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

A new family of signaling intermediates for TGF beta superfamily members an d other growth factors has recently been identified and termed Smads. It ha s been suggested that the Smad1 subfamily is regulated primarily by the TGF beta superfamily member bone morphogenetic protein (BMP). Here we demonstr ate that TGF beta induced phosphorylation of endogenous Smad1 in untransfor med IECs and that the RI and RII TGF beta receptors were detectable in Smad 1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibite d the ability of TGF beta 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, w e demonstrate that the TGF beta receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibite d the ability of RSmad1 to induce the TGF beta-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate tha t TGF beta can regulate Smad1 and that the Ras and MEK signaling components are partially required for the ability of TGF beta to regulate Smad1. J. C ell. Physiol. 178:387-396, 1999. (C) 1999 Wiley-Liss, Inc.