Discrete proteolysis of neuronal calcium sensor-1 (NCS-1) by μ-calpain disrupts calcium binding

SummaryNeuronal calcium sensor-1 (NCS-1) is a high-affinity, low-capacity Ca2+-binding protein expressed in many cell types. We previously showed that NCS-1 interacts with inositol 1,4,5-trisphosphate receptor (InsP3R) and modulates Ca2+-signaling by enhancing InsP3-dependent InsP3R channel activity and intracellular Ca2+ transients. Recently we reported that the chemotherapeutic agent, paclitaxel (taxol) triggers μ-calpain dependent proteolysis of NCS-1, leading to reduced Ca2+-signaling within the cell. Degradation of NCS-1 may be critical in the induction of peripheral neuropathy associated with taxol treatment for breast and ovarian cancer. To begin to design strategies to protect NCS-1, we treated NCS-1 with μ-calpain in vitro and identified the cleavage site by N-terminal sequencing and MALDI mass spectroscopy. μ-Calpain cleavage of NCS-1 occurs within an N-terminal pseudoEF-hand domain, which by sequence analysis appears to be unable to bind Ca2+. Our results suggest a role for this pseudoEF-hand in stabilizing the three functional EF-hands within NCS-1. Using isothermal titration calorimetry (ITC) we found that loss of the pseudoEF-hand markedly decreased NCS-1's affinity for Ca2+. Physiologically, this significant decrease in Ca2+ affinity may render NCS-1 incapable of responding to changes in Ca2+ levels in vivo. The reduced ability of μ-calpain treated NCS-1 to bind Ca2+ may explain the altered Ca2+ signaling in the presence of taxol and suggests a strategy for therapeutic intervention of peripheral neuropathy in cancer patients undergoing taxol treatment.