Functional analysis of human Smad1: role of the amino-terminal domain.
The signals originating from transforming growth factor beta/activin/bone morphogenetic proteins (BMPs) are transduced by a set of evolutionarily conserved family of Smad proteins which, upon activation, directly translocate to the nucleus where they may activate transcription. Smad proteins of different species contain conserved amino- (N) and carboxy- (C) terminal domains separated by a proline-rich linker. Human, Drosophila, and Xenopus Smad1 all have been shown to mediate the biological effects of BMP-4 in Xenopus embryos. We have investigated the functional domains of human Smad1 (hSmad1) using the Xenopus embryo system. Dorsal injection of hSmad1 RNA into the 4-cell-stage embryos results in embryonic ventralization. Since the C-terminus of Smads has been shown to mediate the transcriptional activity, whereas this activity is masked by the presence of the N-terminus, we tested the effect of a hSmad1 construct lacking the C-terminal domain [hSmad1(N)] in the Xenopus embryo system. Surprisingly, we found that hSmad1(N) not only synergizes with hSmad1 in embryonic ventralization, but induces ventralization by itself. Ectopic expression of a dominant negative BMP receptor (DN-BR) as well as neural inducers noggin and chordin induce neurogenesis in the animal cap, which is inhibited by co-expression of either hSmad1 or hSmad1(N). Ventral expression of DN-BR induces formation of a second body axis at tailbud stage, which is also prevented by hSmad1 and hSmad1(N). It has recently been reported that calmodulin interacts with the N-terminal domain of Smad proteins. We demonstrate that the ventralizing activity of hSmad1 and hSmad1(N) is markedly inhibited by calmodulin. Thus, calmodulin acts as a Smad1 inhibitor. A model is proposed to accomodate these findings.
D000818:Animals; D019521:Body Patterning; D002147:Calmodulin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D009420:Nervous System / Q000196:embryology; D051785:Smad Proteins; D051898:Smad1 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000196:embryology