| The type I TGFβ receptor (TβR-I) is a transmembrane serine/threonine kinase of central importance to eukaryotic growth and homeostasis. TβR-I is activated by multiple phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the protein kinase domain in the cytoplasmic portion of the receptor. In this thesis, I describe structural and biochemical studies aimed at understanding the molecular basis of TβR-I regulation by the GS region. The crystal structure of the unphosphorylated cytoplasmic portion of TβR-I containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12, which binds to the GS region an inhibits TGFβ signaling. (TβR-I) adopts a catalytically inactive conformation in this structure that is maintained, in part, by the unphosphorylated GS region. FKBP12 binds to the GS region, capping the activating phosphorylation sites and further stabilizing the inhibited conformation of TβR-I The crystal structure of TβR-I has also been solved in the absence of FKBP12. This structure reveals that certain portions of the GS region are intrinsically unstable, and require FKBP12 to mold them into an inhibitory conformation. In order to better understand the molecular mechanism of TβR-I activation, a novel semisynthetic approach was employed to produce a homogeneously tetraphosphorylated form of TβR-I which was used in biochemical studies. Phosphorylation of the GS region dramatically enhances the specificity of TβR-I for the critical C-terminal serine residues of its physiological substrate, Smad2. In addition, tetraphosphorylated TβR-I is specifically bound by Smad2 in a phosphorylation dependent manner, but is no longer recognized by FKBP12. Thus, phosphorylation activates TβR-I by switching the GS region from a docking site for an inhibitor into part of a docking site for substrate. These observations suggest that phosphoserine/phosphothreonine dependent localization is a key feature of the TβR-I activation process. |
