Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation

Gene 366 (2006) 58 – 66 www.elsevier.com/locate/gene

Review

Phosphorylation, acetylation and ubiquitination: The molecular basis of RUNX regulation Suk-Chul Bae ⁎, Yong Hee Lee Department of Biochemistry, College of Medicine and Institute for Tumor Research, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea Received 9 September 2005; received in revised form 23 September 2005; accepted 15 October 2005 Available online 1 December 2005 Received by A.J. van Wijnen

Abstract The RUNX family members play pivotal roles in normal development and neoplasia. RUNX1 and RUNX2 are essential for hematopoiesis and osteogenesis, respectively, while RUNX3 is involved in neurogenesis, thymopoiesis and functions as a tumor suppressor. Inappropriate levels of RUNX activity are associated with leukemia, autoimmune disease, cleidocranial dysplasia, craniosynostosis and various solid tumors. Therefore, RUNX activity must be tightly regulated to prevent tumorigenesis and maintain normal cell differentiation. Recent work indicates that RUNX activity is controlled by various extracellular signaling pathways, and that phosphorylation, acetylation and ubiquitination are important posttranslational modifications of RUNX that affect its stability and activity. Defining the precise roles, these modifications that play in the regulation of RUNX function may reveal not only how the RUNX proteins are regulated but also how they are assembled into other regulatory machineries. © 2005 Elsevier B.V. All rights reserved. Keywords: RUNX; Phosphorylation; Acetylation; Ubiquitination

1. Introduction The RUNX family of transcription factors plays pivotal roles in normal development and neoplasia. In mammals, the RUNX family genes consist of RUNX1/AML1, RUNX2 and RUNX3 (van Wijnen et al., 2004). RUNX1 is required for hematopoiesis and is genetically altered in leukemia. Indeed, it is the most frequent target of chromosomal translocations associated with human leukemia (Look, 1997; Speck and

Abbreviations: AML, acute myeloid leukemia; BMP, bone morphogenetic protein; CBF-β, core binding factor-β; CBP, CREB-binding protein; Chk, checkpoint protein kinase; ERK, extracellular signal regulated protein kinase; FGF, fibroblast growth factor; HAT, histone acetyltransferase; HDAC, histone deacetylase; IGF-I, insulin-like growth factor I; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; PI3-K, phosphoinositide 3-kinase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12myristate 13-acetate; PST region, proline, serine and threonine-rich region; PTH, parathyroid hormone; SAPK, stress-activated protein kinase; SH2, Src homology 2; SMURF1, Smad ubiquitination regulatory factor 1; TGF-β, transforming growth factor-β ⁎ Corresponding author. Tel.: +82 43 261 2842; fax: +82 43 274 8705. E-mail address: [email protected] (S.-C. Bae). 0378-1119/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.10.017

Gilliland, 2002). Moreover, haploinsufficiency of RUNX1 due to heterozygous loss-of-function mutations is associated with familial platelet disorder with a predisposition to acute myeloid leukemia (FPD-AML) (Song et al., 1999). Sporadic heterozygous mutations of RUNX1 are also leukemogenic (Osato et al., 1999). RUNX1 is also associated with several autoimmune diseases, namely, systemic lupus erythematosus, rheumatoid arthritis and psoriasis (Prokunina et al., 2002; Tokuhiro et al., 2003). RUNX2 is essential for osteogenesis (Ducy et al., 1997) and Runx2-knockout mice display complete bone loss because of arrested osteoblast maturation (Komori et al., 1997; Otto et al., 1997). RUNX2 is also involved in the human disease cleidocranial dysplasia, an autosomal dominant bone disorder, as deletions, insertions or mutations, that inactivates one allele of the RUNX2 gene has been shown to cause this syndrome in humans (Lee et al., 1997; Mundlos et al., 1997). In addition, hypoplasia of the clavicle and the delayed development of membranous bones are observed in Runx2 (±) heterozygous mice (Otto et al., 1997), which are typical features of cleidocranial dysplasia. Runx2 regulates the expression of various bone marker genes during osteoblast differentiation

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

(Stein et al., 2004). These observations demonstrate that inactivation of a single allele of the RUNX2 gene causes the human cleidocranial dysplasia. RUNX3 is required for the development of CD8-lineage T cells (Taniuchi et al., 2002; Woolf et al., 2003) and TrkCdependent dorsal root ganglion neurons (Inoue et al., 2002, 2003; Levanon et al., 2002). It also functions as a tumor suppressor (Bae and Choi, 2004). Targeted deletion of Runx3 in mice was shown to induce hyperplasia of the gastric epithelium (Li et al., 2002). Moreover, lower levels of RUNX3 due to a combination of hemizygous deletion of the gene and hypermethylation of the RUNX3 promoter region have been shown to be associated with human gastric cancer (Li et al., 2002). Subsequent studies have revealed that inactivation of RUNX3 is associated not only with gastric cancer but also with various cancers of the lung, colon, pancreas, liver, prostate, bile duct, breast, larynx, esophagus, endometrium, uterine cervix and testicular yolk sac (Goel et al., 2004; Kang et al., 2004, 2005; Kato et al., 2003; Kim et al., 2004; Ku et al., 2004; Li et al., 2004; Mori et al., 2005; Nakase et al., 2005; Oshimo et al., 2004; Sakakura et al., 2005; Schulmann et al., 2005; Tamura, 2004; Wada et al., 2004; Xiao and Liu, 2004; Yanagawa et al., 2003). These observations together indicate that inappropriate levels of RUNX expression are associated with various human diseases. Consequently, appropriate levels of intracellular RUNX activity must be essential for normal development, which means tight regulatory mechanisms must exist. This review will focus on recent advances made in elucidating how RUNX activity is regulated by extracellular signaling pathways:

59

namely the stabilization and transcriptional activation of RUNXs. 2. Structure of RUNX proteins All RUNX family members share the central Runt domain, which is well conserved and recognizes a specific DNA sequence. In addition, all three mammalian RUNX genes have a highly similar genomic organization, which indicates its preservation throughout evolution. The expression of all three RUNX genes is initiated from two promoters, namely, the distal P1 promoter and the proximal P2 promoter. These promoters give rise to two major protein isoforms that have distinct short amino termini, namely, type II and type I. The type I isoform contains the pentapeptide “MRIPV” at its N-terminus. In place of the pentapeptide of type I, the N-terminal peptide of the type II isoform is somewhat longer (19–32 amino acids) and is initiated with the sequence “MASN /DS”. As a result, the type I and type II human RUNX1 isoforms are composed of 453 (Q01196-1) and 480 (Q01196-8) amino acids, respectively. Similarly, the two isoforms of human RUNX2 contain 507 (Q13950-2) and 521 (Q13950-1) amino acids, respectively, while the human RUNX3 isoforms consist of 415 (Q13761-1) and 429 (Q13761-2) amino acids, respectively. The detailed genomic structures of these two isoforms of the RUNX family members have been summarized by Levanon and Groner (2004), while the functional domains of RUNX1 have been summarized by Ito (1999). Since the type I isoform with the N-terminal pentapeptide MRIPV is expressed more widely than the type II isoform, we used its amino acid numbering throughout this review (Fig. 1).

Fig. 1. Structure of human RUNX proteins and their phosphorylation sites. Phosphorylation sites that have been reported previously are shown above each RUNX protein and are indicated by solid rectangles. Putative phosphorylation sites and the responsible protein kinases predicted by consensus phosphorylation site search analysis (Obenauer et al., 2003; http://scansite.mit.edu) are shown under each RUNX protein. The locations of conserved phosphorylation residues are indicated by dotted arrows, while the conserved regions in RUNX proteins are indicated by dotted lines. AD, transactivation domain of RUNX1 (Ito, 1999); AD3, transactivation domain of RUNX2 (Thirunavukkarasu et al., 1998); ID, inhibitory domain; Q, glutamine tract; A, alanine tract; CK1, casein kinase 1; PST region, proline, serine and threonine-rich region.

60

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

3. RUNX modification by the mitogen-activated protein kinase (MAPK) signaling pathway Cytokines and growth factors regulate the transcriptional activity of RUNX1 by controlling the mitogen-activated protein kinase (MAPK)-mediated phosphorylation of RUNX1. The RUNX1 protein contains 14 serine or threonine residues followed by proline, which is the minimal consensus sequence for MAPK phosphorylation. Three of these sites are located at the amino terminus of the protein, while the remaining 11 sites are distributed in the carboxyl-terminal transactivation domain denoted as the PST region; this region is rich in proline, serine and threonine residues. Hirai and coworkers showed that EGF and IL-3 stimulate the transcriptional activity of RUNX1 by the ERK-mediated phosphorylation of Ser-249 and-266 in the PST region (Tanaka et al., 1996). Later, another group reported that the PMA-induced phosphorylation of four closely spaced serine and threonine PST residues (Ser-249, -266, -276 and Thr-273) by ERK plays a key role in the regulation of RUNX1 activity (Zhang et al., 2004). Mutation of these four sites of RUNX1 to alanine reduced its transcriptional activity, while in contrast their mutation to aspartic acid enhanced its transcriptional activity. These ERK-dependent phosphorylation sites fall in the transactivation domain of RUNX1, more precisely, in the TE3 region (aa 243–291), which by itself shows only weak activity (Ito, 1999; Kanno et al., 1998a). These observations suggest that ERK-dependent phosphorylation is required for the transactivation activity of the RUNX1 protein (Fig. 1). The molecular mechanism by which the transactivation activity of RUNX1 is stimulated by phosphorylation has been elucidated (Imai et al., 2004). The ERK-dependent phosphorylation of Ser-249 and -266 disrupts the interaction of RUNX1 with mSin3A, which is a transcriptional corepressor. The release from mSin3A then potentiates the transactivation and transforming abilities of RUNX1. The mSin3A-interaction domain of RUNX1 has been mapped to the region just Cterminal to the Runt domain since deletion of this region (aa 181–210) impaired the association between the two proteins (Lutterbach et al., 2000). On the other hand, deletion of aa 248– 287, in which the ERK phosphorylation sites reside (Imai et al., 2004), increases the interaction, which is consistent with the observation by Imai et al. (Lutterbach et al., 2000). It remains unknown, however, how the phosphorylation of the activation domain, which is located outside the mSin3A-interacting region, disrupts the interaction of RUNX1 with mSin3A. RUNX1 is degraded by the ubiquitin–proteasome pathway (Huang et al., 2001). The ERK-dependent phosphorylation of the RUNX1 protein appears to destabilize it as EGF treatment markedly diminishes the phosphorylated RUNX1 levels, while the levels of the unphosphorylated form remain unchanged (Imai et al., 2004). These results, together with the observation that ERK-dependent RUNX1 phosphorylation disrupts its interaction with mSin3A, suggest that mSin3A may inhibit the proteasome-mediated degradation of RUNX1 and that its ERK-dependent phosphorylation promotes its degradation by releasing mSin3A. Thus, the ERK-dependent phosphorylation of RUNX1 results in apparently opposite effects, namely, it

increases the transactivation activity of RUNX1 on the one hand and decreases the stability of RUNX1 on the other. This suggests that the increase in transactivation activity of RUNX1 is designed to be transient, although we do not know why, and that negative feed back regulation is required for this purpose. With regard to the other RUNX family members, while all three RUNX proteins interact with mSin3A, RUNX3 does not bear a region containing ERK phosphorylation sites, unlike RUNX1 and RUNX2 (Fig. 1). Thus, ERK-dependent phosphorylation of the RUNX3 protein and its regulatory mechanisms may differ from those of its paralogs. Insulin-like growth factor-1 (IGF-1) is a potent growth factor for many cells and an angiogenic factor that can stimulate endothelial cell proliferation and differentiation through the PI3-K and Akt pathway (Brooks et al., 1997). IGF-1 treatment also leads to MAPK kinase and ERK1/2 activation (Lopaczynski, 1999; Shelton et al., 2004). Increased expression and activation of RUNX2 may be involved in the IGF-1-induced angiogenic differentiation of endothelial cells (Sun et al., 2001). It was also reported that IGF-1 stimulates the DNA-binding activity of RUNX2 in endothelial cells via PI3-K/Pak1/ERKdependent phosphorylation (Qiao et al., 2004). IGF-1 was also shown to stimulate RUNX2 activity during osteoblast and chondrocyte differentiation through the PI3-K pathway (Fujita et al., 2004). Although Akt was suggested to mediate the IGF signaling in osteoblasts (Fujita et al., 2004), long-term inhibition of the MAPK pathway also inhibited IGF-1-induced RUNX2 activation, which suggests that ERK-mediated RUNX2 phosphorylation may be involved in the process (Fujita et al., 2004). Further analysis of the sites in the RUNX2 protein that are phosphorylated during the IGF-1-stimulated differentiation of endothelial cells and osteoblasts will be interesting. 4. RUNX modification by the fibroblast growth factor (FGF) signaling pathway The importance of FGF signaling in skeletal development was revealed when the etiology of craniosynostosis syndromes was discovered (Jabs et al., 1994; Meyers et al., 1995; Muenke et al., 1994; Reardon et al., 1994): these syndromes were found to be associated with mutations in the genes that encode FGF receptors (FGFR) 1, 2 and 3. Craniosynostosis is primarily characterized by the premature fusion of the cranial sutures. In mice, gene replacement of FGFR1 with a constitutively active mutant results in increased Runx2 expression and the premature suture closure that is the hallmark of human craniosynostosis (Zhou et al., 2000). These observations suggest that the aberrant activation of FGF signaling results in the premature differentiation of osteoblasts in the suture space and that Runx2 is a downstream target of FGF/FGFR signaling during bone formation. FGF-2 is a major regulator of intramembranous and endochondral bone formation. In osteoblasts, FGF activates multiple signaling pathways including the ERK and PKC pathways. FGF2 was shown to induce the expression and activation of RUNX2 in osteoblast-like cells and PKCδ plays a

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

role in this FGF-mediated induction of RUNX2 transcription (Kim et al., 2003). Moreover, FGF stimulates the transcriptional activity of RUNX2 and the ERK-dependent transcription of the osteocalcin, which is known to be a target gene of RUNX2 in osteoblast-like cell lines (Xiao et al., 2000). It was also shown that overexpression of MEK1, an upstream kinase of ERK, results in the phosphorylation of endogenous RUNX2 protein and that the purified RUNX2 protein could be phosphorylated by ERK in vitro (Xiao et al., 2000). Moreover, the phosphorylation of RUNX2 was stimulated by FGF2 and suppressed by inhibition of the ERK pathway (Xiao et al., 2002). Since the C-terminal 270 amino acids comprising the PST region of RUNX2 are necessary for its responsiveness to MEK and the ability of FGF-2 to stimulate osteocalcin transcription, the FGF2-mediated phosphorylation sites appear to reside in the PST region of RUNX2 protein. Three ERK phosphorylation sites in the PST region of RUNX1 (Ser-249, -266 and Thr-273) are conserved in RUNX2 (Ser-280, -298 and Thr-305). Further investigation of whether these residues are the FGF-stimulated ERK phosphorylation sites would be interesting (Fig. 1). 5. RUNX modification by the parathyroid hormone (PTH) signaling pathway PTH has been demonstrated to be an anabolic factor in skeletal tissue (Dempster et al., 1993) and in vitro (Partridge et al., 1981). The anabolic effects of PTH have suggested its potential efficacy in the treatment of osteoporosis (Lindsay et al., 1997). It is now clear that prolonged exposure to PTH leads to increased bone resorption while intermittent administration of PTH stimulates bone formation (Chase and Aurbach, 1970; Dobnig and Turner, 1995, 1997; Schiller et al., 1999). PTH was demonstrated to activate the MAPK pathway in osteoblastic cells (Swarthout et al., 2001). The stimulation of MAPK by PTH (at low concentrations) in osteoblast cells is PKCdependent (Swarthout et al., 2001). This activating event is associated with an increase in osteoblast proliferation (Cole, 1999; Swarthout et al., 2001). Protein kinase A (PKA) has also been linked to many of the changes in gene expression that are induced by PTH. PTH activates RUNX2 through PKA since the PTH-dependent induction of collagenase-3 (matrix metalloproteinase-13), which is known to be a target of RUNX2 (Jimenez et al., 1999) and the PTH signaling pathway (Quinn et al., 1990), was abrogated by PKA inhibition. Moreover, a specific PKA consensus site (threonine 341 in the human sequence corresponds to serine 347 in the mouse) within the transactivation domain is phosphorylated by PKA in vitro and this phosphorylation event was shown to be responsible for the PTH-stimulation of RUNX2 transactivationl activity (Selvamurugan et al., 2000). Thus, PTH may regulate RUNX2 activity through PKA (Fig. 1) (although it is not clear yet whether the threonine 341 residue is the major PKAmediated phosphorylation site of RUNX2 in vivo since only a partial fragment of RUNX2 (aa 235–368) has been examined in vitro).

61

6. RUNX modification and the transforming growth factorβ (TGF-β) superfamily signaling pathway Transforming growth factor-β (TGF-β) is a potent multifunctional regulator of cell growth and differentiation. TGF-β1, the prototypic member of the TGF-β super-family, elicits diverse cellular responses and osteogenesis (Massague, 1990). It also stimulates the synthesis of bone matrix proteins and inhibits matrix degradation. Members of the TGF-β superfamily that have important effects on bone cell differentiation are the bone morphogenetic proteins (BMPs) (Hogan, 1996; Reddi, 1994). BMPs were first identified as factors that induce bone formation in vivo when implanted into muscular tissues (Wozney et al., 1988). While TGF-β induces new bone formation when injected in close proximity to bone, BMPs produce bone formation even when injected into ectopic sites. TGF-β and BMPs also bind to distinct receptors: TGF-β recognizes TGF-β type I and II receptors, while the BMPs bind BMP type I and II receptors. Following ligand binding, the receptor-associated kinase is activated and phosphorylates Smads, which then move into the nucleus to stimulate the transcription of a set of target genes (Heldin et al., 1997; Massague, 1998; Zhang and Derynck, 1999). Biochemical analysis has demonstrated that RUNX family transcription factors function not only as a part of TGF-β superfamily signaling pathway but are also regulated by components of the TGF-β pathway. TGF-β/BMP-activated Smads interact with various transcription factors including RUNX and stimulate the transcription of a set of target genes (Hanai et al., 1999; Ito, 2004; Miyazono et al., 2004). BMP induces both the expression of RUNX2 and its interaction with BMP-activated Smads, which facilitates the differentiation of mesenchymal cells into osteoblasts (Ito and Miyazono, 2003; Lee et al., 2000). Apart from activating SMAD proteins, signal transduction by the TGF-β superfamily involves the activation of MAPK cascades. TGF-β/BMP have been shown to activate p38, a member of the stress-activated protein kinases (SAPKs), through MAPK kinase (MKK) 6 or MKK3 (Gallea et al., 2001; Hanafusa et al., 1999). It has been reported that both the Smad and MAPK pathways are essential components of the TGF-β superfamily signaling during osteoblast differentiation (Derynck et al., 2001; Fujii et al., 1999; Gallea et al., 2001; Nishimura et al., 1998; Yamamoto et al., 1997) and for RUNX2 induction (Lee et al., 2002). The tumor suppressor activity of RUNX3 also appears to be associated with TGF-β signaling, as the gastric mucosa of RUNX3 knockout mice, which becomes hyperplastic due to the stimulation of proliferation and the suppression of apoptosis, shows decreased sensitivity to TGF-β (Li et al., 2002). Acetylation of the ε-amino group of lysine residues has recently emerged as an important covalent post-translational modification that regulates protein functions. The level of protein acetylation is controlled by a dynamic equilibrium that is governed by the opposing actions of acetyltransferases and deacetylases. Acetylation by transcriptional coactivator p300 is known to stimulate transcription either by modifying histones in chromatin to produce a transcriptionally active

62

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

conformation, or by directly targeting transcriptional activators (Yang, 2004). It has been shown that RUNX proteins are degraded by an ubiquitin ligase-mediated pathway (Huang et al., 2001) and RUNX2 and RUNX3 are ubiquitinated by Smurfs (Jin et al., 2004; Zhao et al., 2003). The Smurfs recognize the PPxY motif of RUNX through the WW domain and disruption of a single amino acid of the PPxY motif protects RUNX from Smurfmediated degradation (Jin et al., 2004). An important clue regarding the mechanism that regulates ubiquitin-mediated RUNX degradation was provided by the finding that p300 counters Smurf-mediated degradation of RUNX3. p300 stimulates RUNX1-dependent transcription (Kitabayashi et al., 1998), while histone deacetylase (HDAC)-6 represses RUNX2-dependent transcription (Westendorf et al., 2002). Moreover, Jin et al. (2004) demonstrated that RUNX3 activity correlates positively with the increase or decrease in RUNX3 acetylation that is mediated by p300 and HDACs, respectively. Since acetylation and ubiquitination both target lysine residues, increased RUNX3 acetylation appears to suppress its ubiquitination and degradation. Thus, acetylation of RUNX may compete with its ubiquitination. Interestingly, mutations of the lysine residues in RUNX3 significantly increased its stability but abolished its transactivation activity, which suggests that the acetylation of the lysine residues is required for RUNX activity as well as for its stability (Jin et al., 2004). In contrast, HDACs deacetylate RUNX3 and allow the protein to be degraded through the ubiquitin-mediated pathway. These links between p300, HDACs and Smurfs, which control RUNX3 stabilization and degradation, appear to be involved in regulating all three RUNX proteins. The PPxY motif is conserved in RUNX1, RUNX2 and RUNX3, and very recent findings have revealed that RUNX1 (Kitabayashi et al., 1998; Yamaguchi et al., 2004) as well as RUNX2 (our unpublished observation) is acetylated by p300. Notably, RUNX3 acetylation and its subsequent protection from ubiquitin-mediated degradation are stimulated by TGFβ. Although the molecular mechanism by which the TGF-β signaling pathway controls the p300-dependent acetylation of RUNX3 is not clear, Smads appear to play important role in this process since TGF-β/BMP treatment induces the interaction between p300 and Smad (Shen et al., 1998). Moreover, the exogenous expression of Smad increases p300-dependent RUNX3 acetylation (our unpublished observation). The proposed model showing how RUNX activity is regulate by acetylation, deacetylation and ubiquitination is depicted in Fig. 2. However, the acetylation and deacetylation of RUNXs cannot be the only mechanism by which p300 and HDACs regulate RUNX activity. For example, for osteocalcin gene expression, p300-mediated acetylation of RUNX2 appears to be dispensable. This is shown by the fact that a HAT-deficient p300 mutant protein up-regulates the osteocalcin promoter reporter gene expression, indicating that the effect of p300 on the osteocalcin promoter is independent of its intrinsic HAT activity (Sierra et al., 2003). It is likely that p300 and HDACs have other roles in regulating the activity of RUNX proteins.

Fig. 2. Proposed model for the acetylation, deacetylation and ubiquitination of RUNX. The cellular levels of RUNX amounts and activity are controlled by a dynamic equilibrium of RUNX acetylation and deacetylation. In normal situations that require only low levels of RUNX activity, the acetylation of RUNX is decreased by relatively high levels of deacetylase activity and RUNX is exposed to ubiquitin ligase. When a signal comes that aims to induce RUNX activity, for example TGF-β or BMP, RUNX acetylation is increased by relatively high levels of acetyl transferase activity and ubiquitination is inhibited. This activates RUNX and protects it from proteasome-mediated degradation.

Another mechanism that regulates RUNX activity is its phosphorylation upon BMP stimulation. Wee et al. (2002) showed that phosphorylation of the Ser-104 and-451 residues of RUNX2 negatively regulates the protein. Moreover, mutation of Ser-104 to glutamic acid reduced the activity and amount of RUNX2 and inhibited the interaction between RUNX2 and its heterodimerization partner CBF-β, which protects RUNX proteins from proteasome-mediated degradation (Huang et al., 2001). Ser-104 is located in a region that is important for the interaction between CBF-β and RUNX2 (Bravo et al., 2001) and, interestingly, mutation of serine 104 to arginine was discovered in a cleidocranial dysplasia patient (Quack et al., 1999). In addition, the increased phosphorylation of RUNX2 Ser-104 was detected in C2C12 cells that had been differentiated by BMP2. However, the kinase responsible for Ser-104 phosphorylation has not yet been identified (Fig. 1). Ser-451 resides in the transcriptional inhibitory region of RUNX2 (Kanno et al., 1998b; Zhang et al., 2000) and it was suggested that its phosphorylation may inhibit RUNX2 activity. However, mutating serine 451 to glutamic acid had no effect on RUNX2 transcriptional activity and no change in the phosphorylation of serine 451 was detected in C2C12 cells that had been differentiated by BMP2 (Wee et al., 2002). Therefore, it is not known what role the phosphorylation of Ser-451 plays. The same is true for the phosphorylation of Ser-14, which was also found to be a major phosphorylation site, although its mutation had no detectable effect on RUNX2 activity (Wee et al., 2002). 7. Perspectives As discussed above, the RUNX proteins play pivotal roles in normal development and neoplasia and are regulated by posttranslational modifications in response to various signaling pathways that play important roles in given tissues. Undoubtedly, new modifications of RUNX will be identified in the

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

future. Defining the precise effects these modifications have on RUNX function will further reveal how the signaling pathways exert their actions through RUNX proteins. To identify new post-translational modifications, analysis with a consensus sequence searching program (Obenauer et al., 2003; http:// scansite.mit.edu) could be helpful. For example, this program has predicted that Thr-14 in RUNX1 and RUNX3 (Ser-14 in RUNX2) may be phosphorylated by PKA and interestingly, the sequences flanking these residues are conserved in all 3 RUNX proteins. As discussed above, Ser-14 was reported as one of the major phosphorylation sites in RUNX2 (Wee et al., 2002). Since PKA is an important mediator of PTH signaling in osteoblastlike cells, this residue may be phosphorylated by the PTH-PKA pathway. Another example is the Thr-273 residue in RUNX1, which is predicted to be a p38 MAPK phosphorylation site. It was previously reported to be phosphorylated by ERK and to be required for PMA-stimulated RUNX1 activation (Zhang et al., 2004). It is possible that this residue is modified by either ERK or p38 MAPK depending on the extracellular signal involved (Fig. 1). The addition of an acetyl group to a lysine residue creates a new surface for protein association. As the SH2 domain can interact with phospho-Tyr residues, 14-3-3 proteins can interact with phosphor-Ser/Thr (Pawson and Saxton, 1999; Yaffe, 2002), while the bromodomain functions as a structural module specific for acetyl lysine-containing motifs. It has been shown that several chromatin regulators use bromodomains to recognize acetyl lysine (Dhalluin et al., 1999; Hassan et al., 2002; Hudson et al., 2000; Jacobson et al., 2000; Ladurner et al., 2003; Matangkasombut and Buratowski, 2003; Owen et al., 2000). Some proteins contain multiple bromodomains, which may cooperate with each other to increase the affinity for binding partners with multiple acetylated lysine residues (Jacobson et al., 2000). Acetylation is also known to stimulate the association with proteins that do not contain bromodomains (Jeong et al., 2002; Soutoglou et al., 2000). In accordance with this notion, mutations of RUNX3 acetylation sites not only block the ubiquitinationmediated degradation of RUNX3 but also abolish its transactivation activity (Jin et al., 2004). Therefore, an interesting question to be addressed is whether the posttranslational modification of RUNX3 creates a new surface for protein association and thereby facilitates the binding of the protein to other cell regulatory machineries. The regulation of RUNX by competitive acetylation and ubiquitination is reminiscent of p53. p53 is specifically acetylated at multiple lysine residues by CBP/p300 and PCAF (p300/CBP associated factor) (Appella and Anderson, 2001). This acetylation of p53 is critically important for the activation of p53 target genes in vivo (Barlev et al., 2001). These acetylation sites of p53 are also essential for the ubiquitination and subsequent degradation of p53 by Mdm2. In contrast, inhibiting cellular deacetylases (HDACs) prolongs the half-life of endogenous p53 (Ito et al., 2001). The basic mechanism by which acetylation/deacetylation by CBP/p300 and HDACs regulates p53 and RUNX3 protein stability and activity is thus very similar, although this regulatory mechanism is turned on

63

by different stimuli, namely, intracellular stress for p53 and extracellular signals for RUNX3. Thus, an intriguing possibility is that other mechanisms that regulate p53 may also regulate RUNX. For example, it has been shown that the phosphorylation of p53 (Ser-20) by checkpoint kinase 1 (Chk1) and Chk2 in response to ionizing radiation abrogates the interaction between p53 and Mdm2 (Chehab et al., 2000; Hirao et al., 2000; Shieh et al., 2000). It will be of great interest to assess whether any of the RUNX3 phosphorylation events can modulate its ubiquitination and acetylation.

Acknowledgments This work was supported by Creative Research grant R162003-002-01001-0 from the Korea Science and Engineering Foundation to S.-C. Bae. References Appella, E., Anderson, C.W., 2001. Post-translational modifications and activation of p53 by genotoxic stresses. Eur. J. Biochem. 268, 2764–2772. Bae, S.C., Choi, J.K., 2004. Tumor suppressor activity of RUNX3. Oncogene 23, 4336–4340. Barlev, N.A., et al., 2001. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol. Cell 8, 1243–1254. Bravo, J., Li, Z., Speck, N.A., Warren, A.J., 2001. The leukemia-associated AML1 (Runx1)-CBF beta complex functions as a DNA-induced molecular clamp. Nat. Struct. Biol. 8, 371–378. Brooks, P.C., Klemke, R.L., Schon, S., Lewis, J.M., Schwartz, M.A., Cheresh, D.A., 1997. Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo. J. Clin. Invest. 99, 1390–1398. Chase, L.R., Aurbach, G.D., 1970. The effect of parathyroid hormone on the concentration of adenosine 3′,5′-monophosphate in skeletal tissue in vitro. J. Biol. Chem. 245, 1520–1526. Chehab, N.H., Malikzay, A., Appel, M., Halazonetis, T.D., 2000. Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev. 14, 278–288. Cole, J.A., 1999. Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells. Endocrinology 140, 5771–5779. Dempster, D.W., Cosman, F., Parisien, M., Shen, V., Lindsay, R., 1993. Anabolic actions of parathyroid hormone on bone. Endocr. Rev. 14, 690–709. Derynck, R., Akhurst, R.J., Balmain, A., 2001. TGF-beta signaling in tumor suppression and cancer progression. Nat. Genet. 29, 117–129. Dhalluin, C., Carlson, J.E., Zeng, L., He, C., Aggarwal, A.K., Zhou, M.M., 1999. Structure and ligand of a histone acetyltransferase bromodomain. Nature 399, 491–496. Dobnig, H., Turner, R.T., 1995. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 136, 3632–3638. Dobnig, H., Turner, R.T., 1997. The effects of programmed administration of human parathyroid hormone fragment (1–34) on bone histomorphometry and serum chemistry in rats. Endocrinology 138, 4607–4612. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L., Karsenty, G., 1997. Osf2/ Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754. Fujii, M., et al., 1999. Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Mol. Biol. Cell 10, 3801–3813. Fujita, T., et al., 2004. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J. Cell Biol. 166, 85–95.

64

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

Gallea, S., et al., 2001. Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone 28, 491–498. Goel, A., et al., 2004. Epigenetic inactivation of RUNX3 in microsatellite unstable sporadic colon cancers. Int. J. Cancer 112, 754–759. Hanafusa, H., et al., 1999. Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression. J. Biol. Chem. 274, 27161–27167. Hanai, J., et al., 1999. Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter. J. Biol. Chem. 274, 31577–31582. Hassan, A.H., et al., 2002. Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes. Cell 111, 369–379. Heldin, C.H., Miyazono, K., ten Dijke, P., 1997. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465–471. Hirao, A., et al., 2000. DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287, 1824–1827. Hogan, B.L., 1996. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10, 1580–1594. Huang, G., Shigesada, K., Ito, K., Wee, H.J., Yokomizo, T., Ito, Y., 2001. Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitinproteasome-mediated degradation. EMBO J. 20, 723–733. Hudson, B.P., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E., 2000. Solution structure and acetyl-lysine binding activity of the GCN5 bromodomain. J. Mol. Biol. 304, 355–370. Imai, Y., et al., 2004. The corepressor mSin3A regulates phosphorylationinduced activation, intranuclear location, and stability of AML1. Mol. Cell. Biol. 24, 1033–1043. Inoue, K., et al., 2002. Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons. Nat. Neurosci. 5, 946–954. Inoue, K., et al., 2003. Runx3 is essential for the target-specific axon pathfinding of trkc-expressing dorsal root ganglion neurons. Blood Cells Mol. Diseases 30, 157–160. Ito, Y., 1999. Molecular basis of tissue-specific gene expression mediated by the runt domain transcription factor PEBP2/CBF. Genes Cells 4, 685–696. Ito, Y., 2004. Oncogenic potential of the RUNX gene family: ‘overview’. Oncogene 23, 4198–4208. Ito, Y., Miyazono, K., 2003. RUNX transcription factors as key targets of TGFbeta superfamily signaling. Curr. Opin. Genet. Dev. 13, 43–47. Ito, A., et al., 2001. p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO J. 20, 1331–1340. Jabs, E.W., et al., 1994. Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat. Genet. 8, 275–279. Jacobson, R.H., Ladurner, A.G., King, D.S., Tjian, R., 2000. Structure and function of a human TAFII250 double bromodomain module. Science 288, 1422–1425. Jeong, J.W., et al., 2002. Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation. Cell 111, 709–720. Jimenez, M.J., Balbin, M., Lopez, J.M., Alvarez, J., Komori, T., Lopez-Otin, C., 1999. Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation. Mol. Cell. Biol. 19, 4431–4442. Jin, Y.H., et al., 2004. Transforming growth factor-beta stimulates p300dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation. J. Biol. Chem. 279, 29409–29417. Kang, G.H., Lee, S., Lee, H.J., Hwang, K.S., 2004. Aberrant CpG island hypermethylation of multiple genes in prostate cancer and prostatic intraepithelial neoplasia. J. Pathol. 202, 233–240. Kang, S., et al., 2005. Polymorphism in folate-and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer. Gynecol. Oncol. 96, 173–180. Kanno, T., Kanno, Y., Chen, L.F., Ogawa, E., Kim, W.Y., Ito, Y., 1998a. Intrinsic transcriptional activation-inhibition domains of the polyomavirus enhancer binding protein 2/core binding factor alpha subunit revealed in the presence of the beta subunit. Mol. Cell. Biol. 18, 2444–2454. Kanno, Y., Kanno, T., Sakakura, C., Bae, S.C., Ito, Y., 1998b. Cytoplasmic sequestration of the polyomavirus enhancer binding protein 2 (PEBP2)/core binding factor alpha (CBFalpha) subunit by the leukemia-related PEBP2/

CBFbeta-SMMHC fusion protein inhibits PEBP2/CBF-mediated transactivation. Mol. Cell. Biol. 18, 4252–4261. Kato, N., Tamura, G., Fukase, M., Shibuya, H., Motoyama, T., 2003. Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants. Am. J. Pathol. 163, 387–391. Kim, H.J., Kim, J.H., Bae, S.C., Choi, J.Y., Kim, H.J., Ryoo, H.M., 2003. The protein kinase C pathway plays a central role in the fibroblast growth factorstimulated expression and transactivation activity of Runx2. J. Biol. Chem. 278, 319–326. Kim, T.Y., et al., 2004. Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab. Invest. 84, 479–484. Kitabayashi, I., Yokoyama, A., Shimizu, K., Ohki, M., 1998. Interaction and functional cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell differentiation. EMBO J. 17, 2994–3004. Komori, T., et al., 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755–764. Ku, J.L., et al., 2004. Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene 23, 6736–6742. Ladurner, A.G., Inouye, C., Jain, R., Tjian, R., 2003. Bromodomains mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries. Mol. Cell 11, 365–376. Lee, B., et al., 1997. Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nat. Genet. 16, 307–310. Lee, K.S., et al., 2000. Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol. Cell. Biol. 20, 8783–8792. Lee, K.S., Hong, S.H., Bae, S.C., 2002. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene 21, 7156–7163. Levanon, D., Groner, Y., 2004. Structure and regulated expression of mammalian RUNX genes. Oncogene 23, 4211–4219. Levanon, D., et al., 2002. The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons. EMBO J. 21, 3454–3463. Li, Q.L., et al., 2002. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 109, 113–124. Li, Q.L., et al., 2004. Transcriptional silencing of the RUNX3 gene by CpG hypermethylation is associated with lung cancer. Biochem. Biophys. Res. Commun. 314, 223–228. Lindsay, R., et al., 1997. Randomised controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet 350, 550–555. Look, A.T., 1997. Oncogenic transcription factors in the human acute leukemias. Science 278, 1059–1064. Lopaczynski, W., 1999. Differential regulation of signaling pathways for insulin and insulin-like growth factor I. Acta Biochim. Pol. 46, 51–60. Lutterbach, B., Westendorf, J.J., Linggi, B., Isaac, S., Seto, E., Hiebert, S.W., 2000. A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia. J. Biol. Chem. 275, 651–656. Massague, J., 1990. The transforming growth factor-beta family. Annu. Rev. Cell Biol. 6, 597–641. Massague, J., 1998. TGF-beta signal transduction. Annu. Rev. Biochem. 67, 753–791. Matangkasombut, O., Buratowski, S., 2003. Different sensitivities of bromodomain factors 1 and 2 to histone H4 acetylation. Mol. Cell 11, 353–363. Meyers, G.A., Orlow, S.J., Munro, I.R., Przylepa, K.A., Jabs, E.W., 1995. Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans. Nat. Genet. 11, 462–464. Miyazono, K., Maeda, S., Imamura, T., 2004. Coordinate regulation of cell growth and differentiation by TGF-beta superfamily and Runx proteins. Oncogene 23, 4232–4237.

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66 Mori, T., et al., 2005. Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma. Liver Int. 25, 380–388. Muenke, M., et al., 1994. A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat. Genet. 8, 269–274. Mundlos, S., et al., 1997. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89, 773–779. Nakase, Y., et al., 2005. Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography. Br. J. Cancer 92, 562–569. Nishimura, R., Kato, Y., Chen, D., Harris, S.E., Mundy, G.R., Yoneda, T., 1998. Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J. Biol. Chem. 273, 1872–1879. Obenauer, J.C., Cantley, L.C., Yaffe, M.B., 2003. Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31, 3635–3641. Osato, M., et al., 1999. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood 93, 1817–1824. Oshimo, Y., et al., 2004. Frequent loss of RUNX3 expression by promoter hypermethylation in gastric carcinoma. Pathobiology 71, 137–143. Otto, F., et al., 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89, 765–771. Owen, D.J., et al., 2000. The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p. EMBO J. 19, 6141–6149. Partridge, N.C., Alcorn, D., Michelangeli, V.P., Kemp, B.E., Ryan, G.B., Martin, T.J., 1981. Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells. Endocrinology 108, 213–219. Pawson, T., Saxton, T.M., 1999. Signaling networks—do all roads lead to the same genes? Cell 97, 675–678. Prokunina, L., et al., 2002. A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat. Genet. 32, 666–669. Qiao, M., Shapiro, P., Kumar, R., Passaniti, A., 2004. Insulin-like growth factor1 regulates endogenous RUNX2 activity in endothelial cells through a phosphatidylinositol 3-kinase/ERK-dependent and Akt-independent signaling pathway. J. Biol. Chem. 279, 42709–42718. Quack, I., et al., 1999. Mutation analysis of core binding factor A1 in patients with cleidocranial dysplasia. Am. J. Hum. Genet. 65, 1268–1278. Quinn, C.O., Scott, D.K., Brinckerhoff, C.E., Matrisian, L.M., Jeffrey, J.J., Partridge, N.C., 1990. Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells. J. Biol. Chem. 265, 22342–22347. Reardon, W., Winter, R.M., Rutland, P., Pulleyn, L.J., Jones, B.M., Malcolm, S., 1994. Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat. Genet. 8, 98–103. Reddi, A.H., 1994. Bone and cartilage differentiation. Curr. Opin. Genet. Dev. 4, 737–744. Sakakura, C., et al., 2005. Frequent downregulation of the runt domain transcription factors RUNX1, RUNX3 and their cofactor CBFB in gastric cancer. Int. J. Cancer 113, 221–228. Schiller, P.C., D'Ippolito, G., Roos, B.A., Howard, G.A., 1999. Anabolic or catabolic responses of MC3T3-E1 osteoblastic cells to parathyroid hormone depend on time and duration of treatment. J. Bone Miner. Res. 14, 1504–1512. Schulmann, K., et al., 2005. Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk. Oncogene 24, 4138–4148. Shelton, J.G., Steelman, L.S., White, E.R., McCubrey, J.A., 2004. Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells. Cell Cycle 3, 372–379. Shen, X., Hu, P.P., Liberati, N.T., Datto, M.B., Frederick, J.P., Wang, X.F., 1998. TGF-beta-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein. Mol. Biol. Cell 9, 3309–3319.

65

Shieh, S.Y., Ahn, J., Tamai, K., Taya, Y., Prives, C., 2000. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289–300. Sierra, J., et al., 2003. Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity. Mol. Cell. Biol. 23, 3339–3351. Song, W.J., et al., 1999. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat. Genet. 23, 166–175. Soutoglou, E., Katrakili, N., Talianidis, I., 2000. Acetylation regulates transcription factor activity at multiple levels. Mol. Cell 5, 745–751. Speck, N.A., Gilliland, D.G., 2002. Core-binding factors in haematopoiesis and leukaemia. Nat. Rev. Cancer 2, 502–513. Stein, G.S., et al., 2004. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 23, 4315–4329. Sun, L., Vitolo, M., Passaniti, A., 2001. Runt-related gene 2 in endothelial cells: inducible expression and specific regulation of cell migration and invasion. Cancer Res. 61, 4994–5001. Swarthout, J.T., Doggett, T.A., Lemker, J.L., Partridge, N.C., 2001. Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent. J. Biol. Chem. 276, 7586–7592. Tamura, G., 2004. Promoter methylation status of tumor suppressor and tumorrelated genes in neoplastic and non-neoplastic gastric epithelia. Histol. Histopathol. 19, 221–228. Tanaka, T., et al., 1996. The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability. Mol. Cell. Biol. 16, 3967–3979. Taniuchi, I., et al., 2002. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell 111, 621–633. Thirunavukkarasu, K., Mahajan, M., McLarren, K.W., Stifani, S., Karsenty, G., 1998. Two domains unique to osteoblast-specific transcription factor Osf2/ Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfbeta. Mol. Cell. Biol. 18, 4197–4208. Tokuhiro, S., et al., 2003. An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transporter, is associated with rheumatoid arthritis. Nat. Genet. 35, 341–348. van Wijnen, A.J., et al., 2004. Nomenclature for Runt-related (RUNX) proteins. Oncogene 23, 4209–4210. Wada, M., et al., 2004. Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines. Oncogene 23, 2401–2407. Wee, H.J., Huang, G., Shigesada, K., Ito, Y., 2002. Serine phosphorylation of RUNX2 with novel potential functions as negative regulatory mechanisms. EMBO Rep. 3, 967–974. Westendorf, J.J., et al., 2002. Runx2 (Cbfa1, AML-3) interacts with histone deacetylase 6 and represses the p21(CIP1/WAF1) promoter. Mol. Cell. Biol. 22, 7982–7992. Woolf, E., et al., 2003. Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis. Proc. Natl. Acad. Sci. U. S. A. 100, 7731–7736. Wozney, J.M., et al., 1988. Novel regulators of bone formation: molecular clones and activities. Science 242, 1528–1534. Xiao, W.H., Liu, W.W., 2004. Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma. World J. Gastroenterol. 10, 376–380. Xiao, G., et al., 2000. MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J. Biol. Chem. 275, 4453–4459. Xiao, G., Jiang, D., Gopalakrishnan, R., Franceschi, R.T., 2002. Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J. Biol. Chem. 277, 36181–36187. Yaffe, M.B., 2002. Phosphotyrosine-binding domains in signal transduction. Nat. Rev., Mol. Cell Biol. 3, 177–186.

66

S.-C. Bae, Y.H. Lee / Gene 366 (2006) 58–66

Yamaguchi, Y., et al., 2004. AML1 is functionally regulated through p300mediated acetylation on specific lysine residues. J. Biol. Chem. 279, 15630–15638. Yamamoto, N., Akiyama, S., Katagiri, T., Namiki, M., Kurokawa, T., Suda, T., 1997. Smad1 and smad5 act downstream of intracellular signalings of BMP2 that inhibits myogenic differentiation and induces osteoblast differentiation in C2C12 myoblasts. Biochem. Biophys. Res. Commun. 238, 574–580. Yanagawa, N., Tamura, G., Oizumi, H., Takahashi, N., Shimazaki, Y., Motoyama, T., 2003. Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers. Cancer Sci. 94, 589–592. Yang, X.J., 2004. The diverse superfamily of lysine acetyltransferases and their roles in leukemia and other diseases. Nucleic Acids Res. 32, 959–976. Zhang, Y., Derynck, R., 1999. Regulation of Smad signalling by protein associations and signalling crosstalk. Trends Cell Biol. 9, 274–279.

Zhang, Y.W., et al., 2000. A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc. Natl. Acad. Sci. U. S. A. 97, 10549–10554. Zhang, Y., Biggs, J.R., Kraft, A.S., 2004. Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation. J. Biol. Chem. 279, 53116–53125. Zhao, M., Qiao, M., Oyajobi, B.O., Mundy, G.R., Chen, D., 2003. E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation. J. Biol. Chem. 278, 27939–27944. Zhou, Y.X., Xu, X., Chen, L., Li, C., Brodie, S.G., Deng, C.X., 2000. A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures. Hum. Mol. Genet. 9, 2001–2008.