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A biosensor for MAPK-dependent Lin28 signaling

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basa...

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Autores principales: Oldach, Laurel M., Gorshkov, Kirill, Mills, William T., Zhang, Jin, Meffert, Mollie K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935066/
https://www.ncbi.nlm.nih.gov/pubmed/29540527
http://dx.doi.org/10.1091/mbc.E17-08-0500
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author Oldach, Laurel M.
Gorshkov, Kirill
Mills, William T.
Zhang, Jin
Meffert, Mollie K.
author_facet Oldach, Laurel M.
Gorshkov, Kirill
Mills, William T.
Zhang, Jin
Meffert, Mollie K.
author_sort Oldach, Laurel M.
collection PubMed
description Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.
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spelling pubmed-59350662018-07-30 A biosensor for MAPK-dependent Lin28 signaling Oldach, Laurel M. Gorshkov, Kirill Mills, William T. Zhang, Jin Meffert, Mollie K. Mol Biol Cell Brief Reports Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation. The American Society for Cell Biology 2018-05-15 /pmc/articles/PMC5935066/ /pubmed/29540527 http://dx.doi.org/10.1091/mbc.E17-08-0500 Text en © 2018 Oldach et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. http://creativecommons.org/licenses/by-nc-sa/3.0/ This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.
spellingShingle Brief Reports
Oldach, Laurel M.
Gorshkov, Kirill
Mills, William T.
Zhang, Jin
Meffert, Mollie K.
A biosensor for MAPK-dependent Lin28 signaling
title A biosensor for MAPK-dependent Lin28 signaling
title_full A biosensor for MAPK-dependent Lin28 signaling
title_fullStr A biosensor for MAPK-dependent Lin28 signaling
title_full_unstemmed A biosensor for MAPK-dependent Lin28 signaling
title_short A biosensor for MAPK-dependent Lin28 signaling
title_sort biosensor for mapk-dependent lin28 signaling
topic Brief Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935066/
https://www.ncbi.nlm.nih.gov/pubmed/29540527
http://dx.doi.org/10.1091/mbc.E17-08-0500
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