Phosphatases, phosphotyrosine

Madden et al. (1991) developed an assay that measures the activity of a phosphotyrosine phosphatase that is active with a chemically synthesized, dansylated phosphopeptide corresponding to the 1143-1153 region of the human placental insulin receptor fi subunit. 

The substrate, dansyl-Gly-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr(P)-Tyr-Arg-Lys, and its dephosphorylated product were separated at 30°C on a Spherisorb 3 /urn ODS-2 column (4.6 mm x 50 mm). The mobile phase consisted of 20% (w/v) acetonitrile-10 mM sodium phosphate (pH 7.2). The dansyl label was detected by fluorimetry. The limit of detection was 1 pmol/20 fiL. 

Phosphotyrosine phosphatase activity was measured at 30°C in a total volume of 100 /x.L containing 24 mM imidazole (pH 7.2), 1 mM EDTA, 1 mM dithiothreitol, 100 jug of bovine serum albumin, 50 fiM dansyl phosphopeptide, and extracts containing enzyme activity. After 15 minutes, the reaction was terminated by the addition of 20 /x.L of 30% (w/v) trichloroacetic acid. The mixture was centrifuged before injection for HPLC analysis. The reaction was linear with respect to both time and enzyme concentration up to at least 30% substrate conversion. 

The enzyme used was partially purified from human placental membranes. 

Calcineurin is a calcium/calmodulin-dependent protein phosphatase that may be involved in neurotransmission and T-cell proliferation. This assay uses nonlabeled peptides and is comparable in sensitivity to a radioactive assay. 

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Vanadate (sodium orthovanadate or peroxovanadate) exhibits insulin-like effects in vitro (activation of insulin receptor tyrosine kinase, PI 3-kinase, Akt) and in vivo (diabetic rats, humans). These effects can be explained at least in part by the inhibition of phosphotyrosine phosphatases which deactivate the INSR tyrosine kinase. 

Intracellular and extracellular ROS activate tyrosine and serine-threonine kinases (i.e., the MAPK family members). Following TNF-a, TGF-f5 or EGF stimulation, intracellular ROS are generated which stimulate various signaling pathways [73], Tyrosine kinase receptors (e.g., EGF, PDGF and TGF-a) may be activated by ROS directly via protein sulfhydryl group modifications, or inhibition of phosphotyrosine phosphatases (PTPases) and subsequent receptor activation. The latter is possible as PTPases contain a redox-sensitive cysteine at their active site [78], and oxidation of protein sulfhydryl groups results in the inactivation of PTPases. 

Design concepts are now being applied more effectively to mineral supplements. For example, by controlling the redox potential of iron, toxic effects associated with excess Fe(II) during parental supplementation can be avoided. Peroxovanadate complexes can inhibit insulin-receptor-associated phosphotyrosine phosphatase and activate insulin receptor kinase, and both V(IV) and V(V) offer promise as potential insulin mimics. 

It is not clear whether V(V) or V(IV) (or both) is the active insulin-mimetic redox state of vanadium. In the body, endogenous reducing agents such as glutathione and ascorbic acid may inhibit the oxidation of V(IV). The mechanism of action of insulin mimetics is unclear. Insulin receptors are membrane-spanning tyrosine-specific protein kinases activated by insulin on the extracellular side to catalyze intracellular protein tyrosine phosphorylation. Vanadates can act as phosphate analogs, and there is evidence for potent inhibition of phosphotyrosine phosphatases (526). Peroxovanadate complexes, for example, can induce autophosphorylation at tyrosine residues and inhibit the insulin-receptor-associated phosphotyrosine phosphatase, and these in turn activate insulin-receptor kinase. 

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]. 

Receptor Serine/Threonine Receptor Protein Kinase = Receptor Protein Phosphotyrosine Phosphatase = Receptor Tyrosine Kinase = Signal Transducer and Activator of Transcription = Transforming Growth Factor P = Tetradecanoylphorbolacetate = Tetradecanoylphorbolacetate Response Element = Change in Permeability of PM to specific solutes = Change in Transmembrane Potential. 

Krejsa, C.M., S.G. Nadler, J.M. Esselstyn, T. Kavanagh, J.A. Ledbetter, and G.L. Schieven. 1997. Role of oxidative stress in the action of vanadium phosphotyrosine phosphatase inhibitors. J. Biol. Chem. 272 11541-11549. 

Winter, C.L., J.S. Lange, M.G. Davis, G.S. Gerwe, T.R. Downs, K.G. Peters, and B. Kasibhatla. 2005. A nonspecific phosphotyrosine phosphatase inhibitor, bisfmalto-lato)oxovanadium(IV), improves glucose tolerance and prevents diabetes in Zucker diabetic fatty rats. Exp. Biol. Med. 230 207-16. 

Kambayashi, Y., Bardhan, S., Takahashi, K., et al. 1993. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem 268 24543-24546. 

Davis CM, Sumrall KH, Vincent JB. 1996. A biologically active form of chromium may activate a membrane phosphotyrosine phosphatase (PTP). Biochemistry 35 12963-12969. 

Florio T, Pan MG, Newman B, Hershberger RE, Civelli O, Stork PJ (1992) Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity. J Biol Chem 267 24169-24172. 

The regulatory role of protein phosphotyrosine phosphatases received attention rather late, in the 1980, mainly thanks to the work of E. H. Fischer and his laboratory in Seatde. ... 

Phosphotyrosine phosphatases are integrated just like tyrosine Idnases into signalling pathways. They interact with receptors and have recognition motifs that direct diem to their targets. 5 Protein phosphotyrosine phosphatases downregulate tyrosine phosphorylation and play a role in cellular regulation as important as that of protein tyrosine kinases. 

Phosphotyrosine phosphatases are only one of several classes of phosphatases. It has been estimated that about a thousand phosphatases of different specificities exist. Here, we consider only cytosolic and receptor tyrosine phosphatases (Fig. 3.8) (phosphoserine/threonine phosphatases are discussed in Chapter 7). 

Activation of STATs is transient, like that of other transcriptional regulators. When cytokine signalling is terminated, the concentrations of active STATs in the nucleus decline, because STATs are either inactivated by dephosphorylation or removed by proteolysis, or both. Accordingly, the half-life of STAT 1 can be prolonged by preventing dephosphorylation, through inhibition of phosphotyrosine phosphatases. Finally, there are inhibitors of JAK/STAT signalling that are induced by STATs. 

Phosphotyrosine phosphatases dephosphorylate JAKs and silence receptor-JAK complexes. They also inactivate JAKs which have been released from the receptor complex and are out of control. But dephosphorylation of tyrosines may also have positive effects and enhance cytokine signalling. A related phosphotyrosine phosphatase, actually stimulates IL-6 signalling, suggesting that the phosphotyrosines in the IL-6 receptor-JAK complex, which are attacked by the phosphatase, are inhibitory. The phosphatase activi-... 

The biological relevance of negative control of cytokine signalling by dephosphorylation becomes apparent in the phenotype of the mouse mutant, motheaten , which lacks a functional phosphotyrosine phosphatase. This mouse suffers from numerous haematopoietic defects and uncontrolled cell proliferation. 

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