Abstract
Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.
Highlights
Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA and is implicated in cell proliferation and pluripotency control
Lin28a Is Phosphorylated at Ser-200 —To investigate possible post-translational modification of Lin28, we performed transfection-based immunoprecipitation-mass spectrometry analysis and recovered only one phosphorylation of Lin28a at Ser-200 (Fig. 1A and supplemental Fig. S1A), which is highly conserved through evolution
Ser-200 phosphorylation of endogenous and exogenous Lin28a expressed in P19 cells was sensitive to mitogen-activated protein kinase (MAPK) inhibitors including AZD6244 and U0126 (Fig. 2, A, C, and E), whereas co-expression of the constitutively active (CA) form of MEK1 or ERK1 kinase dramatically increased the Ser-200 phosphorylation of both endogenous and exogenous Lin28a in P19 cells (Fig. 2, D and F) and HeLa cells
Summary
Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation*□S. Our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process. Lin28a (cell lineage abnormal 28), an RNA-binding protein highly conserved across eukaryotes from Caenorhabditis elegans to mammals, is an important regulator involved in various physiological processes including cell proliferation, differentiation, and organism development, as well as metabolism. We have identified another regulatory mode of Lin28a-let-7 axis by MAPK signaling and shed new light on how stem cells are regulated between the choices of self-renewal and differentiation
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