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RUNX2, a key transcription factor for osteoblast differentiation, is regulated by ERK1/2 and p38 MAP kinase-mediated phosphorylation. However, the specific contribution of each kinase to RUNX2-dependent transcription is not known. Here we investigate ERK and p38 regulation of RUNX2 using a unique P-RUNX2-specific antibody. Both MAP kinases stimulated RUNX2 Ser319 phosphorylation and transcriptional activity. However, a clear preference for ERK1 versus p38α/β was found when the ability of these MAPKs to phosphorylate and activate RUNX2 was compared. Similarly, ERK1 preferentially bound to a consensus MAPK binding site on RUNX2 that was essential for the activity of either kinase. To assess the relative contribution of ERK1/2 and p38 to osteoblast gene expression, MC3T3-E1 preosteoblast cells were grown in control or ascorbic acid (AA)-containing medium ± BMP2/7. AA-induced gene expression, which requires collagen matrix synthesis, was associated with parallel increases in P-ERK and RUNX2-S319-P in the absence of any changes in P-p38. This response was blocked by ERK, but not p38, inhibition. Significantly, in the presence of AA, BMP2/7 synergistically stimulated RUNX2 S319 phosphorylation and transcriptional activity without affecting total RUNX2 and this response was totally dependent on ERK/MAPK activity. In contrast, although p38 inhibition partially blocked BMP-dependent transcription, it did not affect RUNX2 S319 phosphorylation, suggesting the involvement of other phosphorylation sites and/or transcription factors in this response. Based on this work, we conclude that extracellular matrix and BMP regulation of RUNX2 phosphorylation and transcriptional activity in osteoblasts is predominantly mediated by ERK rather than p38 MAPKs., (© 2012 American Society for Bone and Mineral Research)