We measured the affinity within the four person Nedd4L WW domains for 13 amino acid quick synthetic peptides, containing the T PY motif of Smad2 or Smad3 with either a threonine or even a phosphothreonine residue, Isothermal titration calorimetry examination exposed a high affinity with the WW2 domain for the pT PY motif peptides, This affinity is among the highest reported to date to get a WW PY domain interaction, The affinity of WW2 for your unphosphorylated T PY motif was seven to 15 fold reduce, The Nedd4L WW3 domain also preferentially bound for the phosphorylated T PY motifs, but with decrease affinity than the WW2 domain. The WW1 and WW4 domains bound a lot more weakly and without any preference for that phosphorylated T PY motifs, Interestingly, Smad1 also consist of a conserved T PY motif, Nevertheless, this threonine residue was not phosphorylated in vivo beneath any from the agonist or antagonist stimuli tested, and it had been poorly phosphorylated by CDK89 in vitro, The Smurf1 WW2 domain binds a synthetic peptide of 13 residues such as the T PY motif of Smad1 using a Kd of 32M plus the phosphorylated Smad3 pT PY motif having a Kd of 36M.
These values agree together with the observation that Smurf1 plays a minor part in Smad3 turnover and it calls for contacts with the phosphorylated SerPro cluster for selelck kinase inhibitor targeting Smad1. Applying Smad3 anti phosphopeptide antibodies that specifically understand four individual linker phosphorylation web pages, we observed that TGFB addition induced a quick and pronounced phosphorylation of T179 shortly just after C tail phosphorylation, This was followed by phosphorylation within the SerPro cluster residues S204, S208 and S213, In contrast, EGF addition induced rapid phosphorylation of T208 and T213, followed by phosphorylation of T204 and much less prominently T179, A equivalent preference for phosphorylation of S204 and S208 AR-42 was observed immediately after UV irradiation or NaCl osmotic tension, The anti Smad3 pT179 antibody cross reacts with all the corresponding residue in Smad2, pT220, and this cross response unveiled a quick TGFB induced phosphorylation of this residue likewise, To even further analyze the contribution of these linker web-sites for the Smad3 Nedd4L interaction, we transduced vectors encoding Flag tagged Smad3 into HaCaT cells that were stably depleted of endogenous Smad3 by RNAi mediated knockdown.
The addition of

SB431542 or flavopiridol prevented TGFB induced linker phosphorylation, whereas only SB431542 blocked C tail phosphorylation, U0126 didn’t inhibit these TGFB induced Smad3 phosphorylation events. Flavopiridol and SB431542 at the same time since the linker website mutation abolished the Smad3 Nedd4L interaction, as determined by co immuoprecipitation of Smad3 proteins.