Figure 2. Specific interaction of Nedd4L with linker-phosphorylated Smad2/3.
(A) HEK293T cells were cotransfected with vectors encoding HA-tagged HECT ubiquitin ligases and either wild-type (WT) or linker phosphorylation site mutants (mut) of Flag-Smad3 or Smad1. Whole cell lysates were immunoprecipitated (IP) and subsequently immunoblotted with antibodies as shown. (B) Immobilized GST-Smad3 or GST-Smad1 phosphorylated by CyclinC-CDK8 or CyclinT-CDK9 was incubated with HEK293T cell lysates expressing HA-tagged ubiquitin ligases. Bound proteins were eluted from the beads and immunoblotted with anti-HA antibody. The amounts of GST fusion proteins were shown by Coomassie blue staining. The protein levels of ubiquitin ligases in HEK293T lysates used in (A) and (B) were determined by anti-HA immunoblot in the bottom panel. (C) As in (B), except that immobilized GST-Smad2 protein was phosphorylated in vitro by CyclinT-CDK9 and used as bait. (D) As in (B), immobilized GST-Smad3 protein was phosphorylated in vitro in the presence or absence of ATP and with addition of flavopiridol. (E) Scheme of Smad-E3 ubiquitin ligase interaction induced by TGFβ and BMP signaling. The linker and C-tail phosphorylation are depicted in orange and green, respectively.