Membrane Skeletal Protein Research Articles

RGD-containing ankyrin externalized onto the cell surface triggers α Vβ 3 integrin-mediated erythrophagocytosis

The RGD motif on the extracellular matrix or cell surface, together with its integrin receptors, constitutes a major recognition system for cell adhesion. There are several erythrocyte major membrane skeletal proteins, e.g., α spectrin, ankyrin, and protein 4.2, that bear an RGD motif. However, it is not known whether the RGD/integrin recognition system is utilized in the erythrocyte-macrophage adhesion during erythrophagocytosis. Here we report that the RGD motif of ankyrin, but not others, is recognized by the α vβ 3 integrin receptor. In addition, the RGD motif of ankyrin, a peripheral membrane protein, can be externalized onto the cell surface when erythrocytes are incubated with calcium and sheared both at physiological levels. Furthermore, the erythrocyte-macrophage adhesion can be specifically inhibited by ankyrin and/or α vβ 3. Thus, externalization of ankyrin followed by RGD/integrin recognition may be a novel mechanism by which erythrocytes adhere to macrophages preceding phagocytosis.

Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains.

Immunolocalization of membrane skeletal protein, 4.1G, in enteric glial cells in the mouse large intestine

4.1 family proteins are membrane skeletal proteins that interact with spectrin–actin networks and intramembraneous proteins. We reported that one of them, 4.1G, was immunolocalized in myelinated nerve fibers of the mouse peripheral nervous system, especially along cell membranes of paranodes and Schmidt–Lanterman incisures in Schwann cells. In this study, to examine 4.1G's appearance in unmyelinated peripheral nerve fibers, we focused on the enteric nervous system in mouse large intestines. In intestinal tissues prepared by an “in vivo cryotechnique” followed by freeze-substitution fixation, 4.1G was immunolocalized in Auerbach's myenteric plexus and connecting nerve fiber networks. Its immunostaining was mostly colocalized with glial fibrillar acidic protein, a marker of enteric glial cells, but not with c-Kit, a marker of interstitial cells of Cajal. Using whole-mount preparation after splitting inner and outer muscle layers, the nerve fiber networks including the plexus were clearly detected by the 4.1G immunostaining. By conventional pre-embedding immunoelectron microscopy, 4.1G was detected along cell membranes of enteric glial cells and their processes surrounding axons. These indicate that 4.1G may have some roles in adhesion and/or signal transduction in unmylinated PNS nerve fibers.

Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.

ABNORMAL CALCIUM HOMEOSTASIS IN ARTERIAL MYOCYTES FROM MILAN HYPERTENSIVE STRAIN RATS: PP.29.177

Polymorphisms in the genes encoding adducin, a membrane-skeletal protein, are associated with hypertension in humans and rats (Milan hypertensive strain, MHS). MHS rats, with their control normotensive strain (MNS), are a model for a subgroup of patients with essential hypertension. The relationship between adducin gene polymorphisms and renal dysfunction has been well-studied, but the mechanisms that underlie vascular functional abnormalities are unclear. Ca2+ homeostasis plays a crucial role in the genesis of vascular myogenic tone, and increases in cytosolic Ca2+ concentration ([Ca2+]cyt) appear to underlie at least part of the increased peripheral vascular resistance in hypertension. We hypothesize that Ca2+ signaling mediated by Na/Ca exchanger (NCX) and TRPC-encoded receptor-operated Ca2+ channels is up-regulated in MHS mesenteric artery myocytes compared with that in MNS. The [Ca2+]cyt was determined with digital imaging of Fura-2-loaded freshly dissociated mesenteric artery smooth muscle cells (ASMCs). On average, the MNS rats had a mean systolic BP (SBP) of 113 ± 1 mmHg; the SBP of the MHS rats was significantly higher (144 ± 2 mmHg, P < 0.05). Freshly dissociated MHS ASMCs had significantly higher resting [Ca2+]cyt than did MNS ASMCs (97 ± 2 nM vs. 78 ± 3 nM, P < 0.05). Receptor-operated entry was activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba2+, OAG (100 μM)-induced elevations of cytosolic Ba2+ (Fura-2 340/380 nm ratio) were significantly larger in MHS ASMCs. The ATP (5 μM)-induced Ca2+ responses were also augmented in MHS ASMCs (729 ± 70 nM vs. 533 ± 44 nM, P < 0.05). These changes in MHS ASMCs correlate with ∼3-fold up-regulation of TRPC6, while expression of other TRPCs was not changed. Recently we showed that expression of TRPC6 and NCX1 in ASMCs is inter-related and TRPC6 and NCX1 are both up-regulated in ouabain hypertensive rats (Pulina et al., Amer. J. Physiol. 298(1):H263-H274, 2010). Here, we show that NCX1 is also robustly (∼13-fold) up-regulated in MHS ASMCs. The findings suggest that up-regulation of NCX1 and TRPC6 can represent a general downstream phenomenon intrinsic to various forms of hypertension.

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37 degrees C, or from cholesterol-depleted cells at 4 degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4 degrees C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C(12)E(8) DRMs prepared at 4 degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4 degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs.

Involvement of a membrane skeletal protein, 4.1G, for Sertoli/germ cell interaction

We previously reported that a membrane skeletal protein, 4.1G (also known as EPB41L2), is immunolocalized in mouse seminiferous tubules. In this study, the 4.1G immunolocalizaiton was precisely evaluated at various stages of the mouse seminiferous epithelial cycle with 'in vivo cryotechnique' and also with pre-embedding immunoelectron microscopy in testicular tissues whose ultrastructures were well preserved with glycerol treatment before cryosectioning. In addition, 4.1G-deficient mice were produced, and the morphology of their seminiferous tubules was also evaluated. The 4.1G immunolocalization was different among stages, indicating that it was not only along cell membranes of Sertoli cells, but also those of spermatogonia and early spermatocytes. To confirm the 4.1G immunolocalization in germ cells, in vitro culture of spermatogonial stem cells (SSCs) was used for immunocytochemistry and immunoblotting analysis. In the cultured SSCs, 4.1G was clearly expressed and immunolocalized along cell membranes, especially at mutual attaching regions. In testicular tissues, cell adhesion molecule-1 (CADM1), an intramembranous adhesion molecule, was colocalized on basal parts of the seminiferous tubules and immunoprecipitated with 4.1G in the tissue lysate. Interestingly, in the 4.1G-deficient mice, histological manifestation of the seminiferous tubules was not different from that in wild-type mice, and the CADM1 was also immunolocalized in the same pattern as that in the wild-type. Moreover, the 4.1G-deficient male mice were fertile. These results were probably due to functional redundancy of unknown membrane skeletal molecules in germ cells. Thus, a novel membrane skeletal protein, 4.1G, was found in germ cells, and considering its interaction with CADM family, it probably has roles in attachment of both Sertoli-germ and germ-germ cells.

Analysis of Thermal Stability of Protein 4.1R FERM Domain

[Motivation and Aim]The crystal structure of N-terminal 30kDa domain of protein 4.1R (R30, “FERM” domain), that is a membrane skeletal protein, is three-lobe-clover. The transmembrane proteins, Glycophorin C (GPC) and band 3, and p55 bind to each different lobe of R30. Calmodulin (CaM) also binds to R30 in Ca2+-independent manner. Binding with these proteins may stabilize R30. In the present study, we analyzed temperature dependent changes of R30 structure and its binding affinity to apo-CaM.[Materials and Methods]1) The recombinant proteins (cytoplasmic domains of GPC and band 3, p55, and R30) were purified as GST fusion protein from bacteria lysate. CaM was purified from bovine brain.2) FT-IR (attenuated total reflection (ATR) analysis), with Tensor27 and BIO-ATRII accessory (Bruker Optics K.K.) was used for detecting of secondary structure of R30.3) Dynamic light scattering (DLS) analysis was carried out with Zetasizer Nano ZS® (Sysmex Corp.).4) The binding kinetics of R30 to proteins was analyzed using IAsys (Affinity Sensors). R30 dissolved in 10mM HEPES, pH7.4 containing 0.1M NaCl and 1mM EDTA was incubated at 4°C∼60°C for 30 min and the binding activity was measured.[Results]1) ATR analysis of R30 showed dramatic increase in intensity of β-sheet (1628cm−1 and 1672cm−1) with increase in temperature from 40°C to 45°C. The corresponding change was small in the presence of apo-CaM.2) In DLS measurement, R30 became to be aggregated around 45°C.3) R30 denatured at 50°C lost binding ability to apo-CaM, cytoplasmic domains of GPC and band 3. The binding ability of R30 to p55 did at 40°C.[Discussion]Aggregation of R30 at 45°C may be caused through its β-sheet. FT-IR results suggested increasing of intramolecular β-sheet. Actually, p55 binding site is located at the β-sheet structure rich domain.

Oxidized low-density lipoprotein (Ox-LDL) impacts on erythrocyte viscoelasticity and its molecular mechanism

The oxidized low-density lipoprotein (Ox-LDL) plays an important role in atherosclerosis, yet it remains unclear if it damages circulating erythrocytes. In this study, erythrocyte deformability and its membrane proteins after Ox-LDL incubations are investigated by micropipette aspiration, thiol radical measurement, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results show that Ox-LDL incubation reduces the erythrocyte deformability, decreases free thiol radical contents in erythrocytes, and induces the cross-linking among membrane proteins. SDS-PAGE analysis reveals a high molecular weight (HMW) complex as well as new bands between spectrins and band 3 and reduced ratios between band 3 and other major membrane skeletal proteins. Analyses indicate that Ox-LDL makes erythrocytes harder to deform through a molecular mechanism by which the oxidation of free thiol radicals forms disulfide bonds among membrane skeletal proteins.

Dedifferentiating Spermatogonia Outcompete Somatic Stem Cells for Niche Occupancy in the Drosophila Testis

Differentiating cells can dedifferentiate to replace stem cells in aged or damaged tissues, but the underlying mechanisms are unknown. In the Drosophila testis, a cluster of stromal cells called the hub creates a niche by locally activating Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling in adjacent germline and somatic stem cells. Here, we establish a system to study spermatogonial dedifferentiation. Ectopically expressing the differentiation factor bag-of-marbles (Bam) removes germline stem cells from the niche. However, withdrawing ectopic Bam causes interconnected spermatogonia to fragment, move into the niche, exchange positions with resident somatic stem cells, and establish contact with the hub. Concomitantly, actin-based protrusions appear on subsets of spermatogonia, suggesting acquired motility. Furthermore, global downregulation of Jak-STAT signaling inhibits dedifferentiation, indicating that normal levels of pathway activation are required to promote movement of spermatogonia into the niche during dedifferentiation, where they outcompete somatic stem cells for niche occupancy.

Evidence against a role of ketone bodies in the generation of oxidative stress in human erythrocytes by the application of reliable methods for thiol redox form detection

Aim of this study was to reconsider the previously suggested contribution of ketone bodies in causing oxidative damage in human red blood cells (RBCs) in the light of our recent findings demonstrating some methodological pitfalls that can occur during detection of hematic thiols. RBCs were incubated at 37 °C with 20 mM ketone bodies and analyzed with time for their content of glutathione, glutathione disulfide and S-glutathionylated proteins (in both the hemoglobin and membrane skeletal protein fraction). No changes in the concentrations of glutathione and its related forms were evidenced. Differently from previous reports, our results suggest that ketone bodies do not mediate generation of oxidative stress in human RBCs.

Immunolocalization of phospho-Arg-directed protein kinase-substrate in hypoxic kidneys using in vivo cryotechnique

Protein kinases (PKs) phosphorylate proteins at active regions for signal transduction. In this study, normal and hypoxic mouse kidneys were prepared using an "in vivo cryotechnique" (IVCT) and examined immunohistochemically with specific antibodies against phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) to capture their time-dependent regulation in vivo. Left kidneys were cryofixed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffinized sections were immunostained for P-PK-S, Na(+)/HCO(3) (-) cotransporter NBC1, and a membrane skeletal protein, 4.1B. The P-PK-S was diffusely immunolocalized in the cytoplasm of the proximal tubules in normal kidneys, whereas NBC1 and 4.1B were detected at the basal striations of S1 and S2 segments of the proximal tubule. After 10 or 30 s hypoxia, P-PK-S was still immunolocalized in the cytoplasm of kidneys, but it was detected at the basal striations after 1 or 2 min hypoxia. The immunolocalization of P-Akt-S was the same as P-PK-S in the normal and hypoxic kidneys. Immunoblotting analyses of the kidney tissues under normal or hypoxic condition clearly identified the same 40-kDa bands. The IVCT is useful for time-dependent analysis of the immunodistribution of P-PK-S and P-Akt-S.

Dispensable role of protein 4.1B/DAL-1 in rodent adrenal medulla regarding generation of pheochromocytoma and plasmalemmal localization of TSLC1

Protein 4.1B is a membrane skeletal protein expressed in various organs, and is associated with tumor suppressor in lung cancer-1 (TSLC1) in vitro. Although involvement of 4.1B in the intercellular junctions and tumor-suppression was suggested, some controversial results posed questions to the general tumor-suppressive function of 4.1B and its relation to TSLC1 in vivo. In this study, the expression of 4.1B and its interaction with TSLC1 were examined in rodent adrenal gland, and the involvement of 4.1B in tumorigenesis and the effect of 4.1B deficiency on TSLC1 distribution were also investigated using rodent pheochromocytoma and 4.1B-knockout mice. Although plasmalemmal immunolocalization of 4.1B was shown in chromaffin cells of rodent adrenal medulla, expression of 4.1B was maintained in developed pheochromocytoma, and morphological abnormality or pheochromocytoma generation could not be found in 4.1B-deficient mice. Furthermore, molecular interaction and colocalization of 4.1B and TSLC1 were observed in mouse adrenal gland, but the immunolocalization of TSLC1 along chromaffin cell membranes was not affected in the 4.1B-deficient mice. These results suggest that the function of 4.1B as tumor suppressor might significantly differ among organs and species, and that plasmalemmal retention of TSLC1 would be maintained by molecules other than 4.1B interacting in rodent chromaffin cells.

Oxidative crosslinking, spectrin and membrane interactions of hemoglobin mixtures in HbEβ-thalassemia

The authors have studied the interactions of intact hemoglobin mixtures of HbE and HbA, with the major erythroid membrane skeletal protein, spectrin and tailor-made phospholipids membranes containing aminophospholipids to understand the role of spectrin and phospholipids of erythrocytes in the overall pathophysiology of the hemoglobin disorders. Hemoglobin mixtures were isolated and purified from the peripheral blood samples of HbE carriers and different HbEβ thalassemia patients, taken for diagnosis. Spectrin binding was studied by fluorescence and oxidative crosslinking, by SDS-PAGE. Membrane perturbation experiments were carried out to study the leakage of the self-quenching fluorophore, carboxyfluorescein, entrapped in the phospholipid vesicles. Hemoglobin mixtures with elevated levels of HbE showed stronger interactions with spectrin reflected in the decrease in binding dissociation constant from 17 to 5 μM upon increase in HbE% from about 30 to 90% in the hemolysates. The yield of the spectrin crosslinked complexes of such hemoglobin mixtures also increased with increase in HbE levels. Presence of ATP/Mg and DPG were found to decrease the overall yield of such complexes and the binding affinity of hemoglobins to spectrin. HbE rich hemolysates also induced greater leakage of entrapped carboxyfluorescein (CF) from phospholipid membranes containing aminophospholipids. Results from this study indicate the roles of skeletal proteins and aminophospholipids, particularly under oxidative stress conditions to be important in the premature destruction of erythrocytes in hemoglobin disorders, e.g. HbEβ-thalassaemia.

A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells

It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis in vivo, differentiated N1E-115 neuroblastoma cells were labeled with [35S]-amino acids and were treated with a cleavable crosslinking reagent. The cells were lysed in detergent and the lysates were centrifuged at 100,000 × g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not sediment with them. GAP-43 also did not sediment with the membrane skeleton following nonionic detergent lysis. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitated with GAP-43 from the 100,000 × g supernatant following crosslinker addition to cells or cell lysates. Faint spots at 34 kDa and 60 kDa were also present. Additional GAP-43 was recovered from GAP-43 immunoprecipitation supernatants with anti-calmodulin but not with anti-actin. The results suggest that GAP-43 is not present in complexes with actin or other membrane skeletal or cytoskeletal proteins in these cells, but it is nevertheless possible that a small fraction of the total GAP-43 may interact with other proteins.

RanBPM regulates cell shape, arrangement, and capacity of the female germline stem cell niche in Drosophila melanogaster

Experiments in cultured cells with Ran-binding protein M (RanBPM) suggest that it links cell surface receptors and cell adhesion proteins. In this study, we undertake a genetic study of RanBPM function in the germline stem cell (GSC) niche of Drosophila melanogaster ovaries. We find that two RanBPM isoforms are produced from alternatively spliced transcripts, the longer of which is specifically enriched in the GSC niche, a cluster of somatic cells that physically anchors GSCs and expresses signals that maintain GSC fate. Loss of the long isoform from the niche causes defects in niche organization and cell size and increases the number of GSCs attached to the niche. In genetic mosaics for a null RanBPM allele, we find a strong bias for GSC attachment to mutant cap cells and observe abnormal accumulation of the adherens junction component Armadillo (β-catenin) and the membrane skeletal protein Hu-li tai shao in mutant terminal filament cells. These results implicate RanBPM in the regulation of niche capacity and adhesion.

Archvillin anchors in the Z-line of skeletal muscle via the nebulin C-terminus

Z-Line of skeletal muscle is a complex protein network that likely plays an important role in signaling and muscle homeostasis. We used the yeast two-hybrid system to search for potential novel ligands of the Z-line portion of nebulin. We found that the C-terminal region of nebulin (residues 6457–6528) interacted with the C-terminus of archvillin (residues 1419–1687). Archvillin is a membrane skeletal protein that localizes to costameres, specialized adhesion sites in muscle. The binding sites between nebulin and archvillin were characterized using the yeast two-hybrid system, in vitro pull-down assays, and colocalization experiments in COS-7 cells. Our data suggest a model in which archvillin attaches directly to the Z-line through an interaction with the nebulin C-terminus. The interaction between nebulin and archvillin may provide a direct link between the sarcolemma and myofibrillar Z-lines.

Putting channels in their place

Without voltage-gated sodium channels, heart muscle cells can't keep a beat. Lowe et al. show how these cells, known as cardiomyocytes, direct channels to the right domain of their plasma membrane. Figure 1 Normal levels of a sodium channel (red, top) are reduced in cardiac cells with too little ankyrin-G (green, bottom). Opening Nav1.5 sodium channels allows an inrush of sodium ions that depolarizes a cardiomyocyte. Patients who carry a faulty version of the channel are at risk for potentially lethal heart arrhythmias. Rather than scattering around the membrane, the channels home in on the junctions between cardiomyocytes. In neurons, the membrane skeletal protein ankyrin-G helps direct sodium channels to specific membrane domains, and Lowe et al. tested whether this ankyrin-based pathway performed the same function in heart cells. When the researchers knocked down ankyrin-G using RNAi, they found that Nav1.5 resided around the nucleus instead of in the membrane. The amount of Nav1.5 in the cell also fell, possibly because wayward channels get degraded. After stimulation, cardiac muscle cells lacking ankyrin-G produced a smaller-than-normal current. The knockdown of ankyrin-G had no effect on the location or activity of the cells' primary voltage-gated calcium channel, suggesting that the targeting pathway in heart cells is specific for Nav1.5. By testing mutant forms of ankyrin-G that can't bind to the channel, the team showed that interactions between the two proteins are necessary to get Nav1.5 into position. The big question now is how ankyrin-G does the job. The protein might haul Nav1.5 to the membrane or stabilize channels that have already made it there. Reference: Lowe, J.S., et al. 2008. J. Cell Biol. 180:173–186. [PubMed]

Interactions of Recombinant Mouse Erythrocyte Transglutaminase with Membrane Skeletal Proteins

Transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent gamma-glutamyl-epsilon-lysine crosslinks between glutamine (Q) acyl-donors and lysine (K) acyl-acceptors. Here, we report the cDNA cloning of a TG from mouse reticulocytes, its 4.6-kb message size and high-yield synthesis of recombinant TG in yeast cultures. Its activity was assayed by crosslinking the amine of monodansylcadaverine (DC) onto casein and inside-out vesicles of erythrocytes. The latter contain TG substrates including the anion ion exchanger (AE1) or band 3, and the crosslinking activity was the highest at physiological [GTP] and [ATP] of erythrocytes. To study individually how TG interacts with band 3 and what role P4.2, a pseudo-TG that is normally associated with band 3, may play in their interaction, recombinant cytoplasmic domain of band 3 (cdb3) and P4.2 were also cloned by polymerase chain reaction from mouse reticulocytes, expressed and affinity-purified from Escherichia coli. Enzyme-linked immunosorbent assay and Western blot analysis revealed that increasing [CaCl(2)] enhanced TG-mediated crosslinking of DC to cdb3 but decreased TG binding to cdb3. P4.2 inhibited the TG-mediated crosslinking of cdb3 but stabilized the binding of TG to cdb3 in the presence of calcium. This in vitro study suggests a relationship among TG, cdb3 and P4.2 in erythrocyte membrane during calcium influx.

Phosphorylation of adducin by protein kinase Cδ promotes cell motility

Protein kinase Cdelta (PKCdelta) has been implicated to play a crucial role in cell proliferation, differentiation and apoptosis. In this study, we have investigated the role of PKCdelta in cell motility using Madin-Darby canine kidney cells. Overexpression of PKCdelta promoted membrane protrusions, concomitant with increased cell motility. By contrast, suppression of PKCdelta expression by RNA interference inhibited cell motility. Moreover, a fraction of PKCdelta was detected at the edge of membrane protrusions in which it colocalized with adducin, a membrane skeletal protein whose phosphorylation state is important for remodeling of the cortical actin cytoskeleton. Elevated expression of PKCdelta correlated with increased phosphorylation of adducin at Ser726 in intact cells. In vitro, PKCdelta, but not PKCalpha, directly phosphorylated the Ser726 of adducin. Finally, we demonstrated that overexpression of both adducin and PKCdelta could generate a synergistic effect on promoting cell spreading and cell migration. Our results support a positive role for PKCdelta in cell motility and strongly suggest a link between PKCdelta activity, adducin phosphorylation and cell motility.