The impact of CRMP4 SUMOylation on the Cav1.2 interaction, neurite outgrowth and thermal pain sensitivity.

Abstract

Collapsin response mediator protein 4 (CRMP4) is critical for neuronal development. However, whether CRMP4 could be SUMOylated and how the SUMOylation regulates the interaction with the L-type voltage-gated calcium channel (Cav1.2), neurite outgrowth, and thermal pain sensitivity remain to be elucidated. To determine the SUMOylation of CRMP4, Glutathione S-transferase (GST) - Small Ubiquitin-like Modifier 1 (-SUMO1), -SUMO2, and -SUMO3 proteins were purified for GST-pulldown. Immunofluorescence staining was performed to observe colocalization of CRMP4 and SUMOs. Co-immunoprecipitation (co-IP) was performed to assess the interaction between CRMP4 and SUMO2. GST-pulldown and co-IP were performed to verify the interaction between CRMP4 and Cav1.2. The impact of SUMOylation of CRMP4 on its interaction with Cav1.2 was determined. Then, the effect of CRMP4 SUMOylation on neurite outgrowth was observed. Whole-cell patch clamping revealed the effect of CRMP4 SUMOylation on Cav1.2 mediated calcium influx. Paw withdrawal latency was measured to assess the impact of CRMP4 SUMOylation on thermal pain sensitivity in rats. The data revealed that CRMP4 K374 is a potential site for SUMO modification. SUMO1, SUMO2, and SUMO3 can all interact with CRMP4. SUMO2 interacts with CRMP4, but not a variant of CRMP4 harboring a mutation of K374. CRMP4 and SUMO proteins colocalized in neurites, and CRMP4 deSUMOylation promoted neurite outgrowth. CRMP4 interacted with Cav1.2, and deSUMOylation of CRMP4 strengthened this interaction. CRMP4 promoted calcium influx via Cav1.2, and overexpression of CRMP4 significantly increased thermal pain sensitivity in rats, which CRMP4 deSUMOylation strengthened. In conclusion, these data demonstrate the SUMOylation of CRMP4, elucidate the impacts of SUMOylation on the interaction with Cav1.2 on neurite outgrowth and thermal pain sensitivity.