Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants.

Slã¡Vka Bekeå¡Ovã¡,Anna Kuchaå™Ovã¡,Peter Illã©S,Petra Vyplelovã¡,George Komis,Jozef Å Amaj,Ivan LuptoväIak,Miroslav OveäKa,Pavel Kå™Enek

doi:10.3389/fpls.2015.00336

Abstract

The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

Highlights

Protein post-translational modifications (PTMs) and reversible protein phosphorylation are versatile switches regulating protein structure, interactions, function, and subcellular localization (Müller et al, 2010; Nishi et al, 2013, 2014; Offringa and Huang, 2013; Smékalová et al, 2014) under conditional signaling (Ovecka et al, 2014) or during developmental processes (Beck et al, 2010; Smékalová et al, 2014)
TM As already mentioned Phos-Tag identification of phosphorylated proteins following SDS-PAGE separation and western blotting has been well-established in Arabidopsis thaliana (Bethke et al, 2009; Beck et al, 2010; Mao et al, 2011; Fujita et al, 2013; Li et al, 2014; Smékalová et al, 2014; Kang et al, 2015) while few applications included dicotyledonous and monocotyledonous crops (Panteris et al, 2010; Taylor et al, 2012; Ban et al, 2013)
M. sativa SIMK A prominent mitogen activated protein kinases (MAPKs) of Medicago sativa is SIMK which can be activated by exposure to hyperosmotic salt or sorbitol solutions or by wounding and oxidative stress (Kiegerl et al, FIGURE 1 | Activation of Medicago sativa SIMK by three different stresses (250 mM NaCl, 15 mM H2O2 and 0.8 M sorbitol, all applied for a total of 30 min). (A1,A2) All stresses induce the accumulation of phosphorylated MAPK species compared to untreated

Summary

Introduction

Protein post-translational modifications (PTMs) and reversible protein phosphorylation are versatile switches regulating protein structure, interactions, function, and subcellular localization (Müller et al, 2010; Nishi et al, 2013, 2014; Offringa and Huang, 2013; Smékalová et al, 2014) under conditional signaling (Ovecka et al, 2014) or during developmental processes (Beck et al, 2010; Smékalová et al, 2014). The inducible phosphorylation of few or even a single protein species might have global consequences on cell physiology, if these proteins—e.g., transcription factors—are involved in central processes such as transactivation of gene expression (Ishihama and Yoshioka, 2012; Meng et al, 2013). Phosphopeptides may be identified by mass spectrometric analyses following appropriate phosphopeptide enrichment strategies (St-Denis and Gingras, 2012) and by subtractive motif- or drug-based approaches, while global phosphoproteomic analyses may provide information on kinasespecific phosphoproteome landscapes (Ruse et al, 2008; Boesger et al, 2012)

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