Discrete proteolysis of neuronal calcium sensor-1 (NCS-1) by mu-calpain disrupts calcium binding.
Neuronal calcium sensor-1 (NCS-1) is a high-affinity, low-capacity Ca(2+)-binding protein expressed in many cell types. We previously showed that NCS-1 interacts with inositol 1,4,5-trisphosphate receptor (InsP(3)R) and modulates Ca(2+)-signaling by enhancing InsP3-dependent InsP(3)R channel activity and intracellular Ca(2+) transients. Recently we reported that the chemotherapeutic agent, paclitaxel (taxol) triggers mu-calpain dependent proteolysis of NCS-1, leading to reduced Ca(2+)-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 mu-calpain in vitro and identified the cleavage site by N-terminal sequencing and MALDI mass spectroscopy. mu-Calpain cleavage of NCS-1 occurs within an N-terminal pseudoEF-hand domain, which by sequence analysis appears to be unable to bind Ca(2+). 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 Ca(2+). Physiologically, this significant decrease in Ca(2+) affinity may render NCS-1 incapable of responding to changes in Ca(2+) levels in vivo. The reduced ability of mu-calpain treated NCS-1 to bind Ca(2+) may explain the altered Ca(2+) signaling in the presence of taxol and suggests a strategy for therapeutic intervention of peripheral neuropathy in cancer patients undergoing taxol treatment.
Blachford
Courtney
C
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
Celić
Andjelka
A
Petri
Edward T
ET
Ehrlich
Barbara E
BE
D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002845:Chromatography; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002135:Calcium-Binding Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C101464:frequenin calcium sensor proteins; D002154:Calpain; C449002:mu-calpain; D002118:Calcium