Divergent effects of intrinsically active MEK variants on developmental Ras signaling

Germline mutations in Ras pathway components are associated with a large class of human developmental abnormalities, known as RASopathies, that are characterized by a range of structural and functional phenotypes, including cardiac defects and neurocognitive delays. Although it is generally believed that RASopathies are caused by altered levels of pathway activation, the signaling changes in developing tissues remain largely unknown. We used assays with spatiotemporal resolution in Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish) to quantify signaling changes caused by mutations in MAP2K1 (encoding MEK), a core component of the Ras pathway that is mutated in both RASopathies and cancers in humans. Surprisingly, we discovered that intrinsically active MEK variants can both increase and reduce the levels of pathway activation in vivo. The sign of the effect depends on cellular context, implying that some of the emerging phenotypes in RASopathies may be caused by increased, as well as attenuated, levels of Ras signaling.

[1]  H. Spaink,et al.  Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation. , 2008, Developmental biology.

[2]  E. Wieschaus,et al.  Regulated Expression of nullo Is Required for the Formation of Distinct Apical and Basal Adherens Junctions in the Drosophila Blastoderm , 2000, The Journal of cell biology.

[3]  Pablo Rodriguez-Viciana,et al.  Germline Mutations in Genes Within the MAPK Pathway Cause Cardio-facio-cutaneous Syndrome , 2006, Science.

[4]  S. Thiberge,et al.  Imaging cilia in zebrafish. , 2010, Methods in cell biology.

[5]  Eytan Domany,et al.  A module of negative feedback regulators defines growth factor signaling , 2007, Nature Genetics.

[6]  C. Nüsslein-Volhard,et al.  Function of torso in determining the terminal anlagen of the Drosophila embryo , 1988, Nature.

[7]  Jonathan A. Cooper,et al.  Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. , 1992, The Journal of biological chemistry.

[8]  R. Maeda,et al.  An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases , 2007, Proceedings of the National Academy of Sciences.

[9]  E. Zackai,et al.  Truncating mutations in the last exon of NOTCH3 cause lateral meningocele syndrome , 2015, American journal of medical genetics. Part A.

[10]  Yoosik Kim,et al.  Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo. , 2013, Chaos.

[11]  E. Wieschaus,et al.  Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. , 1986, Developmental biology.

[12]  N. Hacohen,et al.  sprouty Encodes a Novel Antagonist of FGF Signaling that Patterns Apical Branching of the Drosophila Airways , 1998, Cell.

[13]  J. Casanova,et al.  In and out of Torso RTK signalling , 2003, The EMBO journal.

[14]  I. Lax,et al.  The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors. , 2002, Molecular cell.

[15]  George R. Bjorklund,et al.  Layer specific and general requirements for ERK/MAPK signaling in the developing neocortex , 2016, eLife.

[16]  G. Struhl,et al.  Localized surface activity of torso, a receptor tyrosine kinase, specifies terminal body pattern in Drosophila. , 1989, Genes & development.

[17]  R. Lehmann,et al.  A functional antagonism between the pgc germline repressor and torso in the development of somatic cells , 2009, EMBO reports.

[18]  Deborah K. Morrison,et al.  Signaling dynamics of the KSR1 scaffold complex , 2009, Proceedings of the National Academy of Sciences.

[19]  Gareth Baynam,et al.  A germline MTOR mutation in Aboriginal Australian siblings with intellectual disability, dysmorphism, macrocephaly, and small thoraces , 2015, American journal of medical genetics. Part A.

[20]  Michael A. Patton,et al.  Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome , 2001, Nature Genetics.

[21]  N. Perrimon,et al.  A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster. , 1985, Developmental biology.

[22]  T. Schüpbach,et al.  Modulation of gurken translation by insulin and TOR signaling in Drosophila , 2012, Development.

[23]  H. Lipshitz,et al.  Reciprocal effects of hyper- and hypoactivity mutations in the Drosophila pattern gene torso. , 1989, Science.

[24]  N. Perrimon,et al.  Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus. , 1986, Developmental Biology.

[25]  Ming Zhou,et al.  Regulation of Raf-1 by direct feedback phosphorylation. , 2005, Molecular cell.

[26]  J. Olefsky,et al.  Negative Feedback Regulation and Desensitization of Insulin- and Epidermal Growth Factor-stimulated p21ras Activation (*) , 1995, The Journal of Biological Chemistry.

[27]  M. Muda,et al.  Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. , 1998, Science.

[28]  B. Shilo,et al.  MAP kinase in situ activation atlas during Drosophila embryogenesis. , 1997, Development.

[29]  Ioannis G. Kevrekidis,et al.  Dynamics of Inductive ERK Signaling in the Drosophila Embryo , 2015, Current Biology.

[30]  J. Casanova,et al.  Spatially distinct downregulation of Capicua repression and tailless activation by the Torso RTK pathway in the Drosophila embryo , 2006, Mechanisms of Development.

[31]  F. Beuschlein,et al.  PKA catalytic subunit mutations in adrenocortical Cushing’s adenoma impair association with the regulatory subunit , 2014, Nature Communications.

[32]  A. Brunet,et al.  Growth factor‐stimulated MAP kinase induces rapid retrophosphorylation and inhibition of MAP kinase kinase (MEK1) , 1994, FEBS letters.

[33]  Muffy Calder,et al.  The Mammalian MAPK/ERK Pathway Exhibits Properties of a Negative Feedback Amplifier , 2010, Science Signaling.

[34]  L. Gilbert,et al.  The Insect Neuropeptide PTTH Activates Receptor Tyrosine Kinase Torso to Initiate Metamorphosis , 2009, Science.

[35]  R. Davis,et al.  Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669. , 1991, The Journal of biological chemistry.

[36]  S. Shvartsman,et al.  RASopathies: unraveling mechanisms with animal models , 2015, Disease Models & Mechanisms.

[37]  J. den Hertog,et al.  Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish , 2014, Development.

[38]  C. V. Jongeneel,et al.  Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma , 2011, Nature Genetics.

[39]  D. Ornitz,et al.  Analysis of a gain-of-function FGFR2 Crouzon mutation provides evidence of loss of function activity in the etiology of cleft palate , 2010, Proceedings of the National Academy of Sciences.

[40]  H. Yost,et al.  FGF signalling during embryo development regulates cilia length in diverse epithelia , 2010, Nature.

[41]  J. Otlewski,et al.  ERK-Mediated Phosphorylation of Fibroblast Growth Factor Receptor 1 on Ser777 Inhibits Signaling , 2013, Science Signaling.

[42]  D. Morrison,et al.  Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling , 2009, Molecular and Cellular Biology.

[43]  S. Shvartsman,et al.  Nuclear Trapping Shapes the Terminal Gradient in the Drosophila Embryo , 2008, Current Biology.

[44]  J. Fagin,et al.  Endogenous expression of HrasG12V induces developmental defects and neoplasms with copy number imbalances of the oncogene , 2009, Proceedings of the National Academy of Sciences.