Screening for tyrosine phosphatase inhibitors.


Reversible phosphorylation on tyrosine residues of proteins is one of the earliest and important events in the signal transduction pathways leading to the stimulation of cell proliferation. Because many oncogenes encode protein tyrosine kinases (PTKs), much attention had been paid to PTK compared with protein tyrosine phosphatases (PTPases). Recently, it was reported that PTPase played an important role in the signal transduction. Mitosis is initiated following the activation of cdc2-cyclin B kinase complex, so-called M-phase promoting factor or maturation promoting factor (MPF). The key step in the activation of MPF is dephosphorylation on Thr-14 and Tyr-15 of cdc2 protein by cdc25, a kind of dual-specificity protein phosphatases. VHR (VHl-related human protein), which was isolated from a human fibroblast has the dual-specificity phosphatase activity towards phosphotyrosine and phosphoserine/threonine like cdc25. The receptor-type PTPase, CD45 (a leukocyte common antigen) activates protein tyrosine kinase p56 Ick and p59 fyn , which are associated with the intracellular domain of the T cell surface glycoproteins, CD4 and CD8. CD45 is involved in T cell and B cell development and activation. Specific PTPase inhibitors should be useful as a biological tool to reveal the signal transduction. Okadaic acid and tautomycin, which are known to inhibit serine/threonine protein phosphatases (PPases), are valuable tools to test the physiological role of serine/threonine phosphorylation in the signal transduction. On the contrary, only a few PTPase inhibitors, for example, sodium orthovanadate (vanadate), phenylarsine oxide (PAO) and dephostatin have been known. A more potent and selective PTPase inhibitor is still required. To discover specific PTPase inhibitors, we have started the screening of microbial metabolites.
GST-VHR fusion protein (VHR) and GST-cdc25B fusion protein (cdc25B) was expressed in bacteria. Dephosphorylation activity of VHR and cdc25B to p-nitrophenylphosphate (pNpp) was measured in the assay buffer (25 mM MOPS, pH 6.5, 5 mM EDTA, 1 mM dithiothreitol).

The lineweaver-Burk plot of RK-682 (A) and vanadate (B) to pNPP.
The concentrations of RK-682 were 2.7 (●), 4.1 (○), 5.4 (■)uM. VHR (20 ug/ml) hydrolyzed pNPP (1, 2, 3, 4, 5 mM). Vanadate concentrations were 0 (●), 0.16 (○), 0.25 (■)mM. (Ref:2)


  1. T. Hamaguchi, A. Takahashi, A. Manaka, M. Sato, H. Osada
    TU-572, a potent and selective CD45 inhibitor, suppresses IgE-mediated anaphylaxis and murine contact hypersensitivity reactions.
    Int. Arch. Allergy Immunol., 126, 318-324 (2001).

  2. T. Hamaguchi, T. Sudo, H. Osada.
    RK-682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle progression at G1 phase.
    FEBS Lett., 372, 54-58 (1995).

Additional information

Close info