Phosphoprotein phosphatase inhibitor

Increases in cAMP further lead to phosphorylation and activation of phosphoprotein phosphatase inhibitor 1 (PPl-1), which inhibits the activity of phosphoprotein phosphatase and so decreases the conversion (dephosphorylation) of inactive to active HMG-CoA reductase. 

Dephosphorylation of glycogen synthase and glycogen phosphorylase reverses the effects of phosphorylation. This converts glycogen synthase to the independent form and glycogen phosphorylase to a less active form. The primary enzyme responsible for dephosphorylating the glycogen metabolism enzymes is phosphoprotein phosphatase (PP-1). It is regulated by another protein called phosphoprotein phosphatase inhibitor (PI-1). PI-1 is also phosphorylated by active protein kinase. When phosphorylated, PI-1 inhibits PP-1. 

Phosphoprotein phosphatase counters the effects of the kinase cascade by removing phosphates from proteins (Figure 16.11 and Figure 16.12). Thus, it converts the phosphorylated glycogen phosphorylase a (more active) to the dephosphorylated glycogen phosphorylase b (less active) and also converts phosphorylated glycogen synthase D (less active) to the more active glycogen synthase I form (dephosphorylated). The activity of phosphoprotein phosphatase I is controlled by phosphoprotein phosphatase inhibitor. 

Phosphoprotein phosphatase inhibitor regulates the activity of phosphoprotein phosphatase I (Figure... 

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

As outlined above, protein phosphorylation is a key process involved in many signal transduction pathways and reversal of this process is catalyzed by a multiplicity of phosphoprotein phosphatases (PPs). Major PPs catalyzing dephosphorylation of phosphoserine or phosphothreonine residues on proteins include PP1 (inhibited by phosphorylated inhibitor protein I-1 and by okadaic acid and microsystins), PP2 (also inhibited by okadaic acid and microcystins), PP2B or calcineurin (CaM-activated and having a CaM-like regulatory subunit) and PP2C (Mg2+-dependent) [18]. These PPs have been found in all eukaryotes so far examined [18, 19]. In addition, a variety of protein phosphotyrosine phosphatases can reverse the consequences of RTK or JAK/STAT receptor activation [20]. 

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.

Glycogen degradation requires phosphorylated (active) phosphorylase kinase to maintain the production of phosphorylated (active) phosphorylase a the a-l,6-glucosidase is required to remove branch molecules from partially degraded glycogen, and phosphorylated (active) inhibitor protein is required to inactivate phosphoprotein phosphatase. 

Hemmings HC, Jr, Nairn AC, Greengard P (1984a) DARPP-32, a dopamine- and adenosine 3 5 -monophosphate-regulated neuronal phosphoprotein. II. Comparison of the kinetics of phosphorylation of DARPP-32 and phosphatase inhibitor 1. J Biol Chem 259 14491-14497. 

Hemmings HC, Jr, Nairn AC, Elliott JI, Greengard P (1990) Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity. J Biol Chem 265 20369-20376. 

The rapid (i.e. less than 4 h) activation of TH in the median eminence by prolactin that constitutes the tonic component of prolactin stimulation does not require protein synthesis, but is probably associated with effects on the catalytic properties of this enzyme. Pasqualini and coworkers (1994) demonstrated in vitro that prolactin acts directly on TH in the mediobasal hypothalamus to trigger the phosphorylation of this enzyme. This effect, possibly mediated by protein kinase C, makes the enzyme less susceptible to inhibition by newly synthesized DA. That is, prolactin-induced short-term activation of TH results from the removal of end-product inhibition of the enzyme. Conversely, the acute reduction in TH activity measured in vitro in median eminence removed from rats 4 h after administration of bromocriptine is prevented by the coadministration of prolactin (Arbogast and Voogt, 1995). This can also be prevented by an inhibitor of phosphoprotein phosphatases, suggesting that rapid suppression of TH activity secondary to the bromocriptine-induced hypoprolactinemia may also result from dephosphorylation of the enzyme. 

Figure 6.5 Regulation of HMG-CoA reductase. HMG-CoA reductase is active in the dephospho-rylated state phosphorylation (inhibition) is catalysed by reductase kinase, an enzyme whose activity is also regulated by phosphorylation by reductase kinase kinase. Hormones such as glucagon and adrenalin (epinephrine) negatively affect cholesterol biosynthesis by increasing the activity of the inhibitor of phosphoprotein phosphatase-1, PPI-1, (by raising cAMP levels) and so reducing the activation of HMG-CoA reductase. Conversely, insulin stimulates the removal of phosphates (and lowers cAMP levels), and thereby activates HMG-CoA reductase activity. Additional regulation of HMG-CoA reductase occurs through an inhibition of synthesis of the enzyme by elevation in intracellular cholesterol levels.

Chemical tools have played a central role in elucidating the function of posttranslational protein phosphorylation. Kinase and phosphatase inhibitors have played a major role in the study of protein phosphorylation because they allow rapid, reversible inactivation of the target in meaningful contexts (cells and organisms) to study its biologic function. Chemical tools have also helped identify the direct substrates of individual kinases and phosphatases, and conversely, they have helped identify which kinase is responsible for phosphorylating a particular phosphoprotein. Here I will focus on chemical tools developed for protein kinases several reviews discuss chemical tools for protein phosphatases (15, 16). 

Phosphoprotein phosphatase 2A (PP2A) inhibitors Tau kinase inhibitors Diaminothiazoles Thiazolidinedione Troglitazone Pioglitazone Rosiglitazone... 

It is important to distinguish clearly between a covalent modification for activation and an allosteric activation. A covalent modification remains with the enzyme until the modification is enzymatically reversed. For example, phosphorylase kinase and phosphorylase will remain active after phosphorylation until the enzymes are dephosphorylated as catalyzed by phosphoprotein phosphatase. In the case of allosteric activation, the enzyme will remain active as long as there is an elevated allosteric effector such as calcium. As soon as calcium is reconcentrated in subcellular fractions or subcellular organelles and/or extruded from the cell, the enzyme becomes relatively inactive because there is no longer an allosteric activator present. The same is true for allosteric inhibitors. Thus, it is advantageous that both of these mechanisms are available to the cell. 

Other work on ethylene developmental responses using a number of inhibitors of protein kinases and phosphoprotein phosphatases indicates that both these processes are involved in ethylene transduction processes [45-48]. Caution is necessary in interpreting such results however, since it is unlikely that effects on phosphorylation cascades are unique to ethylene. Nevertheless, recent work by Kim et al. [49] has shown that in mung beans, while ethylene induced an increase in ACC oxidase mRNA, it suppressed that for ACC synthase. However, staurosporine and okadaic acid reversed both of these processes, indicating that both phosphorylation and dephosphorylation of proteins is necessary for the signalling processes which regulate these genes. 

Cdc, cell division cycle DARPP-32, dopamine and cAMP-regulated phosphoprotein of 32kDa MAPK, mitogen-activated protein kinase NIPP1, nuclear inhibitor of PP1 PP, protein phosphatase Vffl, vaccinia virus. 

Extrapolating from well-characterized enzymatic inhibition in test tubes, numerous mechanistic ideas concerning the in vivo effects of vanadium compounds have been advanced. The effects of vanadium compounds as transition-state analogs of certain enzymes with a phosphoprotein intermediate in their reaction scheme is proposed to account for the action of vanadium [11] in many biological systems. Unfortunately, it is often difficult to determine if the inhibition observed in the test tube occurs in vivo. For example, although vanadate is a potent inhibitor of plasma membrane ion pumps (such as the sodium potassium ATPase) in the test tube, it is difficult to determine if these pumps are actually inhibited in animals exposed to vanadium compounds. Currently, the role of vanadium compounds as protein phosphatase (PTP) inhibitors is believed to be related to the metabolic effects of this... 

Although CsA and FK506 are extremely selective inhibitors of calcineurin, some of the studies above indicate that these drugs could possibly have calcineurin-independent elfects. Moreover, calcineurin shows phosphatase activity towards a wide variety of phosphoprotein substrates, and inhibition of calcineurin by immunosuppressant-immunophilin complexes blocks phosphatase activity toward a broad spectrum of phos-phoproteins. Inhibitors that blocked calcineurin mediated dephosphorylation of a specific substrate (such as NF-AT, or specific isoforms of NF-AT) without affecting the dephosphorylation of other substrates would be of great therapeutic as well as academic interest. The search for... 

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