mediated α3 degradation (n = 4 separate experiments, CHIP vs. Control, **p <0.001; CHIP + DBeQ (5 μM) vs. CHIP + no treatment, #p <0.02, CHIP + DBeQ (10 μM) vs. CHIP + no treatment, ##p <0.001). (d) The amount of α3 that co-immunoprecipitated with p97 was reduced by UBXN2A knockdown (**p <0.001, n = 3 separate experiments). (e) UBXN2A expression was effectively reduced in dPC12 cells transfected with pSuper vector-based UBXN2A shRNA. GAPDH was probed as loading control.

Fig. 5

UBXN2A/p97 complexes are associated with the proteasome mega complex at the ER–Golgi intermediate space. (a) Fractions of mouse prefrontal cortex homogenates subjected to iodixanol gradient centrifugation were analyzed by Western blot analysis. UBXN2A co-sediments with KDEL (an ER, cis-Golgi marker) and GM130 (a cis-Golgi marker), suggesting that UBXN2A can be found in ER–Golgi compartments in neuronal tissues. (b) The right panel displays the strategy we used forthe subcellular fractionation of membrane proteins from mouse PFC tissue. That approachfurther showed that UBXN2A is enriched in ER/Golgi membrane fractions, but not in synaptosomal or vesicular membrane fractions (right panel). (c) IP experiments using anti-ERGIC53 antibody showed that UBXN2A co-immunoprecipitates with ERGIC53, indicating that UBXN2A may exist in the ER/Golgi intermediate compartment. (d) Immunofluorescence staining of rat hippocampal neurons revealed that the majority of UBXN2A localizes in the soma while some is present at dendritic bifurcations and axon initial segments. UBXN2A (green), MAP2 (blue) and neurofascin (red). (e) UBXN2A and p97 associate with the 26S protesome complex purified from mouse prefrontal cortex. Anti-Pan α and anti-S8 ATPase antibodies were used to detect components of the 26S proteasome. The cis-Golgi marker, GM130 could not be detected in the purified proteasome sample, confirming the successful isolation of partially purified proteasomal complexes.

Fig. 6

UBXN2A interacts with p97 via the UBX domain while it interacts with α3 nAChR via the SEP domain. (a) A pull down experiment conducted on mouse PFC homogenates using in vitro-expressed, (His)₆-tagged UBXN2A showed that p97 can be pulled down by UBXN2A. (b) Schematic diagrams of wild type and several truncated UBXN2A cDNAs cloned into a (His)₆-Tyg peptide tagged pcDNA 3.1 vector. (c) Talon metal affinity resin pulldown experiments conducted in differentiated PC12 cell lysates expressing each construct listed in panel b showed that only WT UBXN2A and truncated UBXN2A containing the UBX domain can pull down p97. These results confirm that UBXN2A interacts with p97 by UBX domain. (d) Immunoprecipitation experiments conducted with anti-Tyg antibodies from the same cell lysates used for the experiment inpanel c showed that only WT UBXN2A and the SEP domain truncated UBXN2A interact with α3, suggesting that UBXN2A interacts with α3 via the SEP domain.

Fig. 7

UBXN2A alters CHIP-mediated α3 nAChR subunit ubiquitination and ERAD by interfering with CHIP’s E3 ligase activity. The cartoondepicts a model for the interaction between CHIP and α3. (a) CHIP, as an E3 ligase and co-chaperone, ubiquitinates the α3 subunit. Upon retro-translocation into the cytosol by the p97 complex, α3 undergoes proteasomal degradation. (b) UBXN2A protects α3 from CHIP-mediated ubiquitination and degradation by binding to CHIP and reducing CHIP’s affinity for α3. The result is less ubiquitination and ultimately, less degradation of α3.