GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex.

Abstract

Corresponding author Communicated by J.PouyssegurThe TGF-, type II receptor (T3R-II) is a transmembrane serine/threonine kinase that, upon ligand binding, recruits and phosphorylates a second transmembrane kinase, TPR-I, as a requirement for signal transduction. T3R-I is phosphorylated by TfR-II in the GS domain, a 30 amino acid region preceding the kinase domain and conserved in type I receptors for other TGF-3-related factors. The functional role of seven serines and threonines in the TPR-I GS domain was investigated by mutational analysis. Five of these residues are clustered (TTSGSGSG) in the middle of the GS domain. Mutation of two or more of these residues impairs phosphorylation and signaling activity. Two additional threonines are located near the canonical start of the kinase domain, and their individual mutation to valine strongly inhibits receptor phosphorylation and signaling activity. Replacement of one of these residues, Thr2O4, with aspartic acid yields a product that has elevated in vitro kinase activity and signals anti-proliferative and transcriptional responses in the absence of ligand and Tr3R-II. The identification of constitutively active T3R-I forms confirms the hypothesis that this kinase acts as a downstream signaling component in the TGF-, receptor complex, and its activation by Tf3R-II or by mutation is necessary and sufficient for propagation of antiproliferative and transcriptional responses.

Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function

“…In this mutant, replacement of three L45 loop residues (N265, D267 and N268) with alanine abolishes the ability of the receptor to interact with and phosphorylate its Smad substrates without affecting its kinase activity or its ability to activate other signaling pathways such as the extracellular signal-regulated kinase (ERK) pathway (Yu et al, 2002). In order to avoid activation of the endogenous receptors, we utilized the kinase active mutants ALK5-T204D (ALK5-TD) and (rat) RImL45-T202D (RImL45-TD) (Wieser et al, 1995). Following geneticin selection and isolation, several individual clones were obtained, which displayed considerable and approximately equal expression of the mutant proteins, which were at least fourfold higher than the level of endogenous ALK5 ( Figure 2a).…”

Involvement of programmed cell death 4 in transforming growth factor-β1-induced apoptosis in human hepatocellular carcinoma

“…Antisense PDCD4 plasmid was prepared by connecting the H731 whole sequence which covers PDCD4 whole sequence with EcoRV -XbaI sites of pcDNA3 vector in the anti-sense direction. Human Smad7 plasmid conjugated as the cDNA under the cytomegalovirus (CMV) promoter of pcDNA3 (Nakao et al, 1997), and wild-type (TbR1), constitutively active (TbR1-TD) and kinase-defective (TbR1-KR) TGF-b type I receptor plasmids conjugated with pcDNA3 vector (Wieser et al, 1995) were obtained from Dr Heldin (Ludwig Institute for Cancer Research, Upsala, Sweden). Transfection of HCC cells with plasmids was performed using Lipofectamine and Plus reagents or Lipofectamine 2000 according to the manufacturer's protocols (Invitrogen Corp., Carlsbad, CA).…”

Endoglin inhibits prostate cancer motility via activation of the ALK2-Smad1 pathway

“…Anti-mouse, anti-rabbit IgG-horseradish peroxidase from Amersham Pharmacia Biotech (Piscataway, NJ, USA). (Wrana et al, 1992;Wieser et al, 1995;Ulloa et al, 1999)); ENG-long isoform pcDNA3 (described by us (Liu et al, 2002)); HA-ALK2 pCMV5 (provided by Andreas Lux, University of Applied Sciences Mannheim, Germany and described by Jeff Wrana (Attisano et al, 1993); constitutively active (Q207D) ALK2 pCMV5 (caALK2) was engineered by site-specific mutagenesis using the Quickchange II (Stratagene, La Jolla, CA, USA) system. Constructs were confirmed by sequencing.…”