Phosphorylation of Ser465 and Ser467 in the C Terminus of Smad2 Mediates Interaction with Smad4 and Is Required for Transforming Growth Factor-β Signaling

Abstract

Members of the Smad family of intracellular signal transducers are essential for transforming growth factor-␤ (TGF-␤) to exert its multifunctional effects. After activation of TGF-␤ receptors, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. Thereafter, these activated Smad complexes translocate to the nucleus, where they may direct transcriptional responses. Here we report that TGF-␤ mediates phosphorylation of Smad2 at two serine residues in the C terminus, i.e. Ser 465 and Ser 467 , which are phosphorylated in an obligate order; phosphorylation of Ser 465 requires that Ser 467 be phosphorylated. Transfection of Smad2 with mutation of Ser 465 and/or Ser 467 to alanine residues into Mv1Lu cells resulted in dominantnegative inhibition of TGF-␤ signaling. These Smad2 mutants were found to stably interact with an activated TGF-␤ receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser 465 and Ser 467 in Smad2 abrogated complex formation of this mutant with Smad4 and blocked the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Moreover, peptides from the C terminus of Smad2 containing phosphorylated Ser 465 and Ser 467 were found to bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides were less effective. Thus, phosphorylated Ser 465 and Ser 467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation.

Smad3 is acetylated by p300/CBP to regulate its transactivation activity

“…Receptor activation results in phosphorylation of the two distal serines in the carboxy-terminal SSXS sequence of Smad3 Souchelnytskyi et al, 1997). Replacement of the three serines with alanines (Smad3(3SA)) renders Smad3 biologically inactive, perhaps owing to an impaired release from the receptor, lack of heteromerization with Smad4 and/or an inability to translocate into the nucleus.…”

“…One class of Smads, the receptor-regulated Smads (R-Smads), are directly phosphorylated at the C-terminal SSXS motif by type I receptors. This induces binding of the R-Smad to the common Smad (Co-Smad) Souchelnytskyi et al, 1997). The R-Smad/Co-Smad complexes are then translocated into the nucleus, where they bind to transcriptional co-activators or corepressors and regulate transcription (Attisano and Wrana, 2000;Miyazono, 2000).…”

Functional consequences of tumorigenic missense mutations in the amino-terminal domain of Smad4

“…They initiate cellular responses by binding to and activating speci®c receptors with intrinsic serine/ threonine kinase activity, whereby downstream e ectors, termed Smad proteins, are activated (Attisano and Wrana, 2000; ten Dijke et al, 2000). Smad2 and Smad3 are receptor-regulator Smads (R-Smads) which transiently interact with the activated TGF-b receptor and become phosphorylated at their extreme C-termini on two serine residues (Abdollah et al, 1997;Souchelnytskyi et al, 1997). Subsequently, activated Smad2 and Smad3 form heteromeric complexes with common-partner Smads (Co-Smads) (Lagna et al, 1996), e.g.…”

Smad8B, a Smad8 splice variant lacking the SSXS site that inhibits Smad8‐mediated signalling

“…Pathway-restricted Smads contain at their carboxyl termini a consensus phosphorylation motif, SSXS, recognized by type I serine/threonine kinase receptors Macias-Silva et al 1996;Kretzschmar et al 1997;Souchelnytskyi et al 1997). This class of Smads interacts transiently with speci®c activated type I receptors, and thereby become phosphorylated following ligand stimulation.…”