Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor.
The Runx2 transcription factor is required for commitment of mesenchymal cells to bone lineages and is a major regulator of osteoblast-specific gene expression. Runx2 is subject to a number of post-transcriptional controls including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453-4459). In this study, we used a combination of in vitro and in vivo phosphorylation analysis, mass spectroscopy, and functional assays to identify two sites at Ser(301) and Ser(319) within the proline/serine/threonine domain of Runx2 that are required for this regulation. These sites are phosphorylated by activated ERK1 in vitro and in cell culture. In addition to confirming ERK-dependent phosphorylation at Ser(319), mass spectroscopy identified two other ERK-phosphorylated sites at Ser(43) and Ser(510). Furthermore, introduction of S301A,S319A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression and differentiation after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301E,S319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.
Ge
Chunxi
C
Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109-1078, USA.
Xiao
Guozhi
G
Jiang
Di
D
Yang
Qian
Q
Hatch
Nan E
NE
Roca
Hernan
H
Franceschi
Renny T
RT
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008809:Mice, Inbred C3H; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry
D050659:Core Binding Factor Alpha 1 Subunit; D018076:DNA, Complementary; C493716:Runx2 protein, mouse; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases