A high dose of a novel GATA factor, human RUNX2B, in an active conformation: implications for CBFβ dependence of RUNX2 transactivation. RUNX2 is a master transcription factor for osteoblast differentiation, required for endochondral ossification and bone formation. While a specific CBFβ-SMAD interaction is required for RUNX2 transcriptional activity in vitro, an intact C-terminal RHD region is required for a synergistic effect with a CBFβ-SMAD complex in vivo. In order to gain insights into the function of RUNX2, we developed an antibody that specifically recognizes the highly active and phosphorylated C-terminal epitope-tagged RUNX2(0KQ). We also purified, from serum-starved HEK293 cells, novel isoforms of human RUNX2, whose sequences contained a single mutation resulting in an increase in phosphorylation. Unlike RUNX2(0KQ), the RUNX2 fragments were inactive as transcriptional factors but were still phosphorylated and the mutated N-termini were in a highly active conformation. This is the first report on a highly phosphorylated RUNX2 form and suggests that the level of CBFβ-dependent transcriptional activity of RUNX2 may be controlled by the phosphorylation of N-terminal serines and that the inactive conformation is retained by many, but not all, RUNX2 isoforms.