Phosphorylation of the RNA–Binding Protein HOW by MAPK/ERK Enhances Its Dimerization and Activity
Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA–binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW–mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles.
Vyšlo v časopise:
Phosphorylation of the RNA–Binding Protein HOW by MAPK/ERK Enhances Its Dimerization and Activity. PLoS Genet 8(3): e32767. doi:10.1371/journal.pgen.1002632
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002632
Souhrn
Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA–binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW–mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles.
Zdroje
1. GlisovicTBachorikJLYongJDreyfussG 2008 RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett 582 1977 1986
2. LaskoP 2003 Gene regulation at the RNA layer: RNA binding proteins in intercellular signaling networks. Sci STKE 2003 RE6
3. VernetCArtztK 1997 STAR, a gene family involved in signal transduction and activation of RNA. Trends Genet 13 479 484
4. ParonettoMPMessinaVBianchiEBarchiMVogelG 2009 Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis. J Cell Biol 185 235 249
5. SidmanRLDickieMMAppelSH 1964 Mutant Mice (Quaking and Jimpy) with Deficient Myelination in the Central Nervous System. Science 144 309 311
6. SuzukiKZagorenJC 1977 Quaking mouse: an ultrastructural study of the peripheral nerves. J Neurocytol 6 71 84
7. EbersoleTAChenQJusticeMJArtztK 1996 The quaking gene product necessary in embryogenesis and myelination combines features of RNA binding and signal transduction proteins. Nat Genet 12 260 265
8. LobbardiRLambertGZhaoJGeislerRKimHR 2011 Fine-tuning of Hh signaling by the RNA-binding protein Quaking to control muscle development. Development 138 1783 1794
9. FrancisRBartonMKKimbleJSchedlT 1995 gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics 139 579 606
10. OhnoGHagiwaraMKuroyanagiH 2008 STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo. Genes Dev 22 360 374
11. BaehreckeEH 1997 who encodes a KH RNA binding protein that functions in muscle development. Development 124 1323 1332
12. FyrbergCBeckerJBarthmaierPMahaffeyJFyrbergE 1997 A Drosophila muscle-specific gene related to the mouse quaking locus. Gene 197 315 323
13. LoPCFraschM 1997 A novel KH-domain protein mediates cell adhesion processes in Drosophila. Dev Biol 190 241 256
14. ZaffranSAstierMGratecosDSemerivaM 1997 The held out wings (how) Drosophila gene encodes a putative RNA-binding protein involved in the control of muscular and cardiac activity. Development 124 2087 2098
15. EdenfeldGVolohonskyGKrukkertKNaffinELammelU 2006 The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila. Neuron 52 969 980
16. ReuvenyAElhananyHVolkT 2009 Enhanced sensitivity of midline glial cells to apoptosis is achieved by HOW(L)-dependent repression of Diap1. Mech Dev 126 30 41
17. VolkT 2010 Drosophila star proteins: what can be learned from flies? Adv Exp Med Biol 693 93 105
18. VolohonskyGEdenfeldGKlambtCVolkT 2007 Muscle-dependent maturation of tendon cells is induced by post-transcriptional regulation of stripeA. Development 134 347 356
19. Nabel-RosenHToledano-KatchalskiHVolohonskyGVolkT 2005 Cell divisions in the drosophila embryonic mesoderm are repressed via posttranscriptional regulation of string/cdc25 by HOW. Curr Biol 15 295 302
20. LiuZLuytenIBottomleyMJMessiasACHoungninou-MolangoS 2001 Structural basis for recognition of the intron branch site RNA by splicing factor 1. Science 294 1098 1102
21. MaguireMLGuler-GaneGNietlispachDRaineARZornAM 2005 Solution structure and backbone dynamics of the KH-QUA2 region of the Xenopus STAR/GSG quaking protein. J Mol Biol 348 265 279
22. GarreySMCassDMWandlerAMScanlanMSBerglundJA 2008 Transposition of two amino acids changes a promiscuous RNA binding protein into a sequence-specific RNA binding protein. RNA 14 78 88
23. ChenTRichardS 1998 Structure-function analysis of Qk1: a lethal point mutation in mouse quaking prevents homodimerization. Mol Cell Biol 18 4863 4871
24. BeuckCSzymczynaBRKerkowDECarmelABColumbusL 2010 Structure of the GLD-1 homodimerization domain: insights into STAR protein-mediated translational regulation. Structure 18 377 389
25. MeyerNHTripsianesKVincendeauMMadlTKatebF 2010 Structural basis for homodimerization of the Src-associated during mitosis, 68-kDa protein (Sam68) Qua1 domain. J Biol Chem 285 28893 28901
26. RyderSPFraterLAAbramovitzDLGoodwinEBWilliamsonJR 2004 RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1. Nat Struct Mol Biol 11 20 28
27. SetteC 2010 Post-translational regulation of star proteins and effects on their biological functions. Adv Exp Med Biol 693 54 66
28. WongGMullerOClarkRConroyLMoranMF 1992 Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell 69 551 558
29. ZhangYLuZKuLChenYWangH 2003 Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism. Embo J 22 1801 1810
30. ResnickRJTaylorSJLinQShallowayD 1997 Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis. Oncogene 15 1247 1253
31. MatterNHerrlichPKonigH 2002 Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420 691 695
32. ParonettoMPZalfaFBottiFGeremiaRBagniC 2006 The nuclear RNA-binding protein Sam68 translocates to the cytoplasm and associates with the polysomes in mouse spermatocytes. Mol Biol Cell 17 14 24
33. BurkartCQiuFBrendelSBenesVHaagP 2007 Modular proteins from the Drosophila sallimus (sls) gene and their expression in muscles with different extensibility. J Mol Biol 367 953 969
34. LakeyALabeitSGautelMFergusonCBarlowDP 1993 Kettin, a large modular protein in the Z-disc of insect muscles. EMBO J 12 2863 2871
35. BardwellLThornerJ 1996 A conserved motif at the amino termini of MEKs might mediate high-affinity interaction with the cognate MAPKs. Trends Biochem Sci 21 373 374
36. JacobsDGlossipDXingHMuslinAJKornfeldK 1999 Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev 13 163 175
37. SharrocksADYangSHGalanisA 2000 Docking domains and substrate-specificity determination for MAP kinases. Trends Biochem Sci 25 448 453
38. RebayIRubinGM 1995 Yan functions as a general inhibitor of differentiation and is negatively regulated by activation of the Ras1/MAPK pathway. Cell 81 857 866
39. Nabel-RosenHDorevitchNReuvenyAVolkT 1999 The balance between two isoforms of the Drosophila RNA-binding protein how controls tendon cell differentiation. Mol Cell 4 573 584
40. DunciaJVSantellaJB3rdHigleyCAPittsWJWityakJ 1998 MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorg Med Chem Lett 8 2839 2844
41. CastagnaMTakaiYKaibuchiKSanoKKikkawaU 1982 Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257 7847 7851
42. SchonwasserDCMaraisRMMarshallCJParkerPJ 1998 Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 18 790 798
43. IsraeliDNirRVolkT 2007 Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target. Development 134 2107 2114
44. GabayLSegerRShiloBZ 1997 In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277 1103 1106
45. SchnorrerFSchonbauerCLangerCCDietzlGNovatchkovaM 2010 Systematic genetic analysis of muscle morphogenesis and function in Drosophila. Nature 464 287 291
46. ClarkKABlandJMBeckerleMC 2007 The Drosophila muscle LIM protein, Mlp84B, cooperates with D-titin to maintain muscle structural integrity. J Cell Sci 120 2066 2077
47. VolkT 1992 A new member of the spectrin superfamily may participate in the formation of embryonic muscle attachments in Drosophila. Development 116 721 730
48. BernsteinSIMogamiKDonadyJJEmersonCPJr 1983 Drosophila muscle myosin heavy chain encoded by a single gene in a cluster of muscle mutations. Nature 302 393 397
49. RozekCEDavidsonN 1983 Drosophila has one myosin heavy-chain gene with three developmentally regulated transcripts. Cell 32 23 34
50. LeeMHSchedlT 2010 C. elegans star proteins, GLD-1 and ASD-2, regulate specific RNA targets to control development. Adv Exp Med Biol 693 106 122
51. ArtztKWuJI 2010 STAR trek: An introduction to STAR family proteins and review of quaking (QKI). Adv Exp Med Biol 693 1 24
52. TisserantAKonigH 2008 Signal-regulated Pre-mRNA occupancy by the general splicing factor U2AF. PLoS ONE 3 e1418 doi:10.1371/journal.pone.0001418
53. WangLLRichardSShawAS 1995 P62 association with RNA is regulated by tyrosine phosphorylation. J Biol Chem 270 2010 2013
54. HaasKFWoodruffE3rdBroadieK 2007 Proteasome function is required to maintain muscle cellular architecture. Biol Cell 99 615 626
55. MichelsonAMGisselbrechtSZhouYBaekKHBuffEM 1998 Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm. Dev Genet 22 212 229
56. ShishidoEOnoNKojimaTSaigoK 1997 Requirements of DFR1/Heartless, a mesoderm-specific Drosophila FGF-receptor, for the formation of heart, visceral and somatic muscles, and ensheathing of longitudinal axon tracts in CNS. Development 124 2119 2128
57. ProutMDamaniaZSoongJFristromDFristromJW 1997 Autosomal mutations affecting adhesion between wing surfaces in Drosophila melanogaster. Genetics 146 275 285
58. StronachBESiegristSEBeckerleMC 1996 Two muscle-specific LIM proteins in Drosophila. J Cell Biol 134 1179 1195
59. MillerKGKarrTLKelloggDRMohrIJWalterM 1985 Studies on the cytoplasmic organization of early Drosophila embryos. Cold Spring Harb Symp Quant Biol 50 79 90
60. KimYCoppeyMGrossmanRAjuriaLJimenezG 2010 MAPK substrate competition integrates patterning signals in the Drosophila embryo. Curr Biol 20 446 451
61. TatsumiRHattoriA 1995 Detection of giant myofibrillar proteins connectin and nebulin by electrophoresis in 2% polyacrylamide slab gels strengthened with agarose. Anal Biochem 224 28 31
62. SubramanianAProkopAYamamotoMSugimuraKUemuraT 2003 Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction. Curr Biol 13 1086 1095
63. XueYRenJGaoXJinCWenL 2008 GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Mol Cell Proteomics 7 1598 1608
64. RenJWenLGaoXJinCXueY 2009 DOG 1.0: illustrator of protein domain structures. Cell Res 19 271 273
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2012 Číslo 3
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