Inhibition of phosphatase activity

Boavida, M.-J., and R. G. Wetzel. 1998. Inhibition of phosphatase activity by dissolved humic substances and hydrolytic reactivation by natural UV. Freshwater Biology 40 285—293. 

The lysosomal and the soluble fractions of acid phosphatase were compared in several other ways. The values (milf) for the lysosomal fractions on various substrates were -glycerophosphate, 1.6 fructose 1,6-diphosphate, 2.0 p-nitrophenyl phosphate, 1.6 AMP, 0.43 they were not significantly different from those obtained with the soluble fraction. The pH-activity curves with these substrates were similar for the two fractions. Inhibition of phosphatase activity of the lysosomal and soluble fractions occurred at approximately the same concentration of fluoride or n- (+) -tartrate when j8-glycerophosphate, AMP, or fructose 1,6-diphosphate were used as substrates. However, with p-nitrophenyl phosphate... 

Since disturbed acid phosphatase activity has been associated with pathological conditions, the research has focused on the development of diagnostic methods for detection of activity as a marker for the onset of the disease, and in some extent to the development of inhibitors rather than activators to treat those conditions in which the increase in enzyme activity has a direct effect on the evolution of the disease. In particular, only the development of bisphospho-nate derivatives as inhibitors for tartrate-resistant acid phosphatase found their way to the market for treatment of osteoporosis [41], Typical inhibition of phosphatase activity includes anionic species such as L-(+)-tartrate, phosphate, vanadate, molybdate, and fluoride and neutral molecules such as formaldehyde. Vanadate ion,, is a competitive unspecific inhibitor for acid phosphatases by forming transition state analogs. Other oxoanions such as molybdate and tungstate also show inhibitory effects on... 

Several phosphatase inhibitors are available and have been useful in many aspects of smooth muscle research. Okadaic acid was the first inhibitor to be widely used and inhibition of phosphatase activity by this compound was demonstrated using smooth muscle (Hartshorne et al., 1989). Calyculin A was also used in several earlier studies. Since 1990 other compounds have been described as phosphatase inhibitors. These are remarkable in that despite considerable differences in structure, they are relatively specific as phosphatase inhibitors. These inhibitors include tau-tomycin, as antifungal antibiotic produced by Strep-tomyces (MacKintosh and Klumpp, 1990 Hori et al.,... 

P-myosin is higher than that of the isolated catalytic subunit. Thus dissociation of the trimeric phosphatase could be a regulatory mechanism. The factors involved in dissociation of PPlc are not established, although there is the suggestion that arachidonic acid is implicated. It is possible that dissociation of the trimeric phosphatase is the end result of the G-protein-linked inhibition of phosphatase activity, observed in several systems. Another possibility is that the trimeric phosphatase binds only to phosphorylated myosin and has a lower affinity for the dephosphory-lated form. Whatever the scenario imagined, it is clear that much remains to be learned about regulation of myosin phosphatase activity. 

Like cyclosporine, tacrolimus inhibits T-cell activation by inhibiting calcineurin. Tacrolimus binds to an intracellular protein, FK506-biruiing protein-12 (FKBP-12), an immunophilin structurally related to cyclophilin. A complex of tacrolimus-FKBP-12, Ca, calmodulin, arui calcineurin then forms, arui calcineurin phosphatase activity is inhibited. described for cyclosporine and depicted in Figure 52—1, the inhibition of phosphatase activity prevents dephosphorylation and nuclear translocation of NFAT arui inhibits T-cell activation. Thus, although the intracellular receptors differ, cyclosporine and tacrolimus target the same pathway for immunosuppression. 

Ichikawa K, Ito M, Hartshorne DJ (1996a) Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem 271 4733-4740... 

Mortland and Gieseking (1952) found that all the clays that they studied exerted an inhibiting effect upon the enzymatic hydrolysis of organic phosphorus c ompounds. The effect of the clays was as follows montmorillonite > Cisne > illite > kaolinite. The inhibition of phosphatase activity corresponded closely to the base exchange capacity of the clays, and was due primarily to their effect on the enzyme and riot to adsorption of the organic phosphorus compounds by the clays. 

Miller, C., Zhang, M., He, Y, Zhao, J., Pelletier, J.P., Martel-Pelletier, J., and Di Battista, J.A. (1998) Transcriptional Induction of Cyclooxygenase-2 Gene by Okadaic Acid Inhibition of Phosphatase Activity in Human Chondrocytes Co-Stimulation of AP-1 and CRE Nuclear Binding Proteins, J. Cell. Biochem. 69, 392 13. 

Calcium-dependent regulation involves the calcium-calmodulin complex that activates smooth muscle MLCK, a monomer of approximately 135 kDa. Dephosphorylation is initiated by MLCP. MLCP is a complex of three proteins a 110-130 kDa myosin phosphatase targeting and regulatory subunit (MYPT1), a 37 kDa catalytic subunit (PP-1C) and a 20 kDa subunit of unknown function. In most cases, calcium-independent regulation of smooth muscle tone is achieved by inhibition of MLCP activity at constant calcium level inducing an increase in phospho-rMLC and contraction (Fig. 1). 

K.H. Lau, T.K. Freeman, D.J. Baylink, A proposed mechanism of the mitogenic action of fluoride on bone cells. Inhibition of the activity of an osteoblastic acid phosphatase, Metabolism 38 (1989) 858-868. 

The immimosuppressive effect of cyclosporin and FK506 could not initially be explained by these observations. Only with the discovery that cyclosporin and FK506 achieve their immunosuppressive effect via inhibition of calcineurin did it become clear that the immimosuppression is mediated by a complex reaction chain involving calcineurin. It was shown that the complexes of cyclosporin/cyclophilin and FK506/ FK506 binding protein bind to calcineurin and inhibit the phosphatase activity of the latter. 

Another class of transcription-modulating drugs is the immunosuppressants such as cyclosporin A (CsA), which inhibit T cell activation and proliferation, events playing a central role in the immune response and therefore in the inflammatory process. CsA blocks transcriptional induction of cytokines by inhibiting the phosphatase calcineurin, and by the subsequent inhibition of the activation of NF-AT and NF-AT-dependent activation genes. 

In 1954, Beaufay and de Duve (27) first suggested a relationship between microsomal phospholipid and glucose-6-phosphatase. They observed a loss of enzymic activity from phospholipid-rich microsomal preparations concomitant with extraction with such organic solvents as butanol or treatment with lecithinase. Various studies were carried out to demonstrate that the latter effect was not produced through inhibition of enzymic activity by accumulated products of the hydrolysis of phospholipids. On the basis of their observations that deoxycholate treatment labilized microsomes to phospholipase action, they concluded that . . . the detergent did not exert its primary effect on the dissociation of phospholipids from microsomal protein, but that it probably disrupted... 

Tacrolimus suppresses peptidyl-prolyl isomerase activity by binding to the immuno-philin FK506-binding protein-12 (FKBP-12), and the tacrolimus-FKBP-12 complex binds to calcineurin and inhibits calcineurin phosphatase activity. As a result, calcineurin is unable to dephosphorylate NFATc and thus its migration to nucleus is blocked where its association with NFATn is necessary for the activation of key cytokine genes. Therefore, its mechanism of action is similar to cyclosporine although tacrolimus binds to a separate set of immunophilins in the cytoplasm. Tacrolimus, like cyclosporine, inhibits the secretion of key cytokines and inhibits T-cell activation (Fig. 4.2). 

Hulley, P. A., F. Gordon, and F.S. Hough. 1998. Inhibition of mitogen-activated protein kinase activity and proliferation of an early osteoblast cell line (MBA 15.4) by dexamethasone Role of protein phosphatases. Endocrinology 139 2423-31. 

Very recendy a new, physiologic inhibitor of calcineurin has been described by Liu and colleagues at MIT and OriGene (Sun et al., 1998). This protein, called cabin-1 (calcineurin binding protein) is a 2220-residue nuclear protein, which was isolated from a murine T-cell cDNA library in a yeast two-hybrid system with a truncated, catalydcally inac-dve calcineurin used as the bait. Cabin-1 was shown to be a phospho-protein capable of binding to and inhibiting the phosphatase activity of calcineurin, and the interaction between cabin-1 and calcineurin was shown to be sensitive to disruption by FK506 or CsA. In a mammalian... 

Bedecs, K., Elbaz, N., Sutren, M., Masson, M., Susini, C., Strosberg, A.D., and Nahmias, C. 1997. Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase. Biochem. J. 325 449-454. 

Figure 18.14 Glycogen biosynthesis and degradation regulation. cAMP activates cAMP-dependent protein kinases. They cause the phosphorylation of glycogen synthase (inactivation), phosphorylase kinase (activation), and the inhibitory protein. The last inhibits phosphoprotein phosphatase. Activated phosphorylase kinase causes the phosphorylation of phosphorylase b, thus activating it to phosphorylase a. Phosphoprotein phosphatase is inhibited by the phosphorylated inhibitor protein. Such inhibition is released when the inhibitor protein is dephosphorylated. The phosphatase then reactivates glycogen synthase and inactivates phosphorylase kinase and phosphorylase a.

Further study of this synthesis in partly purified extracts did not reveal any requirements for cofactor or metal. Shikimate 5-phosphate (alone) was extensively hydrolyzed in these extracts to shikimate and orthophosphate, but, when equimolar amounts of enolpyruvate phosphate were also added, only insignificant quantities of shikimate were produced, and, for every mole of Zl formed, two equivalents of orthophosphate were released. The phosphatase action on shikimate 5-phosphate was strongly inhibited when the amount of enolpyruvate phosphate present was equivalent to only 10 per cent of the shikimate 5-phosphate, but Zl added initially to shikimate 5-phosphate did not inhibit the phosphatase activity. 

Cyclosporine binds to an intracellular protein, cyclophilin. Cyclophilins and similar binding proteins are now referred to collectively as immunophilins and their enzymatic activities are relevant to the actions of immunosuppressants such as cyclosporine and tacrolimus. This complex inhibits the phosphatase activity of calcineurin, which in turn prevents dephosphorylation and translocation of NFAT. NFAT is required to induce a number of cytokine genes, including that for interleukin-2, which serves as a T-cell growth and differentiation factor (Krensky et al., 2005 Matsuda and Koyasu, 2000). Cyclosporine also increases expression of TGF-p, which is a potent inhibitor of IL-2 stimulated cell proliferation and generation of cytotoxic T lymphocytes (Khanna et al., 1994). 

The mechanism of action of tacrolimus is similar to that of cyclosporine at the molecular level (Krensky et al., 2005 Spencer et al., 1997). Tacrolimus exerts potent inhibitory effects on T lymphocyte activation. It binds to an intracellular protein, FKBP-12. A complex is formed which inhibits the phosphatase activity of calcineurin. This in turn prevents dephosphorylation and translocation of nuclear factor of activated T lymphocytes (NFAT) which results in inhibition of T-lymphocyte activation. 

Inhibition of PKC by a-tocopherol in vascular smooth muscle cells is observed to occur at concentrations of a-tocopherol close to those measured in healthy adults [24]. P-Tocopherol per se is not very effective but prevents the inhibitory effect of a-tocopherol. The mechanism involved is not related to the radical scavenging properties of these two molecules, which are essentially equal [25]. In vitro studies with recombinant PKC have shown that inhibition by a-tocopherol is not caused by tocopherol-protein interaction, a-Tocopherol does not inhibit PKC expression as well. Inhibition of PKC activity by a-tocopherol occurs at a cellular level by producing dephosphorylation of the enzyme, whereby P-tocopherol is much less potent [26]. Dephosphorylation of PKC occurs via protein phosphatase PP2A, which is activated by the treatment with a-tocopherol [26-28]. 

---