Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and it has been demonstrated that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibits actin capping measured using pyrene-actin polymerization and abolishes activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures (Matsuoka, 1998).
Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. This study identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. Antibody was prepared that specifically recognizes alpha-adducin phosphorylated at Thr445, and it was found by use of this antibody that Rho-kinase phosphorylates alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulate in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracts the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility (Fukata, 1999).
Pleiotrophin (PTN) was found to regulate tyrosine phosphorylation of beta-adducin through the PTN/receptor protein tyrosine phosphatase (RPTP)beta/zeta signaling pathway. PTN stimulates the phosphorylation of serines 713 and 726 in the myristoylated alanine-rich protein kinase (PK) C substrate domain of beta-adducin through activation of either PKC alpha or beta. PTN also stimulates translocation of phosphoserine 713 and 726 beta-adducin either to nuclei, where it associates with nuclear chromatin and with centrioles of dividing cells, or to a membrane-associated site, depending on the phase of cell growth. Furthermore, PTN stimulates the degradation of beta-adducin in PTN-stimulated cells. Phosphorylation of serines 713 and 726 in beta-adducin is known to markedly reduce the affinity of beta-adducin for spectrin and actin and to uncouple actin/spectrin/beta-adducin multimeric complexes needed for cytoskeletal stability. The data thus suggest that the PTN-stimulated phosphorylation of serines 713 and 726 in beta-adducin disrupts cytoskeletal protein complexes and integrity, features demonstrated in both PTN-stimulated cells and of highly malignant cells that constitutively express the endogenous Ptn gene. The data also support the important conclusion that PTN determines the cellular location of beta-adducin phosphorylated in serines 713 and 726 and raise the possibility that beta-adducin functions in support of structure of heterochromatin and centrioles during mitosis (Pariser, 2005).
Protein kinase Cdelta (PKCdelta) has been implicated to play a crucial role in cell proliferation, differentiation and apoptosis. In this study, the role of PKCdelta in cell motility was investigated using Madin-Darby canine kidney cells. Overexpression of PKCdelta promotes membrane protrusions, concomitant with increased cell motility. By contrast, suppression of PKCdelta expression by RNA interference inhibits cell motility. Moreover, a fraction of PKCdelta is detected at the edge of membrane protrusions in which it colocalizes with adducin, a membrane skeletal protein whose phosphorylation state is important for remodeling of the cortical actin cytoskeleton. Elevated expression of PKCdelta correlates with increased phosphorylation of adducin at Ser726 in intact cells. In vitro, PKCdelta, but not PKCalpha, directly phosphorylates the Ser726 of adducin. Finally, it has been demonstrated that overexpression of both adducin and PKCdelta generate a synergistic effect on promoting cell spreading and cell migration. These results support a positive role for PKCdelta in cell motility and strongly suggest a link between PKCdelta activity, adducin phosphorylation and cell motility (Chen, 2007).
Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. This study demonstrates a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which have been shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. These experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells (Kuhlman, 1996).
Adducin is a protein associated with spectrin and actin in membrane skeletons of erythrocytes and possibly other cells. Adducin has activities in in vitro assays of association with the sides of actin filaments, capping the fast growing ends of actin filaments, and recruiting spectrin to actin filaments. This study presents evidence that adducin exhibits a preference for the fast growing ends of actin filaments for recruiting spectrin to actin and for direct association with actin. beta-Adducin-(335-726) promotes recruitment of spectrin to gelsolin-sensitive sites at fast growing ends of actin filaments with half-maximal activity at 15 nM and to gelsolin-insensitive sites with half-maximal activity at 75 nM. beta-Adducin-(335-726) also exhibits a preference for actin filament ends in direct binding assays; the half-maximal concentration for binding of adducin to gelsolin-sensitive sites at filament ends wau 60 nM, and the Kd for binding to lateral sites is 1.5 microM. The concentration of beta-adducin-(335-726) of 60 nM required for half-maximal binding to filament ends is in the same range as the concentration of 150 nM required for half-maximal actin capping activity. All interactions of adducin with actin require the myristoylated alanine-rich protein kinase C substrate-related domain as well as a newly defined oligomerization site localized in the neck domain of adducin. Surprisingly, the head domain of adducin is not required for spectrin-actin interactions, although it could play a role in forming tetramers. The relative activities of adducin imply that an important role of adducin in cells is to form a complex with the fast growing ends of actin filaments that recruits spectrin and prevents addition or loss of actin subunits (Li, 1998).
Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, the ubiquitous expression of alpha- and gamma-adducins was shown in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, beta-adducin null mice were created by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo (Gilligan, 1999).
The adducin family of proteins interacts with the actin cytoskeleton and the plasma membrane in a calcium- and cAMP-dependent manner. Thus, adducins may be involved in changes in cytoskeletal organization resulting from synaptic stimulation. beta-Adducin knock-out mice were examined in physiological and behavioral paradigms related to synaptic plasticity to elucidate the role the adducin family plays in processes underlying learning and memory. In situ hybridization for alpha- and beta-adducin demonstrates that these mRNAs are found throughout the brain, with high levels of expression in the hippocampus. Schaffer collateral-CA1 tetanic long-term potentiation decayed rapidly in acute hippocampal slices from beta-adducin knock-out mice, although baseline spine morphology and postsynaptic density were normal. Interestingly, the input-output relationship was significantly increased in hippocampal slices from beta-adducin knock-out mice. Furthermore, beta-adducin knock-out mice were impaired in performance of fear conditioning and the water maze paradigm. The current results indicate that beta-adducin may play an important role in the cellular mechanisms underlying activity-dependent synaptic plasticity associated with learning and memory (Rabenstein, 2005).
A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. Promoter fragments of the cognate gene, ahhA (adducin head homologue A), were fused to a lacZ reporter and their expression pattern was determined. The proximal promoter region is necessary for the expression of ahhA at an early (pre-aggregative) stage of development and this expression is Dd-STATa independent. The distal promoter region is necessary for expression at later stages of development in pstA cells, of the slug and in upper cup and pstAB cells during culmination. The distal region is partly Dd-STATa-dependent. The ahhA-null mutant develops almost normally until culmination, but it forms slanting culminants that tend to collapse on to the substratum. The mutant also occasionally forms fruiting bodies with swollen papillae and with constrictions in the prestalk region. The AhhA protein localizes to the stalk tube entrance and also to the upper cup cells and in cells at or near to the constricted region where an F-actin ring is localized. These findings suggest that Dd-STATa regulates culmination and may be necessary for straight downward elongation of the stalk, via the putative actin-binding protein AhhA (Aoshima, 2006).
Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins are phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also leads to adducin proteolysis; inhibition by calpeptin suggests that the protease is calpain. The kinase inhibitor staurosporine inhibits PKC phosphorylation of adducin and also inhibits proteolysis of adducin. Experiments with recombinant alpha adducin demonstrates that the PKC-phosphorylated form is proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets is estimated at 6 microM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. These results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation (Gilligan, 2002).
A spectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. alpha-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activate through protease-activated receptor 1, FcgammaRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. It is concluded that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form (Barkalow, 2003).
The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. Rho targets protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. This study purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacts with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. It was also found that Rho-kinase phosphorylates alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhances the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments (Kimura, 1998).
Fyn is a Src family tyrosine kinase expressed abundantly in neurons and believed to have specific functions in the brain. To understand the function of Fyn tyrosine kinase, attempts were made to identify Fyn Src homology 2 (SH2) domain-binding proteins from a Nonidet P-40-insoluble fraction of the mouse brain. beta-Adducin, an actin filament-associated cytoskeletal protein, was isolated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. beta-Adducin was tyrosine phosphorylated by coexpression with wild type but not with a kinase-negative form of Fyn in COS-7 cells. Cell staining analysis showed that coexpression of beta-adducin with Fyn induced translocation of beta-adducin from the cytoplasm to the periphery of the cells where it was colocalized with actin filaments and Fyn. These findings suggest that tyrosine-phosphorylated beta-adducin associates with the SH2 domain of Fyn and colocalizes under plasma membranes (Shima, 2001).
Adducins are a family of membrane skeleton proteins composed of alpha-, beta- and gamma-subunits that promote actin and spectrin association in erythrocytes. The alpha- and gamma-subunits are expressed ubiquitously, while the beta-subunit is found in brain and erythropoietic tissues. The brain beta-adducin protein is similar in size to that of spleen, but the mRNA transcript is a brain-specific one that has not been yet characterized, having an estimated length of 8-9 kb instead of the 3-4 kb of spleen mRNA. This study shows the molecular basis for these differences by determining the structure of the brain-specific beta-adducin transcript in rats, mice and humans. A brain-specific promoter was identified in rodents that, apparently, is not conserved in humans. In addition, evidence is presented that the brain-mRNAs are formed by a common mechanism consisting in the tissue-specific use of alternative polyadenylation sites generating unusually long 3'-untranslated region of up to 6.6 kb. This hypothesis is supported by the presence of highly-conserved regions flanking the brain-specific polyadenylation site that suggest the involvement of these sequences in the translational regulation, stability and/or subcellular localization of the beta-adducin transcript in the brain (Costessi, 2006).
Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. This study evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. Phospho-adducin immunoreactivity is found in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. Since adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival (Shan, 2005).
Discovering the genetic and cellular mechanisms that drive cardiac morphogenesis remains a fundamental goal, as three-dimensional architecture greatly impacts functional capacity. During development, accurately contoured chambers balloon from a primitive tube in a process characterized by regional changes in myocardial cell size and shape. How these localized changes are achieved remains elusive. This study shows in zebrafish that microRNA-143 (miR-143) is required for chamber morphogenesis through direct repression of adducin3 (add3), which encodes an F-actin capping protein. Knockdown of miR-143 or disruption of the miR-143-add3 interaction inhibits ventricular cardiomyocyte F-actin remodeling, which blocks their normal growth and elongation and leads to ventricular collapse and decreased contractility. Using mosaic analyses, it was found that miR-143 and add3 act cell-autonomously to control F-actin dynamics and cell morphology. As proper chamber emergence relies on precise control of cytoskeletal polymerization, Add3 represents an attractive target to be fine-tuned by both uniform signals, such as miR-143, and undiscovered localized signals. Together, these data uncover the miR-143-add3 genetic pathway as essential for cardiac chamber formation and function through active adjustment of myocardial cell morphology (Deacon, 2010).
Two recent reports provide evidence that miR-143 regulates smooth muscle (SM) cell phenotypic switching based on data drawn from expression studies, cell culture experiments and a postnatal arterial injury model (Cordes, 2009; Xin, 2009). Both studies demonstrate that miR-143 is expressed in mouse aortic smooth muscle (SM) cells on embryonic day 9.5, consistent with a role for miR-143 in SM biology. However, miR-143 is also expressed much more robustly in progenitor cells of the cardiac crescent on embryonic day 7.5, 2 days prior to SM differentiation, and very highly in all myocardial and endocardial cells of the linear heart tube 1 day prior to SM differentiation. As SM precursors and differentiated cells are not observed in the zebrafish heart until 60 hpf and 21 dpf, respectively, a potential SM deficiency cannot explain the profound defect that is evident in MO143 embryos by 48 hpf. Moreover, these expression data in mouse support an SM-independent role for miR-143 during cardiogenesis. Although mice deficient for miR-143 are viable and display no cardiac abnormalities (Xin, 2009), zebrafish miR-143 loss-of-function data suggest that other programs might compensate for the absence of miR-143 during murine development. Thus, in addition to a role in SM biology, the current findings show that myocardial miR-143 acts to drive the morphogenetic events of heart ontogeny by autonomously regulating cytoskeletal dynamics (Deacon, 2010).
The most compelling argument that a primary function for miR-143 is to regulate myocyte growth and elongation is the identification of a cytoskeletal component, Add3, as one of its required targets. In agreement with the current findings, add3 transcripts have been shown to be 2-fold more abundant in adult SM samples from miR-143;miR-145 double-knockout mice (Xin, 2009). In these studies, miR-143 and miR-145 were shown to actively repress numerous transcripts involved in actin dynamics. Interestingly, two additional miRNAs, miR-1 and miR-133, were recently shown to predominantly target actin-related and actin-binding proteins of the sarcomere in skeletal muscle. Since Adducin proteins are known to alter actin cytoskeletal dynamics, Add3 represents an attractive target for converting both uniform and localized genetic signals into precise cell growth and shape changes (Deacon, 2010).
One model for chamber emergence can be envisioned based on the collective results from a variety of model organisms. The linear heart tube consists of smaller, more circular cells that show polymerized F-actin at the periphery. In order for the chamber myocardium to balloon from the tube, outer curvature (OC) cardiomyocytes (CMs) must break down their actin cytoskeleton to allow cell growth and elongation, a process that is dependent upon fine-tuning of Add3 function. It is hypothesized that miR-143 acts throughout the heart tube to limit the production of new, intracellular Add3 protein, thus providing one level of Add3 regulation. Concomitantly, it is speculated that other regionalized signals, such as a kinase, might act to post-translationally promote the dissociation of Add3 from F-actin, thereby allowing the cytoskeleton to be redistributed throughout the cell. In the absence of miR-143 function, continued Add3 production might overwhelm other modes of post-translational regulation, leading to sustained Add3-actin complex formation and cytoskeletal polymerization. Discerning whether miR-143 acts in concert with other regionalized factors to influence cell morphology will be vital to appreciating the full role of the miR-143-add3 pathway in cardiac morphogenesis and, possibly, disease (Deacon, 2010).
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