BMP signaling proteins play an essential role in bone development and postnatal bone formation. In cells of osteoblast linage including mesenchymal stem cells (MSCs), osteoblast precursors, and perhaps mature osteoblasts, BMP binds to BMP receptor leading to Smad1 phosphorylation. Phosphorylated Smad1 then binds Smad4 and the Smad1–4 complex translocates to the nucleus to modulate transcription. Smad1 interacts with Runx2 on the promoter of target genes and coordinately controls osteoblast gene expression and differentiation [Jonason et al., 2009
]. The expression of Smad1 and Runx2 is tightly regulated at mRNA and protein levels. Over the last decade, ubiquitination and proteasomal degradation was revealed as an important regulatory mechanism to control Smad and Runx2 protein levels in osteoblasts.
Zhu et al. 
, using Xenopus Smad1 as bait, identified Smurf1 as an E3 ligase interacting with Smad1 and subsequently inducing Smad1 ubiquitination and proteasome degradation. Investigation of Drosophila
Smurf (DSmurf) demonstrated that mutations of DSmurf result in down-regulation of signals from DPP, the ortholog of BMP2/4, indicating that Smurf1 is likely to regulate BMP signaling pathways in humans [Podos et al., 2001
]. In 2003, our group first decribed the function of Smurf1 in Runx2 degradtion and osteoblast function. We demonstrated that overexpression of Smurf1 induces proteasomal degradation of Smad1 and Runx2 proteins in 2T3 osteoblast precursor cells and in C2C12 myoblast/osteoblast precursor cells. Through its WW domain, Smurf1 specifically recognizes the PY motif of Smad1 and Runx2, resulting in their poly-ubiquitination and degradation through 26S proteasome. Smurf1-induced Smad1 and Runx2 degradation is prevented by treating cells with proteasome inhibitor. Thus, Smurf1 targets ubiquitination of Smad1 and Runx2, and is the first E3 ligase identified in the BMP signaling pathway as a negative regulator of bone cell function [Zhao et al., 2003
]. Smurf1 also induces the degradation of Smad5, facilitating myogenic differentiation of C2C12 cells at the expense of reduced BMP-induced osteogenic differentiation [Ying et al., 2003
Smad1 consists of three distinct domains: two highly conserved N- and C-terminal domains, referred to as mad homology 1 (MH1) and MH2, respectively, and a more divergent intervening linker region. In the inactive state, MH1 and MH2 bind to one another, mutually inhibiting the function of each domain. Binding of BMP receptor with BMP triggers C-terminal phosphorylation of MH2 domain of Smad1, which opens up this structure to allow association with Smad4 or with other DNA-binding proteins via the MH2 domain [Tsukazaki et al., 1998
; Whitman, 1998
] and activates the downstream target genes. In contrast, mitogen-activated protein kinases (MAPKs) catalyze phosphorylation in the linker region leading to an inhibition of Smad1 translocation into the nucleus [Kretzschmar et al., 1997
]. Although MAPK-induced inhibition of BMP signal has been known for more than 10 years, the molecular mechanisms involved remain unknown. A recent study demonstrated that MAPK-induced phosphorylation of the linker region restricts Smad1 activity by enabling Smad1 recognition by the Smurf1 leading to Smad1 ubiquitination and degradation. In addition, Smurf1 binding also blocks the interaction of Smad1 with the nuclear translocation factor Nup214. Thus, MAPK-dependent Smurf1 binding has two negative effects on Smad1 activity: proteasomal degradation and cytoplasmic retention [Sapkota et al., 2007
]. Interestingly, the phosphorylation of the linker region of Smad1 is triggered also by BMP, which is considered a feedback control mechanism. In a similar fashion, TGF-β induces C-terminal phosphorylation of Smads 2 and 3 proteins to activate the TGF-β signaling pathway. TGF-β also promotes the phosphorylation of the linker regions of Smads 2 and 3. However, instead of Smurfs, another member of C2-WW-HECT subfamily E3 ligase, Nedd4-2 is responsible for linker region phosphorylation and Smad2/3 poly-ubiquitination and degradation [Gao et al., 2009
]. Nedd4-2 was previously identified as a regulator of renal sodium channels. Nedd4-2−/−
mice are born normally and survived into adulthood. The animals have hypertension and cardiac hypertrophy. The bone phenotype has not been reported in these mice [Shi et al., 2008
]. These new findings indicate that location of phosphorylation of Smad protein determines the fate of Smad proteins, which may explain how cells respond to various stimuli under different conditions.
To determine whether Smurf1 induces Runx2 degradation through the interaction with the PY motif of Runx2, we created a mutant Runx2 with a PY motif deletion and found that Smurf1 retained some of its ability to induce the degradation of the mutant Runx2, suggesting that Smurf1 could also induce Runx2 degradation through an indirect mechanism. Smurf1 has been shown to interact with Smads 1, 5, 6, and 7 [Moren et al., 2005
] and Smads 1 and 5 also interact with Runx2. We found that Smads 1 and 5 had no effect on Smurf1-induced Runx2 degradation. Smad6 but not Smad7, binds Runx2, enhancing Smurf1-induced Runx2 degradation. These results demonstrate that in addition to its interaction with the PY motif of Runx2, Smurf1 induces Runx2 degradation in a Smad6-dependent manner [Shen et al., 2006b
]. Smad6 does not increase Smurf1-induced JunB degradation and this is probably because that Smad6 does not interact with JunB [Zhao et al., 2010
]. Smad6 gene transcription is up-regulated by BMP-2 in osteoblasts through Smad1 and Runx2 binding to the OSE2 sequences in the Smad6 promoter. Chromatin immunoprecipitation demonstrated that Smurf1 binds the OSE2 site through Runx2 and inhibits Smad6 gene transcription. Treatment with BMP-2 and transfection of Smad1 abolish Smurf1 binding to the OSE2 site, suggesting that Smad1 binding excludes Smurf1 interaction with the OSE2 site and promotes Smad6 gene transcription [Wang et al., 2007
]. Although the ubiquitin-proteasome system functions in the cytoplasm and in nuclear compartments [von Mikecz, 2006
], whether Smurf1 affects Runx2 protein stability in the nucleus is currently unknown.
Runx2 is a member of the Runt domain transcription factor family, which is comprises Runx-1, -2, and -3. The PY motif is conserved among all three Runx proteins, which are targeted by Smurf1 for ubiquitination and degradation in vitro [Jin et al., 2004
; Shen et al., 2006b]. Genetic analyses of animals and humans revealed the involvement of Runx1 in hematopoiesis and leukemia, and Runx3 in the development of T-cells and dorsal root ganglion neurons and in the genesis of gastric cancer. There is no information on the involvement of Smurf1 in these systems and the biologic significance of Smurf1-induced Runx-1 and -3 degradation needs to be determined. Our recent results demonstrate that cyclin D1 induces both Runx-2 and -3 degradation through ubiquitination. Parathyroid hormone-related protein (PTHrP) might prevent premature hypertrophy in chondrocytes partially through inducing the degradation of Runx-2 and -3 in a cyclin D1-dependent manner [Zhang et al., 2009
]. However, which of the E3 ligases are involved in this process remains unknown.