[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.


CONCLUSIONS
MKP-1 inhibits the in vitro interaction between HIF-1alpha and p300.
Liu
Chang-jiang
CJ
Department of Gastroenterology, General Hospital of Jinan Military Region, Jinan 250031, China.
Shi
Yong-quan
YQ
Du
Yu-lei
YL
Pan
Yang-lin
YL
Liang
Jie
J
Han
Shuang
S
Fan
Dai-ming
DM
D015687:Cell Hypoxia; D054638:Dual Specificity Phosphatase 1 / Q000494:pharmacology*; D050881:E1A-Associated p300 Protein / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015533:Transcriptional Activation
D005419:Flavonoids; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011993:Recombinant Fusion Proteins; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; D054638:Dual Specificity Phosphatase 1; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one