(A) HEK293T cells were cotransfected with vectors encoding Myc-tagged ubiquitin, HA-tagged Nedd4L, and Flag-tagged Smad3 (WT or mutant). Whole cell extracts were subjected to anti-Flag immunoprecipitation, followed by immunoblotting with anti-HA and anti-Myc antibodies. (B) As in (A), except that wild-type (WT) or catalytically inactive mutant (DD) form of HA-Nedd4L was cotransfected with WT Flag-Smad3 and Myc-ubiquitin. (C) As in (A), except that full-length (FL), MH1+linker (NL), or linker+MH2 (LC) fragments of Smad3 was cotransfected with WT HA-Nedd4L and Myc-ubiquitin. (D) Wild-type (WT) or catalytically inactive (mut) HA-SCP2 was cotransfected with HA-Nedd4L, Flag-Smad3, and Myc-ubiquitin in HEK293T cells. Whole cell extract and Flag immunoprecipitates were immunoblotted with indicated antibodies. (E) Control HaCaT cells or cells expressing shRNA targeting Nedd4L were transfected with Myc-tagged ubiquitin. 24 h post-transfection cells were stimulated with TGFβ for 1 h. The Smad2/3 immunoprecipitates or whole cell lysates were subjected to western immunoblotting with the indicated antibodies. (F) As in (E), except that HaCaT cells stably expressing Flag-tagged Smad3 in wild-type, AY, or mut forms were used.

(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.

(A) HaCaT cells stably expressing shRNA targeting Nedd4L or pretreated with proteasome inhibitor MG132 were stimulated with TGFβ for 1 h. TGFβ was then removed from the culture medium and cells were lysed at the indicated time points after ligand removal. Cytosolic and nuclear fractions were immunoblotted with the indicated antibodies. (B) Control HaCaT cells and cells stably expressing an shRNA targeting Nedd4L were immunostained with anti-Nedd4L antibody. DAPI staining was used to visualize the nuclei. (C) Stable HaCaT cells described in (B) were harvested and subjected to cytosolic and nuclear fractionation. Cytosolic (C) and nuclear (N) fractions were immunoblotted for endogenous Nedd4L. Histone 1B and α-Tubulin were used as controls for nuclear and cytosolic fractions, respectively. (D) HaCaT cells were treated with TGFβ. Cytosolic and nuclear fractions of the cells were immunoblotted for endogenous Nedd4L. (E) HaCaT cells stably expressing HA-tagged Nedd4L were treated with TGFβ. Whole cell extract (WCE) or the cytosolic (C) and nuclear (N) fractions were subjected to anti-HA immunoprecipitation. The immunoprecipitates or lysates were immunoblotted with the indicated antibodies. (F) Activated, linker-phosphorylated Smad2/3 is mostly nuclear, but Nedd4L and the Nedd4L-Smad2/3 complex are mostly cytoplasmic.

(A) Scheme of the Nedd4L-Smad3 interaction. Brackets indicate the critical interacting domains of Nedd4L and Smad3. (B) The dissociation constants of recombinant WW domains and synthetic Smad linker peptides were measured by isothermal titration calorimetry. Data were derived from triplicate experiments. Errors correspond to deviations from theoretical binding curves. (C) HaCaT cells were treated with TGFβ or EGF for the indicated length of time and lysed. Whole cell extracts were analyzed by western immunoblotting with the indicated antibodies. Anti-Smad3 pT179 cross-reacts with Smad2 pT220, allowing analysis of both phosphorylated species. (D) HaCaT cells stably expressing wild-type Flag-Smad3 were treated with TGFβ in the presence of SB431542, flavopiridol, or U0126. Whole cell extracts and Flag immunoprecipitates were immunoblotted with the indicated antibodies. (E) HaCaT cells stably expressing Flag-tagged Smad3, WT or mutant form, were treated with TGFβ or EGF. Whole cell extracts and Flag immunoprecipitates were immunoblotted with the indicated antibodies. (F) Summary of the phosphorylation events on Smad3 upon TGFβ and EGF treatments. The thickness of the arrows and the darkness of the colors indicate the strength of the phosphorylation of the specific sites. T_1/2 for each phosphorylation is indicated in minutes.

(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.

(A) HaCaT cells were treated with different stimuli. Whole cell extracts were immunoblotted with the indicated antibodies. (B) HaCaT cells were transfected with small interfering RNA (siRNA) targeting indicated genes and treated with BMP or TGFβ for one hour. The agonists were then removed from the culture medium (zero time point). Whole cell extracts were harvested at indicated time points and immunoblotted with the indicated antibodies. (C) Alignment of the linker regions of the receptor activated Smads (R-Smad) in human (h) and Drosophila (d). The conserved PX(S/T)P MAPK kinase sites, proline-directed kinase sites (S/T)-P, and PPXY (PY) motifs are indicated with boxes of different colors. (D) HeLa-S3 cell extracts were subjected to affinity purification with the indicated bait. Some identified proteins were indicated. Contaminant heat-shock and cytoskeletal proteins (not marked) were also detected. (E) Schematic representation of four most closely related HECT domain E3 ubiquitin ligases. Three functional domains are shown: the N-terminal C2 domain, the protein-binding WW domains that vary in number and position among the proteins, and the C-terminal catalytic HECT domain. The numbers on the right indicate the sequence similarity to the Nedd4L protein when queried with full-length (FL) or WW domains of Nedd4L (NP_001138439) against the UniProtKB/Swiss-Prot database using FASTA program on the EMBL-EBI website. (F) HaCaT cells were treated with TGFβ for 1 h and lysed. The Smad2 immunoprecipitates or whole cell lysates were subjected to western immunoblotting with the indicated antibodies.

(A) Control HaCaT cells, cells expressing shRNA targeting Nedd4L, pretreated with proteasome inhibitor MG132, or pretreated with EGF, were stimulated with TGFβ for 1 h. TGFβ was then removed from the culture medium and cells were lysed at the indicated time points. Whole cell extracts were immunoblotted with the indicated antibodies. (B) The Smad2 pTail immunoblots of (A) were quantified using ImageJ. The intensity of each band was normalized to that of the band at zero time point after TGFβ removal. (C) Quantitative real-time PCR (qRT-PCR) analysis of the indicated TGFβ target genes. HaCaT cells expressing control shRNA or shRNA targeting Nedd4L were treated with TGFβ for 3 h, and total RNA was isolated at indicated time points for qRT-PCR analysis. Data show the mean ± S.D of quadruplicates and are representative of three independent experiments. (D) Mouse embryonic stem cells (mESCs) were infected with lentiviruses expressing control shRNA or two independent shRNAs against mNedd4L. These mESCs were treated with activin A or SB431542 for 3 h. Whole cell extracts were immunoblotted with the indicated antibodies. (E) Wild-type mESCs and cells stably expressing an shRNA targeting mNedd4L were immunostained with Nedd4L antibody. DAPI staining was used to visualize nuclei. (F) Control or mNedd4L knockdown mESCs were allowed to differentiate for four days on collagen IV-coated plates in the absence or presence of activin A. Expression levels of activin direct target gene, mLefty1, were measured by qRT-PCR. The bar graphs showed the fold induction by activin in each cell line. Data show the mean ± S.D of quadruplicates and are representative of two independent experiments. (G) Schematic of mouse embryonic stem cell differentiation under serum-free conditions. Addition of activin A specifically induces differentiation towards the definitive endoderm, anterior mesoderm, and axial mesoderm lineages. (H) As in (F), except that the fold induction of specific lineage markers are depicted in the bar graphs.