Tubulin–Na + , K + ‐ATPase interaction: Involvement in enzymatic regulation and cellular function (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.
Molecular and Cellular Biochemistry, 2006
In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na + ,K +-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na + ,K +-ATPase and consequent inhibition of enzyme activity.
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.
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.
activity of the Na+/K+-ATPase in human erythrocytes
2015
ANTOLOVIĆ, R.: Low nanomolar concentrations of ouabain may induce higher activity of the Na+/K+-ATPase in human erythrocytes. Vet. arhiv 76, 489-495, 2006. The inhibitor g-Strophantin, also known as ouabain, is a specific inhibitor of the Na+/K+-ATPase. In this work ouabain has been used for the inhibition of the Na+/K+-ATPase in human erythrocytes and isolated enzyme from pig kidney. Enzymatic activity of the Na+/K+-ATPase has been measured in a broad concentration range of ouabain in two different in vitro investigations. As a potent inhibitor of the sodium pump the cardiotonic steroid ouabain inhibits its enzymatic activity on both isolated human red blood cells and on the purified enzyme from pig kidney. Na+/K+-ATPase activity in erythrocyte can be determined by measuring the ouabain-sensitive uptake of 86Rb (as a congener for potassium). This work provides evidence that very low concentrations of ouabain in the nM range can stimulate increase of Na+/K+-ATPase activity in human ...
Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase
Journal of Biological Chemistry, 1994
The objective of this study has been to delineate the side-specific effects of Na' and K+ on the transport kinetics of tissue-specific NaM pumps. Two experimental systems have been used. In one, NaM pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were delivered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individually transfected into HeLa cells as in previous studies (Jewell, E. A, and Lingrel, J. B (1991) J. Biol. Chern. 266, 16925-16930), with the a, and a, isoforms rendered relatively resistant to ouabain by site-directed mutagenesis. Whereas the kidney microsomes comprise the a1 catalytic isoform, the axolemma microsomes were predominantly as (4 0 %) with lesser amounts of a2 (4 5 %) and a1 (-15%) as measured by the ouabain-sensitive profile of phosphoenzyme as well as by immunoblotting with isoform-specific antibodies using membranes of known specific activity as standards (a1 of kidney, a, and a, of muscle). Both systems were analyzed with respect to the effects of varying concentrations of cytoplasmic Na' and extracellular K' on pump-mediated ssRb+(K+) influx. With the individual isoform-transfected HeLa cells and monensin added to vary and control the intracellular Na+ concentration, differences in apparent affinities of the a, isoform compared with the a1 and a2 isoforms were observed, i.e. a &-fold higher affinity for extracellular K' and 4-fold lower affinity for cytoplasmic Na+. Thus, in the presence of 10 m~ extracellular Na' , apparent values for extracellular K' activation of K'(Rb+) influxes were 0.22 * 0.02 m~ for aI, 0.20 t 0.02 m~ for a2, and 0.09 * 0.01 m~ for a,. At high intracellular K+ (2100 m~) and saturating extracellular K+ concentrations, apparent KO, values for cytoplasmic Na+ activation were 17.6 2 1.1 m~ for al, 19.7 = 1.0 m~ for a,, and 63.5 9.1 m~ for a ,. The functional differences observed with the individual isoform-transfected cells were completely consistent with the kinetic differences observed with the axolemma and kidney pumps fused into erythrocytes. kvolemma pumps had a 4-f o l d lower for extracellular K+ and a &-fold higher for cytoplasmic Na'. In the HeLa transfectants, differences in affinities for cytoplasmic Na' were associated with differences in the steady-state intracellular Na' concentration, Le. 27.5 m~ in a,-transfected cells compared with 15.7 and 19.7 m~ in al-and a,-transfected cells, respectively. Research Council of Canada (to R. B.) and ROHIL28573 from the Na-*This work was supported by Grants MT-3876 from the Medical tional Institutes of Health (to J. B. L.). The costs of publication of this article must therefore be hereby marked "aduertisement" in accordance article were defrayed in part by the payment of page charges. This The abbreviations used are: Na,K-ATPase or Na/K pump, sodiumpotassium adenosine triphosphatase; EP, the phosphorylated form of the Na,K-ATPase enzyme;
Febs Journal, 2005
In the yeast Saccharomyces cerevisiae, plasma membrane H+-ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H+-ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H+-ATPase–tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H+-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H+-pumping activity. The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H+-ATPase) immunoglobulins indicated a physical interaction between H+-ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H+-ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H+-ATPase–tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H+-ATPase activity by acetylated tubulin and concomitant increase of the H+-ATP ase–tubulin complex.