Binding and Inhibition of Cdc25 Phosphatases by Vitamin K Analogues

Abstract

A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304-28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317-1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with Cdc25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-Cdc25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated cells. PD 49 also bound to protein phosphatases other than Cdc25. We found that the new analogue also inhibited Hep3B human hepatoma cell growth. This growth inhibition involved ERK1/2 phosphorylation and was inhibited by a MEK antagonist. The results demonstrate a direct interaction and binding between this growth-inhibiting K vitamin derivative with both purified as well as with cellular Cdc25A, Cdc25B, and Cdc25C.

PM-20, a novel inhibitor of Cdc25A, induces extracellular signal–regulated kinase 1/2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo

“…We previously found that PD 49, a biotin-tagged derivative of Cpd 5, interacted with the catalytic site of Cdc25Bcat (20). We explored whether PD 49 and PM-20 might bind to the same catalytic domain in Cdc25Bcat.…”

“…They especially antagonize the action of extracellular signal -regulated kinase (ERK) phosphatases, resulting in prolonged ERK phosphorylation (14 -19). One of these compounds, Cpd 5, was also found to bind to Cdc25 at its catalytic cysteine (20). Cpd 5 was also found to block hepatoma cell growth both in vitro and in vivo (16, 21 -23).…”

Chemical Knockdown of Protein-tyrosine Phosphatase 1B by 1,2-Naphthoquinone through Covalent Modification Causes Persistent Transactivation of Epidermal Growth Factor Receptor

“…Of the multiple PTPs, PTP1B is ubiquitously expressed (20) and negatively regulates receptor tyrosine kinases such as EGFR (21), so that inhibition of this PTP would lead to EGFR activation. Although 1,2-and 1,4-NQ and their derivatives are reported to be potent inhibitors for PTP1B (22,23), their detailed mechanism of action still remains unclear. This report describes results of a study of 1,2-NQ-PTP1B interaction, which demonstrates that 1,2-NQ-mediated covalent modification of PTP1B, accompanied by reduction of its enzyme activity, results in the transactivation of EGFR in human epithelial A431 cells.…”

A sesquiterpene quinone, dysidine, from the sponge Dysidea villosa, activates the insulin pathway through inhibition of PTPases

“…Covalent addition [41,42] and irreversible oxidation [43] of cysteine in the active site are the main mechanisms. Since p-benzoquinone was contained in the structure of the three sesquiterpene quinones, the inhibition of dysidine against PTP1B was measured in the presence of either DTT (5 mmol/L) or catalase (1000 U/mL) to investigate whether these quinone-dependent mechanisms were involved.…”

“…For example, a number of ortho-quinone-containing compounds inhibited PTPα in a catalase-sensitive manner [43] , suggesting that these compounds inhibited PTPα via generation of ROS (including H 2 O 2 ) and subsequent oxidation of cysteine in the active site. Moreover, menadione (vitamin K3) [41] and other vitamin K analogues [42] containing a benzoquinone in their structure were reported to inhibit the CDC25 phosphatase by covalent interaction between the compound and the enzyme. This covalent bond can be blocked by DTT, GSH or other thiol-containing reductants.…”