The mitogen-activated protein kinase (MAPK) pathway is a conserved eukaryotic signaling module that converts receptor signals into various outputs. MAPK is activated through phosphorylation by MAPK kinase (MAPKK), which is first activated by MAPKK kinase (MAPKKK). A genetic selection based on a MAPK pathway in yeast was used to identify a mouse protein kinase (TAK1) distinct from other members of the MAPKKK family. TAK1 was shown to participate in regulation of transcription by transforming growth factor-beta (TGF-beta). Furthermore, kinase activity of TAK1 was stimulated in response to TGF-beta and bone morphogenetic protein. These results suggest that TAK1 functions as a mediator in the signaling pathway of TGF-beta superfamily members.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. Here, we show that murine Wnt-3a is also acylated at a conserved serine residue (Ser209). Of note, we demonstrated that this residue is modified with a monounsaturated fatty acid, palmitoleic acid. Wnt-3a defective in acylation at Ser209 is not secreted from cells in culture or in Xenopus embryos, but it is retained in the endoplasmic reticulum (ER). Furthermore, Porcupine, a protein with structural similarities to membrane-bound O-acyltransferases, is required for Ser209-dependent acylation, as well as for Wnt-3a transport from the ER for secretion. These results strongly suggest that Wnt protein requires a particular lipid modification for proper intracellular transport during the secretory process.

Bone morphogenetic proteins (BMPs) are secreted proteins that interact with cell-surface receptors and are believed to play a variety of important roles during vertebrate embryogenesis. Bmpr, also known as ALK-3 and Brk-1, encodes a type I transforming growth factor-~ (TGF-[3) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development, we generated Bmpr-mutant mice. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than normal. Morphological and molecular examination demonstrated that no mesoderm had formed in the mutant embryos. The growth characteristics of homozygous mutant blastocysts cultured in vitro were indistinguishable from those of controls; however, embryonic ectoderm (epiblast) cell proliferation was reduced in all homozygous mutants at E6.5 before morphological abnormalities had become prominent. Teratomas arising from E7.0 mutant embryos contained derivatives from all three germ layers but were smaller and gave rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the differentiation of a subset of mesodermal cell types.

Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Wnt signaling controls a variety of developmental processes. The canonical Wnt/β-catenin pathway functions to stabilize β-catenin, and the noncanonical Wnt/Ca2+ pathway activates Ca2+/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca2+ pathway activated by Wnt-5a antagonizes the Wnt/β-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/β-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (β2AR-Rfz-2) containing the ligand-binding and transmembrane segments from the β2-adrenergic receptor (β2AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the β-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits β-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca2+ pathway and antagonizes canonical Wnt/β-catenin signaling.

Members of the Wnt and TGF-beta superfamilies regulate both cell fate and proliferation during development and tissue maintenance. In the early amphibian embryo, the Wnt and TGF-beta superfamily signalling cascades are required for the establishment of a dorsal signalling centre, Spemann's organizer. Intracellular proteins of both pathways, upon activation, translocate to the nucleus to participate in transcription. Here we show that beta-catenin and Lef1/Tcf, which are downstream components of the Wnt signalling cascade, form a complex with Smad4, an essential mediator of signals initiated by members of the TGF-beta growth factor superfamily. In Xenopus, this interaction directly and synergistically affects expression of the twin (Xtwn) gene during formation of the organizer. This is, to our knowledge, the first demonstration of a physical interaction between TGF-beta and Wnt signalling components in vivo.

The two major mitogenic polypeptides for endothelial cells have been purified to homogeneity. The complete primary structure of bovine pituitary basic fibroblast growth factor (FGF) and the amino-terminal amino acid sequence of bovine brain acidic FGF have been established by gas-phase sequence analyses. Homogeneous preparations of these polypeptides are potent mitogens (basic FGF, ED50 60 pg/ml; acidic FGF ED50 6000 pg/ml) for many diverse cell types including capillary endothelial cells, vascular smooth muscle cells, and adrenocortical and granulosa cells; in vivo, basic FGF is a powerful angiogenic agent in the chick chorioallantoic membrane assay. The available protein sequence data demonstrate the existence of significant structural homology between the two polypeptides.