Abstract
Signal transduction by the Smad pathway elicits critical biological responses to many extracellular polypeptide factors, including TGFβ and bone morphogenetic protein. Regulation of Smad signaling imparts several cytoplasmic and nuclear mechanisms, some of which entail protein phosphorylation. Previous work established a protein complex between Smad4 and the scaffolding protein LKB1-interacting protein 1 (LIP1). LKB1 is a well studied tumor suppressor kinase that regulates cell growth and polarity. Here, we analyzed the LKB1-LIP1 and the Smad4-LIP1 protein complexes and found that LIP1 can self-oligomerize. We further demonstrate that LKB1 is capable of phosphorylating Smad4 on Thr77 of its DNA-binding domain. LKB1 inhibits Smad4 from binding to either TGFβ- or bone morphogenetic protein-specific promoter sequences, which correlates with the negative regulatory effect LKB1 exerts on Smad4-dependent transcription. Accordingly, LKB1 negatively regulates TGFβ gene responses and epithelial-mesenchymal transition. Thus, LKB1 and LIP1 provide negative control of TGFβ signaling.
Highlights
TGF and related polypeptides, such as bone morphogenetic proteins (BMPs),4 signal via serine/threonine kinase receptors; upon ligand binding, a type II receptor phosphorylates a type I receptor to activate its kinase activity [1]
In order to study the protein complexes formed between LKB1, LKB1-interacting protein 1 (LIP1), and Smad4, we generated a rabbit polyclonal antibody that recognizes human LIP1
Co-immunoprecipitation assays using this antibody and all three proteins transfected in 293-T cells indicated the formation of complexes between the transfected LKB1, LIP1, and Smad4, with traces of endogenous LKB1 observed in the LIP1 co-precipitates (Fig. 1A, lane 1)
Summary
TGF and related polypeptides, such as bone morphogenetic proteins (BMPs),4 signal via serine/threonine kinase receptors; upon ligand binding, a type II receptor phosphorylates a type I receptor to activate its kinase activity [1]. In order to demonstrate that LKB1 can negatively regulate transcriptional events induced by TGF at the endogenous level, we employed again the Smad4-null MDA-MB-468 cells and adenovirus-mediated transfection of control (GFP) or Smad4 protein in the absence or presence of the three components of the LSM enzymatic complex (Fig. 3D).
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