Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells

Abstract

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

“…MS and MS-MS Analysis-In-gel digests were purified with homemade microcolumns packed with Poros reversed-phase 20 R2 resin (Applied Biosystems) using GELoader tips (Eppendorf) as described previously (13,14). The Poros 20 R2 column was washed with 20 l of 0.1% TFA, and the bound peptides were eluted directly onto the MALDI target using 0.5 l of a matrix solution consisting of ␣-cyano-4-hydroxycinnamic acid (5 mg/ml) in 50% acetonitrile, 0.1% TFA.…”

The Evolutionary Conserved Oil Body Associated Protein OBAP1 Participates in the Regulation of Oil Body Size

“…Maize OBAP1 (LOC 100193701) and Arabidopsis OBAP1a (At1g05510) proteins appeared in proteomic analysis of maize embryos and Arabidopsis seeds, and in both cases, their concentration increased during artificial ageing (Rajjou et al, 2008;Xin et al, 2011). Arabidopsis OBAP1a also appeared in a proteomic analysis focused in the identification of proteins whose Tyr phosphorylation varied significantly in seeds imbibed for 2 d in abscisic acid (Ghelis et al, 2008). These proteomic analysis did not provide any experimental data on the possible subcellular distribution of AtOBAP1a; however, the protein is defined by Ghelis et al (2008) as a "putative lipoprotein."…”

Signal processing by protein tyrosine phosphorylation in plants

“…50 PAO was used to show the implication of PTPs in the ABA-induced expression of RAB16 in barley aleurone cells 51 and of RAB18 expression in A. thaliana suspension cells. 33 The same inhibitor allowed to implicate PTPs in ABAactivated stomatal closure in Arabidopsis. 33,50,52 A PTP activity inhibited by dephostatin has been detected in soybean.…”

“…33 The same inhibitor allowed to implicate PTPs in ABAactivated stomatal closure in Arabidopsis. 33,50,52 A PTP activity inhibited by dephostatin has been detected in soybean. 53 Using sodium ortho-vanadate, it has been shown that a PTP is implicated in the copper-dependent transduction pathway leading to cell death 34 and in the dynamics and organization of microtubules.…”

“…23 Immunoblotting with anti-phospho-Tyr antibodies have permitted detection of plant proteins phosphorylated on Tyr in several other species. 17,19,[24][25][26][27][28][29][30][31][32][33][34][35][36][37] Involvement of PTKs and PTPs by pharmacological approaches. One way to probe specifically elements of signal transduction pathways is by perturbating these pathways through the loss of function of one of these elements.…”