FGF2-activated ERK mitogen-activated protein kinase enhances Runx2 acetylation and stabilization.
Runx2 is a key transcription factor regulating osteoblast differentiation and skeletal morphogenesis, and FGF2 is one of the most important regulators of skeletal development. The importance of the ERK mitogen-activated protein (MAP) kinase pathway in cranial suture development was demonstrated by the findings that the inhibition of FGF/FGF receptor (FGFR) signaling by a MEK blocker prevents the premature suture closure caused by an Fgfr2 mutation in mice. We previously demonstrated that ERK activation does not affect Runx2 gene expression but that it stimulates Runx2 transcriptional activity. However, the molecular mechanism underlying Runx2 activation by FGF/FGFR or ERK was still unclear. In this study, we found that FGF2 treatment increased the protein level of exogenously overexpressed Runx2 and that this increase is reversed by ERK inhibitors. In contrast, overexpression of constitutively active MEK strongly increased the Runx2 protein level, which paralleled an increase in Runx2 acetylation. As Runx2 protein phosphorylation mediated by ERK directly correlates with Runx2 protein stabilization, acetylation, and ubiquitination, we undertook to identify the ERK-dependent phosphorylation sites in Runx2. Analysis of two C-terminal Runx2 deletion constructs showed that the middle third of the protein is responsible for ERK-induced stabilization and activation. An in silico analysis of highly conserved ERK targets indicated that there are three relevant serine residues in this domain. Site-directed mutagenesis implicated Ser-301 in for ERK-mediated Runx2 stabilization and acetylation. In conclusion, the FGF2-induced ERK MAP kinase strongly increased the Runx2 protein level through an increase in acetylation and a decrease in ubiquitination, and these processes require the phosphorylation of Runx2 Ser-301 residue.
Park
Ok-Jin
OJ
From the Department of Molecular Genetics, School of Dentistry and Dental Research Institute, BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, and.
Kim
Hyun-Jung
HJ
the Bone Research Institute BioRunx Co., Ltd. Seoul 110-749, Korea.
Woo
Kyung-Mi
KM
From the Department of Molecular Genetics, School of Dentistry and Dental Research Institute, BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, and.
Baek
Jeong-Hwa
JH
From the Department of Molecular Genetics, School of Dentistry and Dental Research Institute, BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, and.
Ryoo
Hyun-Mo
HM
From the Department of Molecular Genetics, School of Dentistry and Dental Research Institute, BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, and. Electronic address: hmryoo@snu.ac.kr.
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D047468:Immunoprecipitation; D009154:Mutation; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects
D002070:Butadienes; D050659:Core Binding Factor Alpha 1 Subunit; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009570:Nitriles; D011725:Pyridines; C113580:U 0126; D025801:Ubiquitin; D016222:Fibroblast Growth Factor 2; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases; C093642:SB 203580