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PubMed1995

Adducin: a physical model with implications for function in assembly of spectrin-actin complexes.

Adducin binds to spectrin-actin complexes, promotes association of spectrin with actin, and is subject to regulation by calmodulin as well as protein kinases A and C. Adducin is a heteromer comprised of homologous alpha and beta-subunits with an NH2-terminal protease-resistant head domain, connected by a neck region to a COOH-terminal hydrophilic, protease-sensitive region. This study provides evidence that adducin in solution is a mixture of heterodimers and tetramers. CD spectroscopy of COOH-terminal domains of alpha- and beta-adducin bacterial recombinants provides direct evidence for an unstructured random coil configuration. Cross-linking, proteolysis, and blot-binding experiments suggest a model for the adducin tetramer in which four head domains contact one another to form a globular core with extended interacting alpha- and beta-adducin tails. The site for binding to spectrin-actin complexes on adducin was identified as the COOH-terminal tail of both the alpha- and beta-adducin subunits. The capacity of native adducin to recruit spectrin to actin filaments is similar to that of adducin tail domains. Thus, adducin tail domains alone are sufficient to interact with F-actin and a single spectrin and to recruit additional spectrin molecules to the ternary complex.

Actins↗
PubMed1996

Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C.

Adducin promotes association of spectrin with actin and caps the fast growing end of actin filaments. Adducin contains N-terminal core, neck, and C-terminal tail domains, is a substrate for protein kinases A (PKA) and C (PKC), and binds to Ca2+/calmodulin. Ser-726 and Ser-713 in the C-terminal MARCKS-related domains of alpha- and beta-adducin, respectively, were identified as the major phosphorylation sites common for PKA and PKC. PKA, in addition, phosphorylated alpha-adducin at Ser-408, -436, and -481 in the neck domain. Phosphorylation by PKA, but not PKC, reduced the affinity of adducin for spectrin-F-actin complexes as well as the activity of adducin in promoting binding of spectrin to F-actin. The myristoylated alanine-rich protein kinase C substrate-related domain of beta-adducin was identified as the dominant Ca2+-dependent calmodulin-binding site. Calmodulin-binding was inhibited by phosphorylation of beta-adducin and of a MARCKS-related domain peptide by PKA and PKC. Calmodulin in turn inhibited the rate, but not the extent, of phosphorylation of beta-adducin, but not alpha-adducin, by PKA and that of each subunit by PKC. These findings suggest a complex reciprocal relationship between regulation of adducin function by calmodulin binding and phosphorylation by PKA and PKC.

Amino Acid Sequence↗
PubMed1987

Erythrocyte adducin: a calmodulin-regulated actin-bundling protein that stimulates spectrin-actin binding.

Adducin is an erythrocyte membrane skeletal phosphoprotein comprised of two related subunits of 105,000 and 100,000 Mr. These peptides form a functional heterodimer, and the smaller of the two binds calmodulin in a calcium-dependent fashion. Although this protein has been physicochemically characterized, its function remains unknown. We have examined the interaction of human adducin with actin and with human erythrocyte spectrin using sedimentation, electrophoretic, and morphologic techniques. Purified adducin binds actin at physiologic ionic strength and bundles it into arrays of laterally arranged filaments, the adducin forming cross-bridges between the filaments at 35.2 /- 3.8 (2 SD) nm intervals. The stoichiometry of high affinity adducin binding to actin at saturation is 1:7, corresponding to a dimer of adducin for every actin helical unit. Adducin also promotes the binding of spectrin to actin independently of protein 4.1. At saturation, each adducin promotes the association of one spectrin heterodimer. The formation of this ternary spectrin-actin-adducin complex is independent of the assembly path, and the complex exists in a readily reversible equilibrium with the free components. The binding of adducin to actin and its ability to stimulate spectrin-actin binding is down-regulated by calmodulin in a calcium-dependent fashion. These results thus identify a putative role for adducin, and define a calcium- and calmodulin-dependent mechanism whereby higher states of actin association and its interaction with spectrin in the erythrocyte may be controlled.

Actins↗
PubMed1988

Erythrocyte adducin. Comparison of the alpha- and beta-subunits and multiple-site phosphorylation by protein kinase C and cAMP-dependent protein kinase.

Two major substrates for human erythrocyte protein kinase C (PK-C) of Mr 120,000 and 110,000, previously named PKC-1 and PKC-2 [Palfrey, H. C. & Waseem, A. (1985) J. Biol. Chem. 260, 16021-16029] have been found to be identical to CaM-BP 103/97 or 'adducin', recently described by K. Gardner and V. Bennett [(1986) J. Biol. Chem. 261, 1339-1348; (1987) Nature (Lond.) 328, 359-362]. These proteins have been purified from the membrane skeleton by high-salt extraction, ion-exchange and gel filtration chromatography. The two proteins co-fractionate in a ratio of approximately 1:1 under a number of conditions suggesting that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of alpha and beta subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca2+-dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the alpha and beta subunits using three different proteases yields 16-37% overlapping peptides, indicating limited similarity between the two polypeptides. Affinity-purified polyclonal antibodies against each protein show little or no cross-reactivity with the other, indicating that the beta subunit is not derived from the alpha subunit or vice versa. Proteins reactive with both anti-(alpha-adducin) and anti-(beta-adducin) antibodies are found in erythrocytes from rat, rabbit, pig, ferret and duck. Immunoblots of adducin after non-ionic detergent extraction of ghosts reveal that a significant fraction of the protein may associate with non-skeleton membrane components. The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in intact erythrocytes. Fingerprinting suggests that protein kinase C preferentially phosphorylates four distinct sites on the two proteins. Phosphopeptide maps of alpha-adducin are virtually identical to those of beta-adducin after phorbol ester stimulation of intact cells, or after PK-C-catalyzed phosphorylation of the purified protein, indicating strong local similarities in the two proteins. Such maps also suggest that cAMP-dependent protein kinase (cAMP-PK) modifies adducin at some similar and some distinct sites as those modified by PK-C. In vitro phosphorylation of isolated adducin by purified PK-C results in rapid incorporation of phosphate to a final level of approximately 1.5 mol/mol in both alpha and beta subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Adducin in platelets: activation-induced phosphorylation by PKC and proteolysis by calpain.

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 were 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 led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was 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. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.

Adult↗
PubMed1991

Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization.

Adducin is a calmodulin-binding protein involved in the assembly of the erythrocyte membrane skeleton. To investigate the expression of adducin during human erythropoiesis, we performed immunofluorescence studies on smears of cultured human erythroblasts. Adducin immunoreactivity was found in the early stages of erythropoiesis. Proerythroblasts were the first erythroid precursor cells positive for adducin. The adducin signal was very similar to the signal of erythroid beta-spectrin in that both proteins lined the membrane of erythroid precursor cells. Cell fractionation experiments were performed to further analyze the intracellular distribution of adducin in erythroid cells. In erythroblasts, about 60% of total cellular adducin appeared in the Triton-soluble fraction. In reticulocytes, the Triton-soluble fraction decreased to 30% of total reticulocyte adducin. Erythrocytes had no detectable amount of adducin in the Triton-soluble pool. Instead, adducin was quantitatively bound to the Triton-insoluble erythrocyte cytoskeleton. Our results suggest that adducin is expressed early in the development of the erythrocyte membrane skeleton, whereas stable assembly onto the membrane skeleton does not occur before the final stages of mammalian erythropoiesis.

Animals↗
PubMed1995

35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5' rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to alpha-and beta-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to alpha- and beta-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that alpha-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with alpha-adducin. We propose naming this adducin homologue gamma-adducin.

Amino Acid Sequence↗
PubMed1998

Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase.

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted 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. We also found that Rho-kinase phosphorylated alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhanced 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.

Actins↗
PubMed1991

Primary structure and domain organization of human alpha and beta adducin.

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.

Amino Acid Sequence↗
PubMed1990

Identification and protein kinase C-dependent phosphorylation of alpha-adducin in human fibroblasts.

A protein of Mr approximately 120,000, related to the human erythrocyte membrane skeletal protein alpha-adducin, has been identified by immunological criteria in human fibroblasts. Using similar methods, beta-adducin (an Mr approximately 110,000 protein that forms a dimeric complex with alpha-adducin in the erythrocyte) is not present in fibroblasts. Subcellular distribution studies reveal that fibroblast alpha-adducin is largely associated with the particulate fraction and is most effectively solubilized from that fraction by a combination of nonionic detergent and high salt. Immunocytochemistry of quiescent fibroblasts shows that alpha-adducin is clustered in large perinuclear arrays that may correspond to vesicular structures; weak staining was also found in the sub-plasma membrane region. As in erythrocytes, the phosphorylation of fibroblast alpha-adducin is elevated on exposure of cells to phorbol esters that activate protein kinase C (PK-C). In addition, various mitogens such as serum, bradykinin and vasopressin also stimulate alpha-adducin phosphorylation by a PK-C-dependent pathway. The elevation in alpha-adducin phosphorylation is maintained for up to 30 min after mitogen addition. Peptide maps of phospho-alpha-adducin from both fibroblasts and erythrocytes after PK-C-mediated phosphorylation showed multiple phosphorylated peptides but with dissimilar migration patterns, suggesting divergence of structure around the phosphorylation sites. Adducin appears to play an important role in the regulation of spectrin-actin interactions in the red cell and may play a role in cytoskeletal function in the fibroblasts.

Calmodulin-Binding Proteins↗
PubMed1988

Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction.

Erythrocyte adducin is a membrane skeletal protein that binds to calmodulin, is a major substrate for protein kinase C, and associates preferentially with spectrin-actin complexes. Erythrocyte adducin also promotes association of spectrin with actin, and this activity is inhibited by calmodulin. This study describes the isolation and characterization of a brain peripheral membrane protein closely related to erythrocyte adducin. Brain and erythrocyte adducin have at least 50% antigenic sites in common, each contains a protease-resistant core of Mr = 48,000-48,500, and both proteins are comprised of two partially homologous polypeptides of Mr = 103,000 and 97,000 (erythrocytes) and Mr = 104,000 and 107,000-110,000 (brain). Brain and erythrocyte adducin associate preferentially with spectrin-actin complexes as compared to spectrin or actin alone, and both proteins also promote binding of spectrin to actin. Brain adducin binds calmodulin in a calcium-dependent manner, although the Kd of 1.3 microM is weaker by 5-6-fold than the Kd of erythrocyte adducin for calmodulin. Brain adducin is a substrate for protein kinase C in vitro and can accept up to 2 mol of phosphate/mol of protein. Adducin provides a potential mechanism in cells for mediating site-directed assembly of additional spectrin molecules and possibly other proteins at the spectrin-actin junction. Brain tissue contains 12 pmol of adducin/mg of membrane protein, which is the most of any tissue examined other than erythrocytes, which have 50 pmol/mg. The presence of high amounts of adducin in brain suggests some role for this protein in specialized activities of nerve cells.

Actins↗
PubMed1998

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats.

Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na+,K+-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA+,K+-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting.

Animals↗
PubMed2006

Fyn-induced phosphorylation of beta-adducin at tyrosine 489 and its role in their subcellular localization.

Fyn is a Src-family tyrosine kinase involved in neuronal development, transmission, and plasticity in mammalian central nervous system. We have previously reported that Fyn binds to a cytoskeletal protein, beta-adducin, in a phosphorylation-dependent manner. In the present report, we show that Fyn phosphorylates beta-adducin at tyrosine 489 located in its C-terminal tail domain. Phosphorylation of beta-adducin at Y489 was required for its association with the Fyn-SH2 domain. An antibody specific to the phosphorylated form of beta-adducin was raised in rabbits and showed that Y489 of beta-adducin was phosphorylated in wild type, but not in Fyn(-/-) mice, suggesting that Y489 of beta-adducin is phosphorylated downstream of Fyn in vivo. After phosphorylation at Y489, beta-adducin was translocated to the cell periphery, and colocalized with Fyn. These results suggest that Fyn phosphorylates and binds to beta-adducin at Y489, resulting in translocation of beta-adducin to the Fyn-enriched regions in the plasma membrane.

Animals↗
PubMed1998

Adducin in essential hypertension.

In Milan hypertensive rats (MHS) the sequence of events going from renal function to cell membrane ion transport abnormalities and finally to the molecular defect responsible of hypertension has been established. A polymorphism of the cytoskeletal protein adducin has been identified as a likely culprit for hypertension in these rats. Two point mutations in MHS alpha- (F316Y) and beta- (Q529R) adducin genes have been shown to be associated with hypertension in genetic crosses of MHS and MNS rats. Also in humans, a polymorphism of alpha-adducin gene (Gly460Trp) has been found to be significantly associated both to hypertension and salt sensitivity. Studies aimed at clarifying the functional role of alpha-adducin variants have shown that adducin from the MHS rats is able to stimulate Na-KATPase activity both after transfection in renal tubular cells and after incubation with the enzyme in a cell-free system. Also the human hypertensive alpha-adducin variant displays the same activity of MHS adducin in a cell-free system. Therefore, both in humans and in rats, adducin polymorphisms may affect blood pressure and kidney function by modulating the overall capacity of tubular epithelial cells to transport ions, through variations of the Na-KATPase activity. However adducin polymorphisms account for only a portion of hypertension both in humans and rats. Therefore additive or epistatic interactions with other genes involved in renal sodium handling need to be studied.

Animals↗
PubMed1997

Polymorphisms of alpha-adducin and salt sensitivity in patients with essential hypertension.

BACKGROUND: Abnormalities in renal sodium transport may be involved in hypertension. Adducin, an alpha/beta heterodimeric protein found in the renal tubule is thought to regulate ion transport through changes in the actin cytoskeleton. We investigated whether an alpha-adducin polymorphism (Gly 460 Trp) is involved in essential hypertension in two separate populations. METHODS: Linkage analysis of three DNA markers at different distances from the alpha-adducin locus (20-2500 kb) was done in 137 hypertensive sibling-pairs. 477 hypertensive and 322 normotensive individuals were genotyped for the alpha-adducin polymorphism. The blood-pressure response to acute and chronic changes in sodium balance was studied in hypertensive individuals with and without the 460 Trp alpha-adducin allele. FINDINGS: Significant linkage was found for all three markers in the sibling-pair study. The extra shared alleles (9.1%, 6.5%, and 4.7%) and the significance level for linkage (p = 0.0006, p = 0.0119, and p = 0.0211) both decreased with increasing distance from the alpha-adducin locus. There was a significant association between the 460 Trp mutation and hypertension (p = 0.0003). In the salt-sensitivity test, to assess the acute blood-pressure response to changes in body sodium in 86 hypertensive patients, the decrease in mean arterial pressure was greater in 65 patients who were heterozygous for the mutant allele (Gly/Trp) than in 21 wild-type homozygotes (Gly/Gly) (mean decrease 15.9 [SE 2.0] vs 7.4 [1.3] mm Hg; p = 0.001). Similarly, 21 heterozygous hypertensive patients showed a greater fall in mean arterial pressure in response to 2 months' treatment with hydrochlorothiazide than did 37 wild-type homozygous hypertensive patients (mean decrease 14.7 [2.2] vs 6.8 [1.4] mm Hg; p = 0.002). INTERPRETATION: Our findings of significant linkage of the alpha-adducin locus to essential hypertension and greater sensitivity to changes in sodium balance among patients with the mutant allele suggest that alpha-adducin is associated with a salt-sensitive form of essential hypertension. We suggest the alpha-adducin polymorphism may identify hypertensive patients who will benefit from diuretic treatment or manoeuvres to reduce total body sodium.

Aged↗
PubMed1998

Utilization of an 86 bp exon generates a novel adducin isoform (beta 4) lacking the MARCKS homology domain.

A novel isoform of beta-adducin has been amplified and characterized from a human bone marrow cDNA library (GenBank #U43959). This isoform arises from the insertion of an 86 bp alternatively spliced and previously unrecognized exon (now termed exon 15) within codon 581 of the human red blood cell beta-adducin sequence. This results in an insertion of 28 novel amino acids. The remainder of the red cell beta-adducin mRNA is then translated in a different reading frame, adding an additional 35 novel amino acids prior to the stop codon. This new isoform, thus, replaces beta 1-adducin sequence after residue 580 with a total of 63 new amino acids. Sequences from genomic clones of the human beta-adducin gene show that this alternate exon is flanked by splice consensus sequences and is appropriately located in the genomic map between exons encoding up-stream and down-stream sequences, thus defining a new exon. The COOH-terminus of this new isoform, which we designate beta 4, lacks a 22 amino acid lysine-rich sequence common to both the human red cell alpha- and beta-adducin subunits and homologous to a highly conserved region in MARCKS, a filamentous actin-cross linking protein regulated by protein kinase C and calcium/calmodulin. beta 4-adducin preserves a previously identified calmodulin binding domain. PCR analysis indicates that this new beta-adducin isoform is expressed in fetal brain and liver, bone marrow, and NT-2 (neuroepithelial) cells, but is not detected in several other tissues. We anticipate that this new beta 4 isoform of beta-adducin will display unique and tissue-specific functional properties.

Alternative Splicing↗
PubMed2005

Pleiotrophin regulates serine phosphorylation and the cellular distribution of beta-adducin through activation of protein kinase C.

Pleiotrophin (PTN) was found to regulate tyrosine phosphorylation of beta-adducin through the PTN/receptor protein tyrosine phosphatase (RPTP)beta/zeta signaling pathway. We now demonstrate that 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. We also demonstrate that PTN 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, we demonstrate that 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.

Biological Transport, Active↗
PubMed2000

Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.

Actins↗