Abstract
TGFβ signaling via SMAD proteins and protein kinase pathways up- or down-regulates the expression of many genes and thus affects physiological processes, such as differentiation, migration, cell cycle arrest, and apoptosis, during developmental or adult tissue homeostasis. We here report that NUAK family kinase 1 (NUAK1) and NUAK2 are two TGFβ target genes. NUAK1/2 belong to the AMP-activated protein kinase (AMPK) family, whose members control central and protein metabolism, polarity, and overall cellular homeostasis. We found that TGFβ-mediated transcriptional induction of NUAK1 and NUAK2 requires SMAD family members 2, 3, and 4 (SMAD2/3/4) and mitogen-activated protein kinase (MAPK) activities, which provided immediate and early signals for the transient expression of these two kinases. Genomic mapping identified an enhancer element within the first intron of the NUAK2 gene that can recruit SMAD proteins, which, when cloned, could confer induction by TGFβ. Furthermore, NUAK2 formed protein complexes with SMAD3 and the TGFβ type I receptor. Functionally, NUAK1 suppressed and NUAK2 induced TGFβ signaling. This was evident during TGFβ-induced epithelial cytostasis, mesenchymal differentiation, and myofibroblast contractility, in which NUAK1 or NUAK2 silencing enhanced or inhibited these responses, respectively. In conclusion, we have identified a bifurcating loop during TGFβ signaling, whereby transcriptional induction of NUAK1 serves as a negative checkpoint and NUAK2 induction positively contributes to signaling and terminal differentiation responses to TGFβ activity.
Highlights
TGF signaling via SMAD proteins and protein kinase pathways up- or down-regulates the expression of many genes and affects physiological processes, such as differentiation, migration, cell cycle arrest, and apoptosis, during developmental or adult tissue homeostasis
We found that TGF-mediated transcriptional induction of NUAK family kinase 1 (NUAK1) and NUAK2 requires SMAD family members 2, 3, and 4 (SMAD2/3/4) and mitogen-activated protein kinase (MAPK) activities, which provided immediate and early signals for the transient expression of these two kinases
Protein analysis confirmed that NUAK1 and NUAK2 were induced in a time-dependent manner in human fibroblasts (Fig. 1C), mammary cells, and HaCaT and human cervical carcinoma HeLa cells (Fig. S1B)
Summary
TGF, transforming growth factor ; TRI and TRII, TGF type I and II receptor, respectively; WCL, whole-cell lysate(s); MAPK, mitogen-activated protein kinase; SIK, salt-inducible kinase; AMPK, AMP-activated protein kinase; LKB1, liver kinase B1; mTOR, mammalian target of rapamycin; MLC, myosin regulatory light chain; TSS, transcription start site; EMT, epithelial–mesenchymal transition; ␣SMA, ␣-smooth muscle actin; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; qPCR, quantitative PCR; qRT-PCR, quantitative RT-PCR. Transcriptional induction of NUAK1 and NUAK2 by TGF generates signaling loops in a way that NUAK1 inhibits, whereas NUAK2 promotes, biological responses mediated by TGF signaling
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