Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.

Transforming growth factor-beta (TGF-beta) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF-beta activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF-beta. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up-regulation of TGF-beta superfamily-induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl-2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase-negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP-2/4 type IA receptor (BMPR-IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner.     

TAK1-binding protein 1, TAB1, mediates osmotic stress-induced TAK1 activation but is dispensable for TAK1-mediated cytokine signaling

TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.     

Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2

Transforming growth factor-beta-activated kinase 1 (TAK1) mitogen-activated protein kinase kinase kinase has been shown to be activated by cellular stresses including tumor necrosis factor-alpha (TNF-alpha). Here, we characterized the molecular mechanisms of cellular stress-induced TAK1 activation, focusing mainly on the phosphorylation of TAK1 at Thr-187 and Ser-192 in the activation loop. Thr-187 and Ser-192 are conserved among species from Caenorhabditis elegans to human, and their replacement with Ala resulted in inactivation of TAK1. Immunoblotting with a novel phospho-TAK1 antibody revealed that TNF-alpha significantly induced the phosphorylation of endogenous TAK1 at Thr-187, and subsequently the phosphorylated forms of TAK1 rapidly disappeared. Intermolecular autophosphorylation of Thr-187 was essential for TAK1 activation. RNA interference and overexpression experiments demonstrated that TAK1-binding protein TAB1 and TAB2 were involved in the phosphorylation of TAK1, but they regulated TAK1 phosphorylation differentially. Furthermore, SB203580 and p38alpha small interfering RNA enhanced TNF-alpha-induced Thr-187 phosphorylation as well as TAK1 kinase activity, indicating that the phosphorylation is affected by p38alpha/TAB1/TAB2-mediated feedback control of TAK1. These results indicate critical roles of Thr-187 phosphorylation in the stress-induced rapid and transient activation of TAK1 in a signaling complex containing TAB1 and TAB2.     

TAB1beta (transforming growth factor-beta-activated protein kinase 1-binding protein 1beta ), a novel splicing variant of TAB1 that interacts with p38alpha but not TAK1

The mitogen-activated protein kinases (MAPKs) play an important role in a variety of biological processes. Activation of MAPKs is mediated by phosphorylation on specific regulatory tyrosine and threonine sites. We have recently found that activation of p38alpha MAPK can be carried out not only by its upstream MAPK kinases (MKKs) but also by p38alpha autophosphorylation. p38alpha autoactivation requires an interaction of p38alpha with TAB1 (transforming growth factor-beta-activated protein kinase 1-binding protein 1). The autoactivation mechanism of p38alpha has been found to be important in cellular responses to a number of physiologically relevant stimuli. Here, we report the characterization of a splicing variant of TAB1, TAB1beta. TAB1 and TAB1beta share the first 10 exons. The 11th and 12th exons of TAB1 were spliced out in TAB1beta, and an extra exon, termed exon beta, downstream of exons 11 and 12 in the genome was used as the last exon in TAB1beta. The mRNA of TAB1beta was expressed in all cell lines examined. The TAB1beta mRNA encodes a protein with an identical sequence to TAB1 except the C-terminal 69 amino acids were replaced with an unrelated 27-amino acid sequence. Similar to TAB1, TAB1beta interacts with p38alpha but not other MAPKs and stimulates p38alpha autoactivation. Different from TAB1, TAB1beta does not bind or activate TAK1. Inhibition of TAB1beta expression with RNA interference in MDA231 breast cancer cells resulted in the reduction of basal activity of p38alpha and invasiveness of MDA231 cells, suggesting that TauAlphaBeta1beta is involved in regulating p38alpha activity in physiological conditions.     

TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway

The TAK1 MAPKKK mediates activation of JNK and NF-KB in the IL-1-activated signaling pathway. Here we report the identification of TAB2, a novel intermediate in the IL-1 pathway that functionally links TAK1 to TRAF6. Expression of TAB2 induces JNK and NF-kappaB activation, whereas a dominant-negative mutant TAB2 impairs their activation by IL-1. IL-1 stimulates translocation of TAB2 from the membrane to the cytosol where it mediates the IL-1-dependent association of TAK1 with TRAF6. These results define TAB2 as an adaptor linking TAK1 and TRAF6 and as a mediator of TAK1 activation in the IL-1 signaling pathway.     

Expression of TAK1, a mediator of TGF-beta and BMP signaling,during mouse embryonic development

TGF-beta activated kinase 1 (TAK1) is a MAP kinase kinase kinase (MAPKKK) that has been shown to function downstream of BMPs and TGF-beta (J. Biol. Chem. 275 (2000) 17647; EMBO J. 17 (1998) 1019; Science 270 (1995) 2008), as well as in the interleukin-1 (IL-1) signaling pathway (J. Biol. Chem. 276 (2001) 3508; Nature 398 (1999) 252). Using immunohistochemistry (IHC), we demonstrate that TAK1 is expressed ubiquitously during early development. At mid-gestation, TAK1 expression becomes more restricted, with high levels seen specifically during development of diverse organs and tissues including the nervous system, testis, kidney, liver and gut. Additionally, TAK1 expression is seen in the developing lung and pancreas. Our results suggest that TAK1 may play multiple roles in mouse development.     

Xenopus FK 506-binding protein, a novel immunophilin expressed during early development

FK 506-binding proteins (FKBPs) are a family of cytosolic proteins identified by virtue of their ability to bind the immunosuppressants FK 506 and rapamycin. While their function has been extensively studied in the immune system, little is known about their role during early embryonic development. Here we describe the cloning and expression of a new Xenopus FKBP (xFKBP). xFKBP encodes a 63-kDa protein that shares high sequence homology with mouse FKBP65. It is expressed maternally and becomes restricted after the gastrula stage to dorsal mesoderm and notochord. At the tailbud stage expression persists in the notochord and begins to accumulate in epidermis, branchial arches and developing somites. In adults, xFKBP mRNA is confined to the testis.     

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.     

XMAN1, an inner nuclear membrane protein,antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.     

Regulation of nodal and BMP signaling by tomoregulin-1 (X7365) through novel mechanisms

During early vertebrate development, members of the transforming growth factor beta (TGFbeta) family play important roles in a variety of processes, including germ layer specification, patterning, cell differentiation, migration, and organogenesis. The activities of TGFbetas need to be tightly controlled to ensure their function at the right time and place. Despite identification of multiple regulators of Bone Morphogenetic Protein (BMP) subfamily ligands, modulators of the activin/nodal class of TGFbeta ligands are limited, and include follistatin, Cerberus, and Lefty. Recently, a membrane protein, tomoregulin-1 (TMEFF1, originally named X7365), was isolated and found to contain two follistatin modules in addition to an Epidermal Growth Factor (EGF) domain, suggesting that TMEFF1 may participate in regulation of TGFbeta function. Here, we show that, unlike follistatin and follistatin-related gene (FLRG), TMEFF1 inhibits nodal but not activin in Xenopus. Interestingly, both the follistatin modules and the EGF motif contribute to nodal inhibition. A soluble protein containing the follistatin and the EGF domains, however, is not sufficient for nodal inhibition; the location of TMEFF1 at the membrane is essential for its function. These results suggest that TMEFF1 inhibits nodal through a novel mechanism. TMEFF1 also blocks mesodermal, but not epidermal induction by BMP2. Unlike nodal inhibition, regulation of BMP activities by TMEFF1 requires the latter's cytoplasmic tail, while deletion of either the follistatin modules or the EGF motif does not interfere with the BMP inhibitory function of TMEFF1. These results imply that TMEFF1 may employ different mechanisms in the regulation of nodal and BMP signals. In Xenopus, TMEFF1 is expressed from midgastrula stages onward and is enriched in neural tissue derivatives. This expression pattern suggests that TMEFF1 may modulate nodal and BMP activities during neural patterning. In summary, our data demonstrate that tomoregulin-1 is a novel regulator of nodal and BMP signaling during early vertebrate embryogenesis.     

Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity

In Xenopus, ectodermal patterning depends on a mediolateral gradient of BMP signaling, higher in the epidermis and lower in the neuroectoderm. Neural crest cells are specified at the border between the neural plate and the epidermis, at intermediate levels of BMP signaling. We recently described a novel secreted protein, Tsukushi (TSK), which works as a BMP antagonist during chick gastrulation. Here, we report on the Xenopus TSK gene (X-TSK), and show that it is involved in neural crest specification. X-TSK expression accumulates after gastrulation at the anterior-lateral edges of the neural plate, including the presumptive neural crest region. In gain-of-function experiments, X-TSK can strongly enhance neural crest specification by the dorsolateral mesoderm or X-Wnt8 in ectodermal explants, while the electroporation of X-TSK mRNA in the lateral ectoderm of embryos after gastrulation can induce the expression of neural crest markers in vivo. By contrast, depletion of X-TSK in explants or embryos impairs neural crest specification. Similarly to its chick homolog, X-TSK works as a BMP antagonist by direct binding to BMP4. However, X-TSK can also indirectly regulate BMP4 mRNA expression at the neural plate border via modulation of the Delta-Notch signaling pathway. We show that X-TSK directly binds to the extracellular region of X-delta-1, and modulates Delta-dependent Notch activity. We propose that X-TSK plays a key role in neural crest formation by directly regulating BMP and Delta activities at the boundary between the neural and the non-neural ectoderm.     

Endogenous lactate transport in Xenopus laevis oocyte: dependence on cytoskeleton and regulation by protein kinases

Carbon flux in Xenopus laevis oocyte is glycogenic and an endogenous monocarboxylate transporter is responsible for intracellular lactate uptake. The aim of the present study was to determine if direct activation of protein kinases C and A modulates the activity of lactate transporter, as well as to investigate the possible role of cytoskeleton in these regulatory phenomena. The modulation was studied in isolated Xenopus oocytes of stage V–VI by measuring ¹⁴C-lactate uptake, both in the absence and in the presence of cytoskeletal-perturbing toxins. We found that the basal lactate transporter activity depends on the integrity of the cytoskeleton since it is partially inhibited by cytoskeleton disorganisation. Both PKA and PKC activation caused a significant decrease in transport activity and this decrease could be blocked by specific protein kinase inhibitors. The evidenced effects were not additive. Transport inhibition was annulled by agents that destabilize actin filaments or microtubules. We conclude that both protein kinases A and C, whose effects are mediated by cytoskeleton, negatively regulate the endogenous lactate transporter of Xenopus oocyte, suggesting that these kinases may have a role in the control of cytosolic pyruvate/lactate pool in the oocyte. All the experiments in this study comply with the current laws of Italy.     

HMGB1: a novel Beclin 1-binding protein active in autophagy

The autophagosome delivers damaged cytoplasmic constituents and proteins to the lysosome or to the extracellular space. Beclin 1, an essential: autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members and has a critical role in the initiation of autophagy. How the Beclin 1 complex specifically promotes autophagy remains largely unknown. We have found that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage associated molecular pattern molecule (DAMP), is a novel Beclin 1-binding protein important in sustaining autophagy. HMGB1 shares considerable sequence homology with Beclin 1 in yeast, mice and human, representing an evolutionarily conserved regulatory step in early autophagosome formation. Endogenous HMGB1 competes with Bcl-2 for interaction with Beclin 1, and orients Beclin 1 to autophagosomes. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin 1 and sustaining autophagy. Taken together, these findings indicate that endogenous HMGB1 functions as an autophagy effector by regulation of autophagosome formation.     

XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.

Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors. TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor. These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.     

TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.