The phosphorylation of the Smad2/3 linker region by nemo-like kinase regulates TGF-β signaling

Smad2 and Smad3 (Smad2/3) are structurally similar proteins that primarily mediate the transforming growth factor-beta (TGF-beta) signaling responsible for driving cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provide the key mechanism for regulating the TGF-beta signaling pathway, but the details surrounding this phosphorylation remain unclear. Here, using in vitro kinase assay coupled with mass spectrometry, we identified for the first time that nemo-like kinase (NLK) regulates TGF-beta signaling via modulation of Smad2/3 phosphorylation in the linker region. TGF-beta-mediated transcriptional and cellular responses are suppressed by NLK overexpression, whereas NLK depletion exerts opposite effects. Specifically, we discovered that NLK associates with Smad3 and phosphorylates the designated serine residues located in the linker region of Smad2 and Smad3, which inhibits phosphorylation at the C terminus, thereby decreasing the duration of TGF-beta signaling. Overall, this work demonstrates that phosphorylation on the linker region of Smad2/3 by NLK counteracts the canonical phosphorylation in response to TGF-beta signals, thus providing new insight into the mechanisms governing TGF-beta signaling transduction.