Actin cytoskeleton is a vital cellular structure primarily known for controlling cell integrity, division and expansion. Here we present a proteomic dissection of Arabidopsis roots treated by actin depolymerizing agent latrunculin B. Pharmacological disintegration of the actin cytoskeleton by latrunculin B caused downregulation of several proteins involved in the actin organization and dynamics. Moreover, this approach helped to identify new protein candidates involved in gene transcription, due to the altered abundance of proteins involved in mRNA nuclear export. Finally, latrunculin B negatively affected the abundance of abscisic acid (ABA) responsive proteins. SIGNIFICANCE: This article substantially contributes to the current knowledge about the importance of actin organization and dynamics in proteome remodelling. We employed gel based and gel free proteomic analyses and identified several new protein candidates and protein networks linking actin dynamics to the gene transcription and to the ABA response in Arabidopsis.
- MeSH
- aktiny chemie metabolismus MeSH
- Arabidopsis chemie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- kořeny rostlin chemie MeSH
- kyselina abscisová fyziologie MeSH
- mikrofilamenta účinky léků MeSH
- polymerizace účinky léků MeSH
- proteiny huseníčku analýza chemie metabolismus MeSH
- proteom analýza účinky léků MeSH
- proteomika metody MeSH
- thiazolidiny farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.
- MeSH
- akrylamid chemie MeSH
- Arabidopsis enzymologie růst a vývoj MeSH
- bakteriofág lambda enzymologie MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- fenol chemie MeSH
- fosfatasy metabolismus MeSH
- fosfoproteiny izolace a purifikace metabolismus MeSH
- fosforylace MeSH
- kultivační techniky MeSH
- membrány umělé MeSH
- mitogenem aktivované proteinkinasy izolace a purifikace metabolismus MeSH
- polyvinyly chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.
- MeSH
- aktivace enzymů MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- exprese genu MeSH
- geneticky modifikované rostliny genetika růst a vývoj metabolismus MeSH
- Medicago sativa enzymologie genetika MeSH
- mitogenem aktivované proteinkinasy kinas genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semenáček genetika růst a vývoj metabolismus MeSH
- soli metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
