Figure 6
(A) Scheme of Nedd4L domains and locations of two potential SGK1 phosphorylation sites. (B) GST-Nedd4L WW proteins, WT or mutant at the indicated residues, were phosphorylated by activated SGK1 in vitro in the presence of 32P labeled ATP, and subjected to electrophoresis, autoradiography, and Coomassie blue staining. (C) Immobilized GST-Nedd4L WW proteins, WT or mutant, were phosphorylated in vitro by activated SGK1 and incubated with HEK293T lysates expressing Flag-Smad3. Bound proteins were eluted from the beads and immunoblotted with anti-Flag antibody. Protein was monitored by Coomassie blue staining. (D) HEK293T cells were cotransfected with vectors encoding Flag-tagged Smad3, HA-tagged Nedd4L with indicated mutations, and HA-tagged SGK1. Whole cell lysates were immunoprecipitated and immunoblotted with antibodies as shown. (E) HaCaT cells stably expressing Flag-tagged Smad3 were transfected with siRNA targeting endogenous SGK1. 24 h post-transfection cells were stimulated with TGFβ. The Flag immunoprecipitates or whole cell lysates were immunoblotted with the indicated antibodies. (F) Model depicting the molecular interactions and possible fates of linker-phosphorylated Smad2/3. Linker phosphorylation sites are depicted with small circles; red, major phosphorylation sites. The PPXY motifs are indicated with green bars.