Tubulin must be acetylated in order to form a complex with membrane Na+,K+-ATPase and to inhibit its enzyme activity (original) (raw)
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Tubulin–Na + , K + ‐ATPase interaction: Involvement in enzymatic regulation and cellular function
Journal of Cellular Physiology, 2018
A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na + ,K +-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin-NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na +. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na + and K + , and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na + /K + homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.
Tubulin–Na + , K + ‐ATPase interaction: Involvement in enzymatic regulation and cellular function
Journal of Cellular Physiology, 2018
A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na + ,K +-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin-NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na +. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na + and K + , and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na + /K + homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca 2 +-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300 μg/ml) in combination with acidic lipids at concentrations > 10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50 μg/ml tubulin, increased PMCA activity > 12-fold, whereas tubulin alone at high concentration (≥300 μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca 2 + transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (b 50 μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.
Biochem. J, 2009
We showed previously that NKA (Na + /K +-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubuleplasma membrane anchorage site through an interaction between acetylated tubulin and CD5.
Brain Research, 1995
Previous studies of rat cerebral cortex and rat C6 glioma cells have demonstrated that dimeric tubulin is capable of activating the G proteins Gs and Gil via transfer of guanine nucleotide from tubulin to Gscu and Gilcu. To provide further information regarding cytoskeletal modulation of adenylyl cyclase, the present study examined effects of tubulin on the activation of the enzyme in rat striatal membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analog 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of adenylyl cyclase by u 130%. Furthermore, tubulin-GppNHp activated SKF 38393-sensitive adenylyl cyclase and potentiated forskolin-stimulated activity of the enzyme. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analog [ 32p]p3 (4-azidoanilido)-p'-S-GTP ([ 32P]AAGTP), was incubated with striatal membranes, AAGTP was transferred from tubulin to Gscu as well as Gicu with the extents of nucleotide transfers being 7.6 k 0.8% and 17.8 f 1.4% of AAGTP originally bound to tubulin, respectively. These results indicate that, in rat striatum, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gsa, supporting the hypothesis that tubulin contributes to the regulation of neuronal signal transduction. . 0006-8993/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01073-4
The International Journal of Biochemistry & Cell Biology, 2012
Our previous studies demonstrated that acetylated tubulin forms a complex with Na + ,K +-ATPase and thereby inhibits its enzyme activity in cultured COS and CAD cells. The enzyme activity was restored by treatment of cells with l-glutamate, which caused dissociation of the acetylated tubulin/Na + ,K +-ATPase complex. Addition of glucose, but not elimination of glutamate, led to reformation of the complex and inhibition of the Na + ,K +-ATPase activity. The purpose of the present study was to elucidate the mechanism underlying this effect of glucose. We found that exposure of cells to high glucose concentrations induced: (a) microtubule formation; (b) activation of aldose reductase by the microtubules; (c) association of tubulin with membrane; (d) formation of the acetylated tubulin/Na + ,K +-ATPase complex and consequent inhibition of enzyme activity. Exposure of cells to sorbitol caused similar effects. Studies on erythrocytes from diabetic patients and on tissues containing insulin-insensitive glucose transporters gave similar results. Na + ,K +-ATPase activity was >50% lower and membrane-associated tubulin content was >200% higher in erythrocyte membranes from diabetic patients as compared with normal subjects. Immunoprecipitation analysis showed that acetylated tubulin was a constituent of a complex with Na + ,K +-ATPase in erythrocyte membranes from diabetic patients. Based on these findings, we propose a mechanism whereby glucose triggers a synergistic effect of tubulin and sorbitol, leading to activation of aldose reductase, microtubule formation, and consequent Na + ,K +-ATPase inhibition.
Identification and characterization of a tubulin binding protein in rat brain plasma membrane
Neurochemistry International, 1994
Akstraet-Studies on the interaction of FITC-tubulin and ~2~I-tubulin with isolated plasma membrane of neural cells and with primary cultures of neuronal (N) and glial (G) cells of rat brain demonstrate the presence of specific, saturable, high affinity tubulin binding sites in these cells. The positive fluorescence of live unfixed primary cultures of N and G cells following incubation with FITC-tubulin indicate that the tubulin binding sites are located on the outer side of the plasma membrane. Such fluorescence was not observed with FITC-BSA, FITC conalbumin or freshly dissociated cells from rat tissues or established cell lines. Binding of FITC-tubulin or 125I tubulin is competed only by tubulin and not by other proteins. Scatchard analysis of the binding of t25I-tubulin to purified plasma membrane indicates very high affinity (Kd = 85 nM) with a Bmax of 7.4 pmol/mg protein. The putative tubulin receptor was partially purified by affinity chromatography on tubulin-sepharose column. Immunoprecipitation of the solubilized tubulinreceptor complex followed by SDS-PAGE analysis and autoradiography, revealed the presence of two components of molecular weights 70 and 45 kDa respectively, presumably representing the two nonidentical subunits of the putative receptor. In conjunction with several recent reports indicating the secretion of high molecular weight proteins from cultured neural cells and the ability of tubulin to modulate adenyl cyclase in synaptic membranes these findings suggest that the binding of exogenous tubulin to sites external to the plasma membrane may be involved in signal transduction. 290 SLrKANYA CHAIrl)HURY el 0]. of the receptor has been determined by immunoprecipitation of the solubilized ~251_labeled tubulin receptor complex using antitubulin antibodies.
The Journal of Cell Biology, 1983
We describe the interaction of pure brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. We studied the tubulin-binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay. The tubulin-membrane interaction was time- and temperature-dependent, reversible, specific, and saturable. The binding of tubulin to membranes appears to be specific since acidic proteins such as serum albumin or actin did not interfere in the binding process. The apparent overall affinity constant of the tubulin-membrane interaction ranged between 1.5 and 3.0 X 10(7) M-1; similar values were obtained for the three types of membranes. Tubulin bound to membranes was not entrapped into vesicles since it reacted quantitatively with antitubulin antibodies. At saturation of the tubulin-binding sites, the amount of reversibly ...