The calmodulin-binding site in alpha-fodrin is near the calcium-dependent protease-I cleavage site

Fodrin (brain spectrin) binds calmodulin and is susceptible to proteolysis by calcium-dependent protease I (CDP-I, calcium-activated neutral protease I, or calpain I). Both events involve the central region of the alpha-fodrin subunit, and calmodulin binding enhances the sensitivity of fodrin to CDP-I mediated proteolysis. Fragments of fodrin, generated chemically or proteolytically, which retain calmodulin binding activity have been identified and analyzed by two-dimensional peptide mapping and by direct protein sequencing. Both CDP-I and calmodulin interact with the terminal portion of the eleventh repetitive unit in fodrin, which is at the center of the molecule. CDP-I cleavage occurs between Tyr104 and Gly105 and preserves the calmodulin binding activity of the carboxyl-terminal fragment. In contrast, chymotryptic cleavage at Trp120 reduces the ability of this fragment to bind calmodulin, and tryptic cleavage beyond Trp120 completely eliminates calmodulin binding activity. It is concluded that Ser-Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala- Thr- Phe-Asn-Ser-Ile-Lys, a 24-residue peptide which bridges repeats 11 and 12 of brain alpha spectrin contains the high affinity calmodulin binding domain.     

Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.     

Similarities between the Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton and alpha-fodrin

The Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton (D245) has been compared with human erythrocyte spectrin and mammalian brain fodrin [J. Levine and M. Willard (1981) J. Cell Biol. 90, 631-643]. Mammalian erythrocyte alpha-spectrin, brain alpha-fodrin, and D245 are all localized in the cell surface-associated cytoskeleton, and have similar molecular weights. Like mammalian erythrocyte spectrin, D245 was extracted from erythrocyte ghosts under low-ionic-strength conditions. However, D245 failed to bind an antibody which reacted strongly with both subunits of human erythrocyte spectrin. Unlike mammalian erythrocyte alpha- and beta-spectrin, D245 bound calmodulin in the absence of urea both in a "gel-binding" assay and in situ using azidocalmodulin [D.C. Bartelt, R.K. Carlin, G.A. Scheele, and W.D. Cohen (1982) J. Cell Biol. 95, 278-284]. Striking similarities were noted between D245 and alpha-fodrin in that both exhibited (a) comparable calcium-dependent calmodulin binding properties, (b) strong reactivity with two different anti-fodrin antibody preparations, (c) similar reactivity with antibody to brain CBP-I, now believed to be fodrin, (d) proteolytic degradation yielding an Mr 150,000 calmodulin-binding fragment, and (e) lack of reactivity with an anti-spectrin antibody. A protein with calmodulin-binding and anti-fodrin-binding properties similar to D245 was detected in cytoskeletal preparations of chicken erythrocytes. Moderate and consistent cross-reactivity of anti-fodrin with human erythrocyte alpha-spectrin was also observed. The data indicate that D245 is functionally and immunologically more closely related to alpha-fodrin than to alpha-spectrin of the mammalian erythrocyte.     

Calmodulin regulates fodrin susceptibility to cleavage by calcium-dependent protease I

The intracellular calcium-dependent proteolysis of fodrin has been postulated to be central to the regulation of plasticity of the cortical cytoskeleton of many eukaryotic cells. The close proximity of the sites of calmodulin (CaM) binding and calcium-dependent protease I (CDP-I) cleavage in mammalian alpha-fodrin suggested that their action may be linked. In hypotonic and isotonic buffers, CDP-I proteolysis of the beta subunit of fodrin was absolutely dependent upon the presence of active CaM. The stimulation by CaM was inhibited by CaM antagonists. The rate of CDP-I proteolysis of both subunits was enhanced by CaM, while the rate of fodrin proteolysis with other proteases was not influenced by CaM. The increase in the susceptibility of fodrin to CDP-I proteolysis was half-maximal at 80 nM CaM, and maximal at 200 nM CaM. The unusual and differential susceptibility of alpha- and beta-fodrin to proteolysis by CDP-I in the absence of CaM was exploited to investigate the quaternary structure of fodrin in which only the alpha subunit was cleaved. Cleavage of the alpha subunit alone did not destroy the tetrameric form of the molecule, whereas CDP-I cleavage of both subunits rendered the molecule incapable of reforming tetramers. These results provide structural and functional evidence that CaM and CDP-I act synergistically in the regulated proteolysis of fodrin.     

Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations

A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido- calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.     

The Immunogenic SigA Enterotoxin of Shigella flexneri 2a Binds to HEp-2 Cells and Induces Fodrin Redistribution in Intoxicated Epithelial Cells

Background

We have previously shown that the enterotoxin SigA which resides on the she pathogenicity island (PAI) of S. flexneri 2a is an autonomously secreted serine protease capable of degrading casein. We have also demonstrated that SigA is cytopathic for HEp-2 cells and plays a role in the intestinal fluid accumulation associated with S. flexneri infections.

Methods/Principal Findings

In this work we show that SigA binds specifically to HEp-2 cells and degrades recombinant human αII spectrin (α-fodrin) in vitro, suggesting that the cytotoxic and enterotoxic effects mediated by SigA are likely associated with the degradation of epithelial fodrin. Consistent with our data, this study also demonstrates that SigA cleaves intracellular fodrin in situ, causing its redistribution within cells. These results strongly implicate SigA in altering the cytoskeleton during the pathogenesis of shigellosis. On the basis of these findings, cleavage of fodrin is a novel mechanism of cellular intoxication for a Shigella toxin. Furthermore, information regarding immunogenicity to SigA in infected patients is lacking. We studied the immune response of SigA from day 28 post-challenge serum of one volunteer from S. flexneri 2a challenge studies. Our results demonstrate that SigA is immunogenic following infection with S. flexneri 2a.

Conclusions

This work shows that SigA binds to epithelial HEp-2 cells as well as being able to induce fodrin degradation in vitro and in situ, further extending its documented role in the pathogenesis of Shigella infections.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.     

Alpha-fodrin in the adrenal gland: localization by immunoelectron microscopy

Localization of fodrin, the brain equivalent of spectrin (a protein constituent of the erythrocyte membrane cytoskeleton), was investigated at the ultrastructural level in rat adrenal gland. By use of an affinity purified antibody directed against the alpha-fodrin subunit, all chromaffin cells, cortical cells, nerve fibers, and their surrounding Schwann cells were found to be labeled close to the cytoplasmic side of their plasma membranes. The labeling appeared more intense for chromaffin cells, and secretory granules and mitochondria were frequently found to be associated with the zone containing alpha-fodrin in these cells. The immunostained zone was estimated to extend 230 +/- 70 nm into the cytoplasm. This localization is discussed in terms of what is known of the properties of spectrin, and possible roles of the molecule in the chromaffin cell are suggested.     

Mechanism of cytoskeletal regulation (I): functional differences correlate with antigenic dissimilarity in human brain and erythrocyte spectrin

Human erythrocyte and brain spectrin (fodrin, calspectin) have been compared quantitatively with respect to the extent and sites of antigenic and functional similarity. Brain spectrin cross-reacts strongly with approx. 1% of the epitopes in erythrocyte spectrin, but weakly with at least 50%. The distribution of shared determinants is not uniform. Brain spectrin is most deficient in epitopes characteristic of the 80 kDa and 52 kDa domains of the alpha-subunit (alpha-I and alpha-III) and of terminal portions of the 28 kDa and 74 kDa domains of the beta-subunit (beta-I and beta-IV). The functions associated with these domains also differ between the two proteins. Brain spectrin does not undergo extensive polymerization and binds calmodulin at a different site. The unique ability of erythrocyte spectrin to oligomerize beyond the tetramer reflects its role in the membrane skeleton. Non-erythroid spectrins probably function as specific linkers between membrane receptors and the filamentous cytoskeleton. In this sense, they may act as regulated transducers of information flow between the membrane and the cytoplasmic matrix.     

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.     

Structure of the calmodulin alphaII-spectrin complex provides insight into the regulation of cell plasticity

AlphaII-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 alphaII-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 alphaII-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 alphaII-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.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cleavage of cytoskeletal proteins by caspases during ovarian cell death: evidence that cell-free systems do not always mimic apoptotic events in intact cells

Several lines of evidence support a role for protease activation during apoptosis. Herein, we investigated the involvement of several members of the CASP (cysteine aspartic acid-specific protease; CED-3- or ICE-like protease) gene family in fodrin and actin cleavage using mouse ovarian cells and HeLa cells combined with immunoblot analysis. Hormone deprivation-induced apo-ptosis in granulosa cells of mouse antral follicles incubated for 24 h was attenuated by two specific peptide inhibitors of caspases, zVAD-FMK and zDEVD-FMK (50-500 microM), confirming that these enzymes are involved in this paradigm of cell death. Proteolysis of actin was not observed in follicles incubated in vitro while fodrin was cleaved to the 120 kDa fragment that accompanies apoptosis. Fodrin, but not actin, cleavage was also detected in HeLa cells treated with various apoptotic stimuli. These findings suggest that, in contrast to recent data, proteolysis of cytoplasmic actin may not be a component of the cell death cascade. To confirm and extend these data, total cell proteins collected from mouse ovaries or non-apoptotic HeLa cells were incubated without and with recombinant caspase-1 (ICE), caspase-2 (ICH-1) or caspase-3 (CPP32). Immunoblot analysis revealed that caspase-3, but not caspase-1 nor caspase-2, cleaved fodrin to a 120 kDa fragment, wheres both caspases-1 and -3 (but not caspase-2) cleaved actin. We conclude that CASP gene family members participate in granulosa cell apoptosis during ovarian follicular atresia, and that collapse of the granulosa cell cytoskeleton may result from caspase-3-catalyzed fodrin proteolysis. However, the discrepancy in the data obtained using intact cells (actin not cleaved) versus the cell-free extract assays (actin cleaved) raises concern over previous conclusions drawn related to the role of actin cleavage in apoptosis.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Comparison of spectrin isolated from erythroid and non-erythroid sources

Spectrin from erythrocytes and two other tissues (brain and intestine) were isolated from two distant species, pig and chicken; some structural and functional properties were compared. A quantitative antibody inhibition assay was used to determine that antibodies to mammalian red cell spectrin cross-react very poorly, if at all, with their non-erythroid (brain) counterpart and similarly antibodies to pig brain spectrin (fodrin) cross-react very weakly with erythroid spectrin. By contrast, antibodies which were directed against the 240000-Mr subunit of avian fodrin were completely inhibited with avian spectrin and vice versa. To analyze the structural relatedness of these molecules further we compared the chymotryptic iodinated peptide maps generated from each individual subunit. Consistent with the antibody results, we find little (less than 10%) homology between peptides derived from mammalian fodrin and spectrin, but complete homology (100%) of the peptides derived from the 240000-Mr subunits of chicken fodrin, spectrin and another related molecule from intestine, TW260/240. Whereas the peptide maps of fodrin (brain spectrin) revealed striking similarity between divergent species, suggesting a high degree of structural conservation, the peptide maps of erythrocyte spectrin was highly variable between species, indicating that it has diverged considerably in mammalian evolution. In addition we have compared a functional activity of mammalian spectrins, the ability to bind calmodulin, using two different assays. Both results show that, whereas fodrin-calmodulin interaction can be readily demonstrated, the binding to mammalian erythroid spectrin is negligible. This suggests that the high-affinity calmodulin site present on fodrin has been lost from spectrin in mammalian evolution.     

c-Src binds alpha II 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 beta-spectrin on Ser/Thr is well recognized. Less clear is whether alpha-spectrin is phosphorylated in vivo and whether spectrin is phosphorylated on tyrosine (pTyr). We affirmatively answer both questions. In cultured Madin-Darby canine kidney cells, alphaII spectrin undergoes in vivo tyrosine phosphorylation. Enhancement of the steady state level of pTyr-modified alphaII spectrin by vanadate, a phosphatase inhibitor, implies a dynamic balance between alphaII spectrin phosphorylation and dephosphorylation. Recombinant peptides containing the Src homology 3 domain of alphaII spectrin (but not the Src homology 3 domain of alphaI 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 alphaII spectrin is resistant to maitotoxin-induced cleavage by mu-calpain in vivo. In vitro studies of recombinant alphaII spectrin peptides representing repeats 9-12 identify two sites of pTyr modification. The first site is at Tyr(1073), a residue immediately adjacent to a region encoded by alternative exon usage (insert 1). The second site is at Tyr(1176). This residue flanks the major site of cleavage by the calcium-dependent protease calpain, and phosphorylation of Tyr(1176) by c-Src reduces the susceptibility of alphaII spectrin to cleavage by mu-calpain. Calpain cleavage of spectrin, activated by Ca(2+) 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 alphaII spectrin now would appear to also mediate these events. The spectrin skeleton thus forms a point of convergence between kinase/phosphatase and Ca(2+)-mediated signaling cascades.     

No evidence for calpain I involvement in fodrin rearrangements linked to regulated secretion.

Stimulation of secretion in chromaffin and parotid acinar cells is associated with dramatic rearrangements of the subplasmalemmal cytoskeleton, notably of fodrin and F-actin. It has been proposed that a proteolytic cleavage of fodrin resulting from an activation of the neutral calcium activated protease (calpain) could be responsible for these changes. Using an affinity-purified anti-alpha-fodrin antibody, several cleavage products of fodrin could clearly be detected following incubation of total cell homogenates from chromaffin and parotid acinar cells with purified calpain I. On the other hand, maximum stimulation of secretion of chromaffin cells by nicotine, and of parotid acinar cells by carbachol plus isoproterenol, was not associated with an increased appearance of cleavage products of fodrin. This result is not compatible with the 'proteolytic cleavage' hypothesis.     

A calmodulin and alpha-subunit binding domain in human erythrocyte spectrin

Human erythrocyte spectrin binds calmodulin weakly under native conditions. This binding is enhanced in the presence of urea. The site responsible for this enhanced binding in urea has now been shown to reside in a specific region of the spectrin beta-subunit. Cleavage of spectrin with trypsin, cyanogen bromide or 2-nitro-5-thiocyanobenzoic acid generates fragments of the molecule which retain the ability to bind calmodulin under denaturing conditions. The origin of these fragments, identified by two-dimensional peptide mapping, is the terminal region of the spectrin beta-IV domain. The smallest peptide active in calmodulin binding is a 10 000 Mr fragment generated by cyanogen bromide cleavage. Only the intact 74 000 Mr fragment generated by trypsin (the complete beta-IV domain) retains the capacity to reassociate with the isolated alpha-subunit of spectrin. The position of a putative calmodulin binding site near a site for subunit-subunit association and protein 4.1 and actin binding suggests a possible role in vivo for calmodulin regulation of the spectrin-actin membrane skeleton or for regulation of subunit-subunit associations. This beta-subunit binding site in erythrocyte spectrin is found in a region near the NH2-terminus at a position analogous to the alpha-subunit calmodulin binding site previously identified in a non-erythroid spectrin by ultrastructural studies.     

The spectrin super-family

The review is focused on recent data on the primary sequences of erythroid and non-erythroid spectrins. As in other fields, the techniques of molecular genetics have allowed great advances in our knowledge of the structure and the genetic story of these molecules. Comparison of alpha-chains sequences of the non-erythroid (fodrin) and erythroid spectrin demonstrated that the fodrin alpha-genes are strictly conserved across species, while the mammalian spectrin genes have diverged rapidly. Spectrin and fodrin alpha-chains are largely composed of homologous 106-amino-acid repeat units. Spectrin alpha-chain is lacking a 37 amino-acid sequence which bears the calmodulin-binding site of the fodrin alpha-chain. The highest degree of homology between the spectrin alpha-chain and the fodrin alpha-chain lies in a central atypical segment unrelated to the canonical repeat sequence. This region is closely related to the N-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. Like the spectrin alpha-chain, the major central part of the spectrin beta-chain is made up of repeat units of 106 amino-acids. The N-terminal domain of the beta-chain, and especially the actin binding site, is the region of greatest homology among members of the spectrin super-family, including Drosophila spectrin beta-chain, dystrophin and alpha-actinin. The C-terminal extremity of the erythroid beta-chain is also of great interest, since tissue-specific differential processing of 3'beta-spectrin gene pre-mRNA generates a beta spectrin-isoform with a unique C-terminus in human skeletal muscle.     

Tyrosine phosphatase activity of lymphoma CD45 (GP180) is regulated by a direct interaction with the cytoskeleton.

GP180 is one of the major transmembrane glycoproteins in mouse T-lymphoma cells. This molecule is an isoform of CD45 and is known to contain an intrinsic protein tyrosine phosphatase (PTPase) activity. Using several complementary biochemical techniques, we have found that fodrin (a spectrin-like protein) is preferentially co-isolated with CD45 (GP180), suggesting that a complex between CD45 (GP180) and the cytoskeleton exists in mouse T-lymphoma cells. Furthermore, we have determined that this CD45 (GP180)-fodrin complex is dissociated by high salt treatment. Using in vitro binding assays, we have shown that CD45 (GP180) binds directly and specifically to fodrin (Kd approximately 1.1 nM) or spectrin (Kd approximately 3.2 nM) in a saturable manner. Additional analyses indicate that a 48-kDa phosphopeptide of CD45 (GP180) contains the fodrin/spectrin-binding domain. Most importantly, the direct binding of fodrin/spectrin to CD45 (GP180) is found to significantly stimulate the PTPase activity of CD45. Enzyme kinetic analysis indicates that fodrin and spectrin increase the Vmax of CD45 (GP180)-mediated dephosphorylation by 7.5 and 3.2-fold, respectively, without significantly changing the Km value. These results strongly suggest that the cytoskeletal proteins, fodrin and spectrin, play an important role in the regulation of the CD45 (GP180) PTPase activity during lymphocyte activation.