Transforming growth factor-β (TGF-β)/activin/nodal signalling play a critical role in many
physiological and pathophysiological processes such as in embryogenesis, adult tissue
homeostasis as well as in disease onset and progression. They signal via heteromeric
complexes of type I and type II receptors, which phosphorylate and activate downstream
signal effectors- Smad2 and Smad3 (Smad2/3). Phosphorylation of the C-terminal SxS motif
of Smad2/3 by the receptors induces them to form oligomeric complexes with Smad4 and
accumulate in the nucleus where they regulate transcription. In addition to the SxS
phosphorylation, Smad2/3 can also be phosphorylated at their linker region. Smad2/3 are
comprised of two highly conserved MH1 and MH2 domains connected by an unstructured
linker that contains several phosphorylatable serine and threonine residues. Specifically, there
are proline-directed linker threonine (LT) and serine (LS) residues that are phosphorylated by
various kinases such as PI3K/mTORC2, MAPKs, Rho/ROCK, GSK3-β, CDKs etc. to regulate
Smad2/3 activity by altering their subcellular localisation, transcription, and protein stability.
LT and LS are closely linked and are perceived to have similar functions in TGF-β signalling.
However, a study in our lab showed that they are differentially regulated, suggesting they may
also have different functions in TGF-β signalling. Therefore, this study aims to dissect the
individual roles of LS and LT in regulating TGF-β signalling by using various cell models such
as human embryonic stem cells (hESCs), mouse embryonic stem cells (mESCs), and PC3
cells for mechanistic and functional studies. LS was found to be phosphorylated by CDK8/9
while LT was not much affected. Inhibition of LS phosphorylation (pLS) delayed the
deactivation of Smad2, resulting in their nuclear accumulation and augmented transcriptional
activity. Conversely, inhibition of pLS in inactivated Smad2 after withdrawing the agonist
resulted in Smad2 proteasomal degradation. These findings reveal two distinct mechanisms
by which pLS can regulate TGF-β signalling, which will greatly impact our understanding of
the effects of non-canonical Smad signalling on canonical TGF-β signalling and also provides
potential insights into ways TGF-β responses could be manipulated for therapeutic benefits.