Sequential degradation of αII and βII spectrin by calpain in glutamate or maitotoxin-stimulated cells

SB Glantz, CD Cianci, R Iyer, D Pradhan… - Biochemistry, 2007 - ACS Publications
SB Glantz, CD Cianci, R Iyer, D Pradhan, KKW Wang, JS Morrow
Biochemistry, 2007ACS Publications
Calpain-catalyzed proteolysis of αII-spectrin is a regulated event associated with neuronal
long-term potentiation, platelet and leukocyte activation, and other processes. Calpain
proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive
glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis
for these divergent consequences of calpain action, and their relationship to spectrin
proteolysis, is unclear. Calpain preferentially cleaves αII spectrin in vitro in repeat 11 …
Calpain-catalyzed proteolysis of αII-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves αII spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), βII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second αII spectrin cleavage at G1230*S1231. βII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the βII spectrin COOH-terminus. CaM promotes βII spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant βII spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds αII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both αII and βII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates αII spectrin cleavage only at Y1176*G1177, while βII spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of αII and then βII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both αII and βII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of αII spectrin vs βII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses:  one that enhances skeletal plasticity without destroying the spectrin−actin skeleton, characterized by preservation of βII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of βII spectrin and complete dissolution of the spectrin skeleton.
ACS Publications