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.