Cloning and expression of a rat Smad1: Regulation by TGFβ and modulation by the Ras/MEK pathway

Abstract

A new family of signaling intermediates for TGFβ superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFβ superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFβ induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFβ receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFβ to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFβ receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFβ-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFβ can regulate Smad1 and that the Ras and MEK signaling components are partially required for the ability of TGFβ to regulate Smad1.