Histone H3 as a novel substrate for MAP kinase phosphatase-1.
Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.
Kinney
Corttrell M
CM
Dept. of Cell Biology, Lerner Research Institute and Cleveland Clinic Lerner College of Medicine of Case Western Reserve Univ., Cleveland Clinic, NB-21, 9500 Euclid Ave., Cleveland, OH 44195, USA.
Chandrasekharan
Unni M
UM
Yang
Lin
L
Shen
Jianzhong
J
Kinter
Michael
M
McDermott
Michael S
MS
DiCorleto
Paul E
PE
D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D054638:Dual Specificity Phosphatase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D044127:Epigenesis, Genetic; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D012694:Serine; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D006657:Histones; D010446:Peptide Fragments; D042461:Vascular Endothelial Growth Factor A; C082835:thrombin receptor peptide (42-47); D012694:Serine; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate