The N-terminus zinc finger domain of Xenopus SIP1 is important for neural induction, but not for suppression of Xbra expression

Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity.     

Recombinant expression and purification of smad proteins

Smads transduce intracellular signals initiated by members of the transforming growth factor beta (TGF beta) family, including activins, TGF betas, and bone morphogenetic proteins. Recently, various models concerning the mechanism of Smad action have been proposed; however, these models are basically qualitative. Quantitative verification of the validity of the models requires significant amounts of purified Smad proteins, but purification of full-length Smad protein has not been straightforward even using recombinant protein expression systems. Here, we report purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydroxylapatite columns, expressed Smads were purified to near homogeneity as judged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 100 microg/l culture for Smad4. The purified Smad proteins had three known in vitro activities: Smad2 phosphorylation by TGF beta receptor complexes immunoprecipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and Smad2 interaction with calmodulin. The data suggest that purified proteins could be useful for biochemical analyses to evaluate the current models quantitatively.     

Identification of two Smad4 proteins in Xenopus. Their common and distinct properties

Smad family proteins have been identified as mediators of intracellular signal transduction by the transforming growth factor-beta (TGF-beta) superfamily. Each member of the pathway-restricted, receptor-activated Smad family cooperates and synergizes with Smad4, called co-Smad, to transduce the signals. Only Smad4 has been shown able to function as a common partner of the various pathway-restricted Smads in mammals. Here we have identified a novel Smad4-like molecule in Xenopus (XSmad4beta) as well as a Xenopus homolog of a well established Smad4 (XSmad4alpha). XSmad4beta is 70% identical to XSmad4alpha in amino acid sequence. Both of the Xenopus Smad4s can cooperate with Smad1 and Smad2, the pathway-restricted Smads specific for bone morphogenetic protein and TGF-beta, respectively. However, they show distinct properties in terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4beta, but not XSmad4alpha, has the potent ability to induce ventralization when microinjected into the dorsal marginal region of the 4-cell stage of the embryos. These results suggest that the two Xenopus Smad4s have overlapping but distinct functions.