Chang-l Yeo

Nodal Signals to Smads through Cripto-Dependent and Cripto-Independent Mechanisms

Nodal ligands are essential for the patterning of chordate embryos. Genetic evidence indicates that EGF-CFC factors are required for Nodal signaling, but the molecular basis for this requirement is unknown. We have investigated the role of Cripto, an EGF-CFC factor, in Nodal signaling. We find that Cripto interacts with the type I receptor ALK4 via the conserved CFC motif in Cripto. Cripto interaction with ALK4 is necessary both for Nodal binding to the ALK4/ActR-IIB receptor complex and for Smad2 activation by Nodal. We also find that Nodal can inhibit BMP signaling by a Cripto-independent mechanism. Inhibition appears to be mediated by heterodimerization between Nodal and BMPs, indicating that antagonism between Nodal and BMPs can occur at the level of dimeric ligand production.

Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase

Febrifugine, one of the fifty fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity whilst its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis, and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of Th17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response pathway (AAR). Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS) inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural products. This work both explains the molecular mechanism of a promising family of therapeutics, and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

The Role of FAST-1 and Smads in Transcriptional Regulation by Activin during Early Xenopus Embryogenesis

Smads are signal transducers for the transforming growth factor-β superfamily of factors. In early Xenopusembryos, the transforming growth factor-β member activin induces the gene Mix.2 by stimulating the formation of a multiprotein complex, activin-responsive factor (ARF). This complex contains Smad2 or Smad3, Smad4, and a novel forkhead transcription factor, FAST-1, and binds to an enhancer (activin-responsive element; ARE) that confers activin regulation of Mix.2 transcription. Both FAST-1 and Smads can bind directly to the ARE; we have investigated 1) the role of FAST-1 and Smad DNA binding sites in ARF recognition of the ARE, 2) the contributions of FAST-1 and Smad binding to ARF binding in vitro and to ARE regulation in early Xenopus embryos, 3) the extent to which different Smads can replace Smad4 in regulation of the ARE. We find that ARF binds to ARE through both FAST-1 and Smad binding sites. FAST-1 recognition of the ARE is essential both for ARF binding in vitro and activin regulation in vivo. In contrast, Smad binding of ARE is unnecessary for ARF binding or activin regulation but does enhance the binding and regulatory activity of ARF. Also, Smad3 can partially substitute for Smad4 in the regulation of the ARE. These observations elucidate how broadly expressed signal transducers (Smads) regulate a developmentally specific transcriptional response in conjunction with a temporally restricted transcription factor, FAST-1.

Protein kinase A phosphorylates and regulates dimerization of 14‐3‐3ζ

Recognition of phosphorylated serine/threonine‐containing motifs by 14‐3‐3 depends on the dimerization of 14‐3‐3. However, the molecular cues that control 14‐3‐3 dimerization are not well understood. In order to identify proteins that control 14‐3‐3 dimerization, we analyzed proteins that have effects on 14‐3‐3 dimerization and report that protein kinase A (PKA) phosphorylates 14‐3‐3ζ at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14‐3‐3ζ dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14‐3‐3ζ by PKA. A phospho‐mimic mutant of 14‐3‐3ζ, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14‐3‐3ε and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14‐3‐3ζ and the functional interaction of 14‐3‐3ζ with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14‐3‐3.

A mutation in TGFB3 associated with a syndrome of low muscle mass, growth retardation, distal arthrogryposis and clinical features overlapping with marfan and loeys–dietz syndrome

The transforming growth factor β (TGF‐β) family of growth factors are key regulators of mammalian development and their dysregulation is implicated in human disease, notably, heritable vasculopathies including Marfan (MFS, OMIM #154700) and Loeys–Dietz syndromes (LDS, OMIM #609192). We described a syndrome presenting at birth with distal arthrogryposis, hypotonia, bifid uvula, a failure of normal post‐natal muscle development but no evidence of vascular disease; some of these features overlap with MFS and LDS. A de novo mutation in TGFB3 was identified by exome sequencing. Several lines of evidence indicate the mutation is hypomorphic suggesting that decreased TGF‐β signaling from a loss of TGFB3 activity is likely responsible for the clinical phenotype. This is the first example of a mutation in the coding portion of TGFB3 implicated in a clinical syndrome suggesting TGFB3 is essential for both human palatogenesis and normal muscle growth.

A Secreted Tyrosine Kinase Acts in the Extracellular Environment

Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.

Akt phosphorylates and regulates the osteogenic activity of Osterix

Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.

Acetylation of histone deacetylase 6 by p300 attenuates its deacetylase activity.

Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.

Regulation of Activin/Nodal Signaling by Rap2-Directed Receptor Trafficking

We show that Rap2, a member of the Ras GTPase family, positively regulates Activin/Nodal signaling activity by controlling the trafficking of its receptors. In the absence of ligand activation, Rap2 directs internalized Activin/Nodal receptors into a recycling pathway, thereby preventing their degradation and maintaining their levels on the cell surface. Upon ligand activation, Rap2 no longer promotes receptor recycling but delays its turnover. In both cases, Rap2 contributes to upregulation of signaling activity by antagonizing Smad7. In addition, we found that the efficiency of Activin/Nodal receptor recycling is different between dorsal and ventral halves of Xenopus early embryo, which results from the asymmetric expression of Rap2 and Smad7. Consequently, they regulate cell responsiveness to ligands and the spatiotemporally dynamic activation of Smad2 along the dorsoventral axis of the embryo. Therefore, these findings suggest a molecular basis for the regulation of signaling activity and embryonic patterning by Activin/Nodal receptor trafficking.

Metallothionein-III provides neuronal protection through activation of Nuclear Factor-κB via the TrkA/Phosphatidylinositol-3 kinase/Akt signaling pathway

Metallothionein (MT)-III is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. The present study investigated the mechanisms of MT-III protection of neuronal cells from hypoxia or DNA damage-induced cell death. MT-III reduced the hydrogen peroxide- or DNA damage-induced effects on neuronal cells, including the cell death, the activation of caspase-3 and -9, and the release of mitochondrial cytochrome c to the cytoplasm in a dose-dependent manner. MT-III also increased the activation of Akt, the phosphorylation and degradation of IkappaB, the nuclear translocation/accumulation and the transcriptional activity of nuclear factor-kappaB (NF-kappaB) in neuronal cells in a dose-dependent manner. The MT-III-induced antiapoptotic effects and increase in NF-kappaB activity were blocked by specific inhibitors of TrkA, phosphatidylinositol-3 kinase (PI3K), Akt, or NF-kappaB, indicating that MT-III provides neuronal protection by activating NF-kappaB through the TrkA/PI3K/Akt signaling pathway.