Charakterystyka fosfoproteomu jądrowego komórek linii T87 Arabidopsis thaliana
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2013-07-02
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Characterization of the nuclear phosphoproteome of Arabidopsis thaliana T87 suspension culture
Abstract
Odwracalna fosforylacja białek jest procesem dynamicznym, któremu w różnym czasie i różną intensywnością podlega 25%-50% białek komórki. Za katalizę tego procesu odpowiadają kinazy (fosforylacja) i fosfatazy (defosforylacja) białkowe. Współczesne metody identyfikacji PTM oparte są głównie na analizach spektrometrii mass (MS, ang. mass spectrometry), dzięki którym możliwe stało się monitorowanie ogólnego poziomu zmian fosforylacji białek w związku ze zmianami stanu fizjologicznego komórki. W niniejszej pracy materiałem wyjściowym analiz fosfoproteomicznych były jądra komórkowe zawiesiny komórkowej linii T87 A. thaliana. Fosforylowane białka identyfikowano z wykorzystaniem technik wzbogacania fosfopeptydów typu MOAC oraz analizie MS/MS. W wyniku analiz jakościowych zidentyfikowano 356 fosfobiałek jądrowych związanych z procesami dojrzewania pre-mRNA (splicing) i pre-rRNA (biosynteza rybosomów). Wśród tych białek, 60 nie było wcześniej opisanych jako ufosforylowane lub zidentyfikowano dla nich nowe miejsca fosforylacji. Wskazano także, 47 białek, które mogą być de- lub fosforylowane w sygnalizacji stresu solnego. Analizy ilościowe poziomu białek jądrowych po dwugodzinnym stresie solnym wykazały obniżenie ekspresji białek jąderkowych związanych z syntezą i dojrzewaniem cząsteczek rRNA. Ponadto, wykorzystując nadekspresję fosfatazy białkowej ABI1 zidentyfikowano białka podjednostki proteasomu 20S, które mogą być defosforylowane przez tą fosfatazę. Wykazano także, po raz pierwszy, że fosfataza ta może podlegać odwracalnej fosforylacji w części końca N białka.
One of better-known PTM is the reversible protein phosphorylation that influences the major metabolic processes and cellular signaling pathways. Phosphorylation is a dynamic process, which may concern 25-50% of cellular proteins. There are two enzymatic groups which are responsible for protein phosphorylation: kinase and phosphatase for phosphorylation and dephosphorylation respectively. The current methods of PTMs identification are mainly based on mass spectrometry (MS) techniques. In this work the main object of phosphoproteomic analysis were nuclei of the T87 cell suspension culture of Arabidopsis thaliana. The phosphorylated protein were identified using MOAC phosphopeptide enrichment methods and MS analysis. Using qualitative LC-MS/MS analysis there were identified 356 nuclear phosphoproteins mainly related to RNA metabolism processes. Among them 60 proteins have not been previously described as phosphoproteins or there had been identified additional phosphorylation sites for these phosphoproteins. Moreover, 47 phosphoproteins have been indicated as potentially regulated by reversible phosphorylation in salt stress signaling processes. The quantitative, LC-MS analysis of nuclear protein has been showed that the level of nucleolar proteins involved in rRNA maturation processes was reduced after two-hour salt stress treatment. Using overexpression of ABI1 protein phosphatase there have been identified a dephosphorylation of almost all proteins of 20S proteasome subunit. Moreover, for the first time we indicated that ABI1 phosphatase is phosphorylated on N terminal part of sequence.
One of better-known PTM is the reversible protein phosphorylation that influences the major metabolic processes and cellular signaling pathways. Phosphorylation is a dynamic process, which may concern 25-50% of cellular proteins. There are two enzymatic groups which are responsible for protein phosphorylation: kinase and phosphatase for phosphorylation and dephosphorylation respectively. The current methods of PTMs identification are mainly based on mass spectrometry (MS) techniques. In this work the main object of phosphoproteomic analysis were nuclei of the T87 cell suspension culture of Arabidopsis thaliana. The phosphorylated protein were identified using MOAC phosphopeptide enrichment methods and MS analysis. Using qualitative LC-MS/MS analysis there were identified 356 nuclear phosphoproteins mainly related to RNA metabolism processes. Among them 60 proteins have not been previously described as phosphoproteins or there had been identified additional phosphorylation sites for these phosphoproteins. Moreover, 47 phosphoproteins have been indicated as potentially regulated by reversible phosphorylation in salt stress signaling processes. The quantitative, LC-MS analysis of nuclear protein has been showed that the level of nucleolar proteins involved in rRNA maturation processes was reduced after two-hour salt stress treatment. Using overexpression of ABI1 protein phosphatase there have been identified a dephosphorylation of almost all proteins of 20S proteasome subunit. Moreover, for the first time we indicated that ABI1 phosphatase is phosphorylated on N terminal part of sequence.
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Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii
Zakład Biotechnologii
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Keywords
Fosforylacja białek, Protein phosphorylation, Stres solny, Salt stress, Fosfataza białkowa ABI1, Protein phosphatase ABI1