The degradation of ␣II-and ␤II-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both ␣II-and ␤II-subunits. Major C-terminal ␣II-spectrin BDPs were detected at Ϸ150, 145, and 120 kDa (␣II-BDP150, ␣II-BDP145, and ␣II-BDP120, respectively); major C-terminal ␤II-spectrin BDPs were at Ϸ110 and 85 kDa (␤II-BDP110 and ␤II-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that ␣II-BDP150 and ␣II-BDP120 were generated by cleavages at DETD 1185 *S 1186 and DSLD 1478 *S 1479 , respectively. For ␤II-spectrin, a major caspase site was detected at DEVD 1457 *S 1458, and both ␤II-BDP110 and ␤II-BDP85 shared a common N-terminal sequence starting with Ser 1458 . An additional cleavage site near the C terminus, at ETVD 2146 *S 2147 , was found to account for ␤II-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both ␣II-and ␤II-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.The importance of proteases in the expression of mammalian apoptosis has been the subject of many recent studies. The mammalian interleukin-1␤-converting enzyme (ICE) 1 -like protease family (renamed caspase (1) (5), and its deletion by gene knockout blocks neuronal death during brain development with consequential lethality (6). Besides the caspases, a second family of proteases implicated in the initiation and control of apoptosis are the calpains (7, 8), especially in several hematopoietic and neuronal cells (9 -12). The relationship between these two protease families, the consequences of each on their respective substrates and on cellular physiology, or the conditions under which each is activated remain poorly understood. While many proteins are cleaved during apoptosis, a prominent target of both calpain and caspase action is ␣II-spectrin, the major component of the cortical membrane skeleton. In neurons, calcium-activated calpain cleavage of ␣II-spectrin (non-erythroid ␣-spectrin or ␣-fodrin) accompanies N-methyl-D-aspartic acid receptor activation (13), 2 does not directly cause neuronal toxicity (7,15), and is postulated to be necessary for synaptic and neuronal plasticity (16 -18). Indeed, ␣II-spectrin cleavage by calpain appears to be a molecular mechanism by which skeletal plasticity can be enhanced without complete dissolution of the spectrin skeleton since calpain-mediated cleavage of ␣II-spectrin bestows calmodulin regulation on oligomeric spectrin-actin complexes, but does not dissociate them (unless ␤II-spectrin is also c...

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.

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 non-apoptotic cell death following excessive glutamate exposure, hypoxia, HIVgp120/160 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 Y 1176 and G 1177 . 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 G 1230 *S 1231 . 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 Y 1176 *G 1177 , 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 non-apoptotic 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. Spectrin is the major component of the cytoskeletal network associated with the plasma membrane of vertebrate cells (for reviews 1 , 2 , 3). While seven spectrin genes exist, encoding two variants of an alpha spectrin and five beta spectrins, the most common form of this protein is a heterotetramer of αII,βII spectrin. Together with actin and a host of adapter proteins, spectrin controls the distribution of many integral and peripheral membrane proteins, and ...

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