Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription.
The coordinated activity of Runx2 and BMP/TGFbeta-activated Smads is critical for formation of the skeleton, but the precise structural basis for the Runx2/Smad interaction has not been resolved. By deletion mutagenesis, we have defined the Runx2 motif required for physical and functional interaction with either BMP or TGFbeta responsive Smads. Smad responsive transcriptional activity was retained upon deletion of the C-terminus to amino acid (aa) 432 but lost with deletion to aa 391. Thus the Smad interacting domain (SMID) of Runx2 (432-391) is embedded in the well-defined nuclear matrix targeting signal (NMTS) that mediates intranuclear trafficking. The SMID suffices as an interacting module when fused to the heterologous Gal-4 protein. Formation of the Runx2 and Smad complex is dependent on Runx2 phosphorylation through the MAPK signaling pathway, as determined by co-immunoprecipitation studies. We established that all SMID/NMTS deficient Runx2 mutants do not show in situ association with Smad in the nucleus nor do they support BMP2-mediated osteogenic induction of the mesenchymal C2C12 cell line. Thus, we provide direct evidence that the SMID/NMTS domain (391-432) of Runx2 is essential for BMP2-mediated osteoblast differentiation. Our findings suggest that TGFbeta/ BMP2 signaling, MAPK dependent phosphorylation, and Runx2 subnuclear targeting converge to induce the osteogenic phenotype.
Afzal
Faiza
F
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Pratap
Jitesh
J
Ito
Kosei
K
Ito
Yoshiaki
Y
Stein
Janet L
JL
van Wijnen
Andre J
AJ
Stein
Gary S
GS
Lian
Jane B
JB
Javed
Amjad
A
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D019070:Cell Lineage; D002467:Cell Nucleus / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D016296:Mutagenesis; D010011:Osteocytes / Q000166:cytology / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051902:Smad5 Protein; D015534:Trans-Activators / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; C493709:RUNX2 protein, human; C493716:Runx2 protein, mouse; C497797:SMAD5 protein, human; D051902:Smad5 Protein; C497798:Smad5 protein, mouse; D015534:Trans-Activators; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D016212:Transforming Growth Factor beta