Quantitative Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Invasion Motor Complex
Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.
Vyšlo v časopise:
Quantitative Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Invasion Motor Complex. PLoS Pathog 7(9): e32767. doi:10.1371/journal.ppat.1002222
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002222
Souhrn
Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.
Zdroje
1. TenterAMHeckerothARWeissLM 2000 Toxoplasma gondii: from animals to humans. Int J Parasitol 30 1217 1258
2. de CarvalhoKMMinguiniNMoreira FilhoDCKara-JoseN 1998 Characteristics of a pediatric low-vision population. J Pediatr Ophthalmol Strabismus 35 162 165
3. MeissnerMSchluterDSoldatiD 2002 Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298 837 840
4. Herm-GotzAWeissSStratmannRFujita-BeckerSRuffC 2002 Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. EMBO J 21 2149 2158
5. GaskinsEGilkSDeVoreNMannTWardG 2004 Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J Cell Biol 165 383 393
6. JohnsonTMRajfurZJacobsonKBeckersCJ 2007 Immobilization of the type XIV myosin complex in Toxoplasma gondii. Mol Biol Cell 18 3039 3046
7. FrenalKPolonaisVMarqJBStratmannRLimenitakisJ 2010 Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8 343 357
8. BullenHETonkinCJO'DonnellRAThamWHPapenfussAT 2009 A novel family of apicomplexan glideosome associated proteins with an inner-membrane anchoring role. J Biol Chem 284 25353 25363
9. BaumJPapenfussATBaumBSpeedTPCowmanAF 2006 Regulation of apicomplexan actin-based motility. Nat Rev Microbiol 4 621 628
10. CarruthersVBoothroydJC 2007 Pulling together: an integrated model of Toxoplasma cell invasion. Curr Opin Microbiol 10 83 89
11. CarruthersVBSibleyLD 1999 Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 31 421 428
12. BoothroydJCDubremetzJF 2008 Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat Rev Microbiol 6 79 88
13. MoudyRManningTJBeckersCJ 2001 The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii. J Biol Chem 276 41492 41501
14. LovettJLSibleyLD 2003 Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells. J Cell Sci 116 3009 3016
15. SinghSAlamMMPal-BhowmickIBrzostowskiJAChitnisCE 2010 Distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites. PLoS Pathog 6 e1000746
16. LovettJLMarchesiniNMorenoSNSibleyLD 2002 Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores. J Biol Chem 277 25870 25876
17. WetzelDMChenLARuizFAMorenoSNSibleyLD 2004 Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii. J Cell Sci 117 5739 5748
18. BillkerOLouridoSSibleyLD 2009 Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5 612 622
19. LouridoSShumanJZhangCShokatKMHuiR 2010 Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465 359 362
20. OjoKKLarsonETKeylounKRCastanedaLJDerocherAE 2010 Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors. Nat Struct Mol Biol 17 602 607
21. KatoNSakataTBretonGLe RochKGNagleA 2008 Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. Nat Chem Biol 4 347 356
22. GreenJLRees-ChannerRRHowellSAMartinSRKnuepferE 2008 The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. J Biol Chem 283 30980 30989
23. DelormeVCaylaXFaureGGarciaATardieuxI 2003 Actin dynamics is controlled by a casein kinase II and phosphatase 2C interplay on Toxoplasma gondii Toxofilin. Mol Biol Cell 14 1900 1912
24. ThingholmTEJensenON 2009 Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol Biol 527: 47-56, xi
25. CoxJMannM 2008 MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26 1367 1372
26. LarsenMRThingholmTEJensenONRoepstorffPJorgensenTJ 2005 Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4 873 886
27. DosztanyiZCsizmokVTompaPSimonI 2005 IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21 3433 3434
28. CollinsMOYuLCampuzanoIGrantSGChoudharyJS 2008 Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder. Mol Cell Proteomics 7 1331 1348
29. DunkerAKUverskyVN 2008 Signal transduction via unstructured protein conduits. Nat Chem Biol 4 229 230
30. GilkSDGaskinsEWardGEBeckersCJ 2009 GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex. Eukaryot Cell 8 190 196
31. WinterDKugelstadtDSeidlerJKappesBLehmannWD 2009 Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry. Anal Biochem 393 41 47
32. HeaslipATLeungJMCareyKLCattiFWarshawDM 2010 A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity. PLoS Pathog 6 e1000720
33. BoschJTurleySDalyTMBoghSMVillasmilML 2006 Structure of the MTIP-MyoA complex, a key component of the malaria parasite invasion motor. Proc Natl Acad Sci U S A 103 4852 4857
34. DebreczeniJEFarkasLHarmatVHetenyiCHajduI 2005 Structural evidence for non-canonical binding of Ca2+ to a canonical EF-hand of a conventional myosin. J Biol Chem 280 41458 41464
35. PolonaisVFothBJChinthalapudiKMarqJBMansteinDJ 2011 Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii. Traffic 12 287 300
36. HimmelDMMuiSO'Neall-HennesseyESzent-GyorgyiAGCohenC 2009 The on-off switch in regulated myosins: different triggers but related mechanisms. J Mol Biol 394 496 505
37. DonaldRGAlloccoJSinghSBNareBSaloweSP 2002 Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase. Eukaryot Cell 1 317 328
38. DonaldRGZhongTMeijerLLiberatorPA 2005 Characterization of two T. gondii CK1 isoforms. Mol Biochem Parasitol 141 15 27
39. HuynhMHCarruthersVB 2009 Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8 530 539
40. GouldSBKraftLGvan DoorenGGGoodmanCDFordKL 2011 Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates. Mol Biol Evol 28 1319 1331
41. KimKSoldatiDBoothroydJC 1993 Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262 911 914
42. StriepenBHeCYMatrajtMSoldatiDRoosDS 1998 Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. Mol Biochem Parasitol 92 325 338
43. DonaldRGCarterDUllmanBRoosDS 1996 Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J Biol Chem 271 14010 14019
44. McNultyDEAnnanRS 2009 Hydrophilic interaction chromatography for fractionation and enrichment of the phosphoproteome. Methods Mol Biol 527: 93-105, x
45. ThingholmTELarsenMR 2009 The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests. Methods Mol Biol 52757 66, xi
46. WashburnMPWoltersDYatesJR3rd 2001 Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19 242 247
47. EngJKMcCormackALYatesJRIII 1994 An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5 976 989
48. CociorvaDDLTYatesJR 2007 Validation of tandem mass spectrometry database search results using DTASelect. Curr Protoc Bioinformatics Chapter 13 Unit 13 14
49. EliasJEGygiSP 2007 Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4 207 214
50. LuBRuseCXuTParkSKYatesJ3rd 2007 Automatic validation of phosphopeptide identifications from tandem mass spectra. Anal Chem 79 1301 1310
51. CoxJMaticIHilgerMNagarajNSelbachM 2009 A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4 698 705
52. TordaAEProcterJBHuberT 2004 Wurst: a protein threading server with a structural scoring function, sequence profiles and optimized substitution matrices. Nucleic Acids Res 32 W532 535
53. FiserASaliA 2003 Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol 374 461 491
54. WolfEKimPSBergerB 1997 MultiCoil: a program for predicting two- and three-stranded coiled coils. Protein Sci 6 1179 1189
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2011 Číslo 9
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Hostile Takeover by : Reorganization of Parasite and Host Cell Membranes during Liver Stage Egress
- HTLV-1 Propels Thymic Human T Cell Development in “Human Immune System” Rag2 gamma c Mice
- Exploiting and Subverting Tor Signaling in the Pathogenesis of Fungi, Parasites, and Viruses
- A Viral Ubiquitin Ligase Has Substrate Preferential SUMO Targeted Ubiquitin Ligase Activity that Counteracts Intrinsic Antiviral Defence