[Inhibition of the in vitro interaction between hypoxia inducible factor 1alpha and p300 by mitogen-activated protein kinase phosphatase 1].

Objective: To investigate the effects of mitogen-activated protein kinase phosphatase-1 (MKP-1) on the interaction between hypoxia-inducible factor (HIF)-1alpha and coactivator p300.

Methods: Prokaryotic expression vector PGEX-4T1 was constructed based on DNA recombination technology. GST fusion proteins were purified by glutathione-agarose beads and transfected into Escherichia coli BL-21. Effects of MKP-1 or PD98059 on in vitro interaction between HIF-1alpha and p300 was determined by Pull-down assay coupled with Western bloting. Effects of MKP-1 on the p300 expression level was detected using Western bloting.

Results: (1) GST fusion proteins were expressed in BL-21 Escherichia coli and purified proteins were obtained. (2) Less amount of p300 was pulled down by GST fusion protein from SGC7901-MKP-1 cells than from control cells after 12 hours of exposure to hypoxia. (3) 12 hours after hypoxia, Less amount of p300 was pulled down by GST fusion protein from PD98059 treated cells than from DMSO treated cells or SGC7901 cells. (4) There were no significant differences in p300 expression levels between recombinant MKP-1 transfected cells and control cells after 12 hours of exposure to hypoxia.

Conclusion: MKP-1 inhibits the in vitro interaction between HIF-1alpha and p300.