Calpain-truncated CRMP-3 and -4 contribute to potassium deprivation-induced apoptosis of cerebellar granule neurons.
Increasing evidence shows that calpain-mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain-mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation-induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2-D DIGE and MALDI-TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP-1, -2, -3 and -4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain-mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP-1, -2 and -4 were truncated by calpain at the C-terminus, whereas CRMP-3 was cleaved at the N-terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation-triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP-3 and -4, but not CRMP-1 and -2, significantly induced neuronal apoptosis. These findings demonstrated that calpain-truncated CRMP-3 and -4 act as pro-apoptotic players when CGNs undergo apoptosis.
Liu
Wei
W
Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China.
Zhou
Xing-Wang
XW
Liu
Shaojun
S
Hu
Kunhua
K
Wang
Chong
C
He
Qingyu
Q
Li
Mingtao
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D006023:Glycoproteins / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C449760:Dpysl3 protein, rat; C485779:Dpysl4 protein, rat; D006023:Glycoproteins; D009419:Nerve Tissue Proteins; C039304:calpain inhibitors; D002154:Calpain; D011188:Potassium