Carol D. Cianci

Transforming growth factor beta induces caspase 3-independent cleavage of alphaII-spectrin (alpha-fodrin) coincident with apoptosis.

Transforming growth factor β (TGF-β) is a potent growth inhibitor and inducer of cell death in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. In this report the mouse immature B cell line, WEHI 231, was used to examine the mechanisms involved in TGF-β-mediated apoptosis. Induction of apoptosis is detected as early as 8 h after TGF-β administration. Coincident with the onset of apoptosis, the cytoskeletal actin-binding protein, αII-spectrin (α-fodrin) is cleaved into 150-, 115-, and 110-kDa fragments. The broad spectrum caspase inhibitor (Boc-D-fmk (BD-fmk)) completely abolished TGF-β-induced apoptosis and αII-spectrin cleavage. Caspase 3, although present in WEH1 231 cells, was not activated by TGF-β, nor was its substrate, poly(ADP-ribose) polymerase. These results identify αII-spectrin as a novel substrate that is cleaved during TGF-β-induced apoptosis. Our data provide the first evidence of calpain and caspase 3-independent cleavage of αII-spectrin during apoptosis and suggests that TGF-β induces apoptosis and αII-spectrin cleavage via a potentially novel caspase. This report also provides the first direct evidence of caspase 3 activation in WEH1 231 cells and indicates that at least two distinct apoptotic pathways exist.

c-Src Binds αII Spectrin's Src Homology 3 (SH3) Domain and Blocks Calpain Susceptibility by Phosphorylating Tyr1176

Spectrin is a ubiquitous heterodimeric scaffolding protein that stabilizes membranes and organizes protein and lipid microdomains on both the plasma membrane and intracellular organelles. Phosphorylation of β-spectrin on Ser/Thr is well recognized. Less clear is whether α-spectrin is phosphorylatedin vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, αII spectrin undergoesin vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified αII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between αII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of αII spectrin (but not the Src homology 3 domain of αI spectrin) bind specifically to phosphorylated c-Src in Madin-Darby canine kidney cell lysates, suggesting that this kinase is responsible for its in vivo phosphorylation. pTyr-modified αII spectrin is resistant to maitotoxin-induced cleavage by μ-calpain in vivo. In vitro studies of recombinant αII spectrin peptides representing repeats 9–12 identify two sites of pTyr modification. The first site is at Tyr1073, a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr1176. This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr1176 by c-Src reduces the susceptibility of αII spectrin to cleavage by μ-calpain. Calpain cleavage of spectrin, activated by Ca2+ and calmodulin, contributes to diverse cellular processes including synaptic remodeling, receptor-mediated endocytosis, apoptosis, and the response of the renal epithelial cell to ischemic injury. Tyrosine phosphorylation of αII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca2+-mediated signaling cascades.

Structure of the Calmodulin αII-Spectrin Complex Provides Insight into the Regulation of Cell Plasticity

αII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of αII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of αII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, we have solved the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human αII-spectrin (Protein Data Bank ID code 2FOT). The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes.

c-Src binds alpha II spectrin's SH3 domain and blocks calpain susceptibility by phosphorylating Tyr1176

Spectrin is a ubiquitous heterodimeric scaffolding protein that stabilizes membranes and organizes protein and lipid microdomains on both the plasma membrane and intracellular organelles. Phosphorylation of β-spectrin on Ser/Thr is well recognized. Less clear is whether α-spectrin is phosphorylatedin vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, αII spectrin undergoesin vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified αII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between αII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of αII spectrin (but not the Src homology 3 domain of αI spectrin) bind specifically to phosphorylated c-Src in Madin-Darby canine kidney cell lysates, suggesting that this kinase is responsible for its in vivo phosphorylation. pTyr-modified αII spectrin is resistant to maitotoxin-induced cleavage by μ-calpain in vivo. In vitro studies of recombinant αII spectrin peptides representing repeats 9–12 identify two sites of pTyr modification. The first site is at Tyr1073, a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr1176. This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr1176 by c-Src reduces the susceptibility of αII spectrin to cleavage by μ-calpain. Calpain cleavage of spectrin, activated by Ca2+ and calmodulin, contributes to diverse cellular processes including synaptic remodeling, receptor-mediated endocytosis, apoptosis, and the response of the renal epithelial cell to ischemic injury. Tyrosine phosphorylation of αII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca2+-mediated signaling cascades.

Cell organization, growth, and neural and cardiac development require αII-spectrin

Spectrin α2 (αII-spectrin) is a scaffolding protein encoded by the Spna2 gene and constitutively expressed in most tissues. Exon trapping of Spna2 in C57BL/6 mice allowed targeted disruption of αII-spectrin. Heterozygous animals displayed no phenotype by 2 years of age. Homozygous deletion of Spna2 was embryonic lethal at embryonic day 12.5 to 16.5 with retarded intrauterine growth, and craniofacial, neural tube and cardiac anomalies. The loss of αII-spectrin did not alter the levels of αI- or βI-spectrin, or the transcriptional levels of any β-spectrin or any ankyrin, but secondarily reduced by about 80% the steady state protein levels of βII- and βIII-spectrin. Residual βII- and βIII-spectrin and ankyrins B and G were concentrated at the apical membrane of bronchial and renal epithelial cells, without impacting cell morphology. Neuroepithelial cells in the developing brain were more concentrated and more proliferative in the ventricular zone than normal; axon formation was also impaired. Embryonic fibroblasts cultured on fibronectin from E14.5 (Spna2−/−) animals displayed impaired growth and spreading, a spiky morphology, and sparse lamellipodia without cortical actin. These data indicate that the spectrin–ankyrin scaffold is crucial in vertebrates for cell spreading, tissue patterning and organ development, particularly in the developing brain and heart, but is not required for cell viability.

Identification of the Primary Caspase 3 Cleavage Site in alpha II-Spectrin during Apoptosis

Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initally cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögrens syndrome.

Chapter 14 Polarized Assembly of Spectrin and Ankyrin in Epithelial Cells

Publisher Summary The approximate disposition and role of proteins, such as actin, protein 4.1, protein 4.9, adducin, tropomyosin, and ankyrin in the red cell skeleton is now comprehensible. The predominant component is spectrin that is an antiparallel heterodimer of two subunits (α,β). Spectrin heterodimers self-associate through α to β subunit interactions at the amino terminus of the α-subunit and bind F-actin by an interaction near the amino terminus of the β-subunit. Spectrin also binds a number of accessory proteins that modify its interactions, with actin or the bilayer, and all the interactions, within the spectrin skeleton, appear subject to the several levels of post-translational control. The major role of the spectrin skeleton in erythrocytes is to enhance the structural stability and deformability of the plasma membrane. This chapter discusses the unexpected spatial polarization of the nonerythroid spectrin cytoskeleton, the interaction of spectrin and ankyrin, with basolateral NA + /K + -adenosine triphosphate (ATP)ase, whether polarized membrane skeletal assembly require ankyrin, and the models for the role of the nonermhroid spectrin–actin cytoskeleton. The nascent spectrin skeleton, topographically assembled, by either direct or indirect L-CAM interactions, becomes a “nucleating” center, about which other proteins needed for efficient transport or signal transductions are gathered. Thus, the key and most fundamental role of the spectrin skeleton may be that of an organizer of membrane transport and signaling complexes. Spectrin–ankyrin interaction may not be primarily involved with the assembly of the spectrin skeleton at the membrane.