Binding and inhibition of Cdc25 phosphatases by vitamin K analogues.
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.
Kar
Siddhartha
S
Department of Surgery, Liver Cancer Center, Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Lefterov
Iliya M
IM
Wang
Meifang
M
Lazo
John S
JS
Scott
Colleen N
CN
Wilcox
Craig S
CS
Carr
Brian I
BI
D000933:Antifibrinolytic Agents / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014812:Vitamin K / Q000031:analogs & derivatives* / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000933:Antifibrinolytic Agents; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D014812:Vitamin K; D001710:Biotin; D020928:Mitogen-Activated Protein Kinases; D020687:cdc25 Phosphatases