Identification of a Novel Negative Regulator of Activin/Nodal Signaling in Mesendodermal Formation of Xenopus Embryos

Abstract

Phosphotyrosine binding (PTB) domains, which are found in a large number of proteins, have been implicated in signal transduction mediated by growth factor receptors. However, the in vivo roles of these PTB-containing proteins remain to be investigated. Here, we show that Xdpcp (Xenopus dok-PTB containing protein) has a pivotal role in regulating mesendoderm formation in Xenopus, and negatively regulates the activin/nodal signaling pathway. We isolated cDNA for xdpcp and examined its potential role in Xenopus embryogenesis. We found that Xdpcp is strongly expressed in the animal hemisphere at the cleavage and blastula stages. The overexpression of xdpcp RNA affects activin/nodal signaling, which causes defects in mesendoderm formation. In addition, loss of Xdpcp function by injection of morpholino oligonucleotides leads to the expansion of the mesodermal territory. Moreover, we found that axis duplication by ventrally forced expression of activin is recovered by coexpression with Xdpcp. In addition, Xdpcp inhibits the phosphorylation and nuclear translocation of Smad2. Furthermore, we also found that Xdpcp interacts with Alk4, a type I activin receptor, and inhibits activin/nodal signaling by disturbing the interaction between Smad2 and Alk4. Taken together, these results indicate that Xdpcp regulates activin/nodal signaling that is essential for mesendoderm specification. Phosphotyrosine binding (PTB)2 domain-containing proteins are implicated in signal transduction, protein trafficking, and cytoskeletal dynamics (1). The PTB domain is important forprotein-proteininteractionsthatareeitherphosphotyrosinedependent or -independent. As a result, PTB-containing proteins engage in a wide range of cellular functions (2). PTB domain proteins play a pivotal role in signal transduction mediated by various growth factors, including epidermal growth factor (3), insulin (4, 5), nerve growth factor (6), and transforming growth factor-␤ (TGF-␤) (7,8). To mediate TGF-␤ signaling, PTB proteins such as Dab2 (7) and Dok1 (8) act as adaptors to stabilize the interaction between the receptor and Smad2/3. TGF-␤ signaling is implicated in a number of cellular functions such as proliferation, migration, differentiation, and apoptosis. Disturbance of TGF-␤ signaling is related to serious human diseases including cancer, fibrosis, and heritable disorders (9). TGF-␤ signaling is initiated by the binding of ligands to two types of Ser/Thr kinase receptors, called type I and type II receptors. The type I receptor is activated by the type II receptor and propagates intracellular signaling to the nucleus through the phosphorylation of receptor-activated Smads (R-Smad) including Smad1, -2, -3, -5, and -8. Then, the active Smad complex that is formed by the interaction of R-Smad with Smad4 translocates into the nucleus where it regulates the transcription of target genes.TGF-␤ signaling is also crucial for developmental processes including germ-layer specification and patterning during embryogenesis. Xenopus late blastula embryo consists of th...

Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis

“…Western blot assays and co-IP were carried out as previously described 49 , 54 . Briefly, embryos or animal cap explants were homogenized in Triton-X 100 lysis buffer [20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 140 mM NaCl, 10% glycerol, 1 mM EGTA, 1.5 mM , 1 mM DTT, 1 mM sodium vanadate ( ), 50 mM NaF, 20 g/ml aprotinin, 40 g/ml leupeptin, and 0.75 mM phenylmethylsulfonyl fluoride (PMSF)] for western blot assay, or IP buffer [20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM EDTA (pH 7.5), 1% TritonX-100, 1 mM , 100 mM NaF, 25 mM -glycerophosphate, 20 g/ml aprotinin, 40 g/ml leupeptin, and 0.75 mM PMSF] for co-IP assay.…”