Identification and Functional Characterization of ERK/MAPK Phosphorylation Sites in the Runx2 Transcription Factor

• Published in: The Journal of biological chemistry Vol. 284; no. 47; pp. 32533 - 32543
• Main Authors: Ge, Chunxi, Xiao, Guozhi, Jiang, Di, Yang, Qian, Hatch, Nan E., Roca, Hernan, Franceschi, Renny T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.11.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Binding Sites; Cell Line; Chlorocebus aethiops; Core Binding Factor Alpha 1 Subunit - metabolism; COS Cells; DNA, Complementary - metabolism; Extracellular Signal-Regulated MAP Kinases - metabolism; Humans; MAP Kinase Signaling System; Mass Spectrometry - methods; Mice; Mice, Inbred C3H; Phosphorylation; Serine - chemistry; Transcription, Chromatin, and Epigenetics
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M109.040980

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 Ser301 and Ser319 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 Ser319, mass spectroscopy identified two other ERK-phosphorylated sites at Ser43 and Ser510. 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.