Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.     

Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or Rous sarcoma virus-induced transformed cells

With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein.     

Characterization of the interaction between calpactin I and fodrin (non-erythroid spectrin)

Calpactin I, a calcium-binding protein associated with the membrane cytoskeleton, has been reported to bind to a calcium-dependent manner to fodrin, to certain phospholipids, and to F-actin. We have investigated the interaction between calpactin I and fodrin. Using a gel filtration assay, we observed one or more calpactin I molecules were bound calcium-dependently only at high concentrations of calpactin (greater than 1 microM), indicating that the interaction is of only moderate affinity. At higher concentrations of calpactin I, the calpactin coprecipitated with fodrin in a calcium-dependent manner. The molar ratio of calpactin to fodrin tetramer in the precipitate was greater than 25:1, indicating that the calpactin binds to a large number of sites. Moreover, the monomeric form of calpactin I (p36), which did not induce precipitation of fodrin, showed no evidence of saturation in its binding to fodrin even when more than 30 mol of p36 were bound per mole of fodrin tetramer. Several proteins other than fodrin, including clathrin, alpha-actinin, and neurofilament-H, also interacted calcium-dependently with calpactin I in the gel filtration assay. These results demonstrate that the interaction between calpactin and fodrin is not of high affinity, is not readily saturated, and is not specific for fodrin. Our results suggest that calpactin's interaction with fodrin is a particular example of a calcium-dependent, but promiscuous, binding of calpactin to proteins.     

A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.     

Phosphorylation reduces the affinity of protein 4.1 for spectrin

The phosphorylation of protein 4.1 by the membrane kinase and casein kinase A has been investigated. Each of these kinases catalyzed the incorporation of 2 mol of phosphate per mole of protein 4.1. The presence of both kinases in the reaction mixture did not lead to an increase in the incorporation of phosphates into the protein. An analysis of the acid hydrolysis products of the 32P-labeled protein 4.1 indicated that the radioactivities were distributed between phosphothreonine and phosphoserine in a ratio of about 2 to 1. The effects of phosphorylation on the binding of protein 4.1 to spectrin were investigated by using sucrose density gradient centrifugation. The affinity of protein 4.1 for spectrin was reduced about 5-fold, from a KD of 2 X 10(-6) M to a KD of 9.4 X 10(-6) M, by phosphorylation. The phosphorylation of spectrin, on the other hand, appeared to increase slightly its affinity for protein 4.1. The results suggest that phosphorylation may lead to a relaxation of the cytoskeletal network and the formation of a more flexible membrane structure that is important to red cell function.     

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.     

Properties of red cell membrane proteins: mechanism of spectrin and band 4.1 interaction

Interactions between human red cell's band 4.1 and spectrin were studied by fluorescence resonance energy transfer and batch microcalorimetry techniques. The association constant (Ka = 8.6 X 10(7) M-1), the stoichiometry (one molecule of band 4.1 to one molecule of spectrin), the reversibility, and the enthalpy (delta H = -6 kcal/mol) were determined. A proton uptake was observed to take place as a result of the spectrin-band 4.1 complex formation. In addition to the protonation of the reaction products, the entropic contribution (-T delta S) has been observed to be responsible for approximately 50% of the binding free energy. We concluded that the environment plays a significant role in the stabilization of the complex. Since band 4.1 has been required for the maintenance of the cytoskeletal stability, small alterations of the binding energies or the degree of interaction could have a pronounced effect on the structure of the erythrocyte membrane.     

Ubiquitination of spectrin regulates the erythrocyte spectrin-protein-4.1-actin ternary complex dissociation: implications for the sickle cell membrane skeleton.

It has been demonstrated by our laboratory that the irreversibly sickled cell (ISC) spectrin-4.1-actin complex dissociates slowly as compared to ternary complexes formed out of control (AA) and reversibly sickle cell (RSCs) core skeletons. These studies indicated that the molecular basis for the inability of irreversibly sickled cells (ISCs) to change shape is a skeleton that disassembles, and therefore reassembles, very slowly. The present study is based on the following observations: a) alpha-spectrin repeats 20 and 21 contain ubiquitination sites, and b) The spectrin repeats beta-1 and beta-2 are in direct contact with spectrin repeats alpha-20 and alpha-21 during spectrin heterodimer formation, and contain the protein 4.1 binding domain. We demonstrate here that alpha-spectrin ubiquitination at repeats 20 and 21 increases the dissociation of the spectrin-protein-4.1-actin ternary complex thereby regulating protein 4.1's ability to stimulate the spectrin-actin interaction. Performing in vitro ternary complex dissociation assays with AA control and sickle cell SS spectrin (isolated from high-density sickle cells), we further demonstrate that reduced ubiquitination of alpha-spectrin is, in part, responsible for the locked membrane skeleton in sickle cell disease.     

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.     

Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.     

Immunolocalization of a spectrin-like protein (fodrin) in pancreatic acinar cells

A spectrin-like protein (fodrin) was localized in porcine pancreas using an immunoperoxidase procedure with antibodies raised against erythrocyte spectrin. Fodrin was primarily associated with the cell plasma membrane although some was also detectable in the cytoplasm of the acinar cells. The membrane labelling of the acinar cells was uneven such that the lateral and basal membranes were strongly labelled by anti-spectrin antibodies whereas the apical membranes were poorly labelled. The implications of the results to secretion and to the occurrence of specific membrane domains are discussed.     

Alpha-actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.     

In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (calpain I)

1. In whole ghosts, ankyrin, protein 4.1, protein band 3 and spectrin are lysed by purified calpain I in the presence of calcium. 2. Limited calpain lysis of purified ankyrin results in several peptides, including a 85 kD peptide bearing the ankyrin interaction site for the protein band 3 internal fragment (43 kD), and a 55 kD peptide carrying the ankyrin-spectrin interaction site. 3. These peptides are differently phosphorylated: the 85 kD by cytosol casein kinase, and the 55 kD by membrane casein kinase. 4. Protein 4.1 lysis mainly produces a 30 kD peptide resistant to proteolysis. 5. The spectrin beta-chain is more sensitive to calpain cleavage than the alpha chain; both chains seem to be cleaved in a similar sequential manner. 6. Limited proteolysis of spectrin dimer does not impede tetramerization in vitro.     

Functional characterization of human erythrocyte spectrin alpha and beta chains: association with actin and erythrocyte protein 4.1

Human erythrocyte spectrin alpha and beta chains were purified by preparative sodium dodecyl sulfate gel electrophoresis and also by DEAE-cellulose chromatography in the presence of urea. The purified chains behaved as individual monomers on sucrose gradients and did not form homodimers. Recombination of the chains led to the formation of alpha-beta heterodimers with sedimentation characteristics identical with native alpha-beta dimers. The binding of 125I-labeled band 4.1 to alpha and beta chains was measured by sucrose gradient rate zonal sedimentation and by quantitative immunoassay. It was found that both alpha and beta chains associated with 125I-labeled band 4.1 in a nearly identical manner over the range of band 4.1 concentration studied. The association was abolished by heat denaturation of the spectrin chains or by denaturation of band 4.1 with a 40-fold molar excess of N-ethylmaleimide. As expected, purified beta chains but not alpha chains bound to 125I-labeled ankyrin as measured by a quantitative radioimmunoassay. The binding of purified alpha chains, beta chains, and recombinant alpha-beta heterodimers to F-actin was measured in the presence of band 4.1. We found that alpha or beta chains separately exhibited no band 4.1 dependent association with F-actin but that alpha-beta heterodimers formed by recombination of the chains did. We conclude that spectrin binding to F-actin in the presence of band 4.1 requires the participation of both of spectrin's polypeptide chains.     

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (K_d) of .50 and .17?μM, for prodan, and .04 and .02?μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain?>?SH3 domain?>?Self-association domain?>?N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.     

Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1

Spectrin dimers interact weakly with F-actin under physiological solvent conditions (with an association constant of about 5 X 10(3) M-1 at 20 degrees C). In the presence of the membrane skeletal constituent, protein 4.1, strong binding is observed; an analysis of the profiles for formation of a ternary complex leads to an association constant of about 1 X 10(12) M-2. This association becomes weaker at low ionic strength, whereas the opposite applies to the spectrin-actin interaction. The stability of the ternary complex is maximal at physiological ionic strength and somewhat above. The effect of temperature in the range 0-20 degrees C on the formation of the ternary complex is small, whereas the spectrin-actin interaction almost vanishes at low temperature. There is no detectable calcium sensitivity in either the binary or the ternary system within the limits of precision of our assay. The ternary complex resembles the natural system in the membrane in that the actin is resistant to dissociation and unavailable in the deoxyribonuclease assay; after selective proteolytic destruction of spectrin and 4.1, all the actin becomes available. In the absence of 4.1, spectrin dimers do not measurably protect the actin against dissociation.     

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.