We describe a reproducible protocol to explore for the first time the phosphoproteome of a seedless plant, the moss Physcomitrella patens. Following tryptic digestion of a total protein extract, phosphorylated peptides were isolated using the combination of C18 reverse-phase chromatography (RP-C18), immobilized Fe(3+) metal affinity chromatography (IMAC), capillary zone electrophoresis (CZE), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. The total protein extracts were first prepared as usually made for plant two-dimensional gel electrophoresis, the tryptic digest was desalted and concentrated by reverse phase chromatography, and from this mixture the phosphorylated peptides were captured by IMAC. Subsequently, the complex phosphopeptide mixture was separated into ten fractions by RP-C18-HPLC and each analyzed by CZE. This permitted the detection of 253 distinct phosphopeptides. These were identified by nano-LC-MS/MS and MALDI-TOF-MS analysis in conjunction with alkaline phosphatase treatment to remove covalently bound phosphate to specifically identify the phosphopeptides. Among others, several kinases and a transcription factor were identified. This protocol will be taken as a basis to unravel early events in plant signal transduction known to occur via rapid phosphorylation/dephosphorylation of proteins.

Leaf peroxisomes are fragile, low-abundance plant cell organelles that are difficult to isolate from one of the few plant species whose nuclear genome has been sequenced. Leaf peroxisomes were enriched at high purity from spinach (Spinacia oleracea) and approximately 100 protein spots identified from 2-dimensional gels by a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and de novo sequencing. In addition to the predominant enzymes involved in photorespiration and detoxification, several minor enzymes were detected, underscoring the high sensitivity of the protein identification. The tryptic peptides of three unknown proteins shared high sequence similarity with Arabidopsis proteins that carry putative peroxisomal targeting signals type 1 or 2 (PTS1/2). The apparent Arabidopsis orthologues are a short-chain alcohol dehydrogenase (SDRa/IBR1, At4g05530, SRL>) and two enoyl-CoA hydratases/isomerases (ECHIa, At4g16210, SKL>; NS/ECHId, At1g60550, RLx(5)HL). The peroxisomal localization of the three proteins was confirmed in vivo by tagging with enhanced yellow fluorescent protein (EYFP), and the targeting signals were identified. The single Arabidopsis isoform of naphthoate synthase (NS) is orthologous to MenB from cyanobacteria, which catalyses an essential reaction in phylloquinone biosynthesis, a pathway previously assumed to be entirely compartmentalized in plastids in higher plants. In an extension of a previous study, the present in vivo targeting data furthermore demonstrate that the enzyme upstream of NS, chloroplastic acyl-CoA activating enzyme isoform 14 (AAE14, SSL>), is dually targeted to both plastids and peroxisomes. This proteomic study, extended by in vivo subcellular localization analyses, indicates a novel function for plant peroxisomes in phylloquinone biosynthesis.

Summary.  Background: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib–IX complex initiates a signaling cascade leading to integrin αIIbβ3 activation, a key process in hemostasis and thrombosis. Interaction of 14‐3‐3ζ with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. Objective: The aim of our study was to determine whether other members of the 14‐3‐3 family bind to GPIb–IX. Results: In this study, western blot analyses showed that platelets also contain the 14‐3‐3β, 14‐3‐3γ, 14‐3‐3ε, 14‐3‐3η and 14‐3‐3θ isoforms, but lack 14‐3‐3σ. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14‐3‐3 isoforms expressed in platelets, including, as previously reported, 14‐3‐3ζ, bind to GPIb–IX. In addition, their interaction was found to critically require the same GPIbα domains (580–590 and 605–610) already identified as essential for 14‐3‐3ζ binding, in agreement with the conservation of the sequence of the I‐helix among these different isoforms. Pull‐down experiments indicated that all six 14‐3‐3 isoforms present in platelets bind to GPIbβ. In contrast, deletion or mutation of the GPIbβ intracytoplasmic tail did not affect the interaction of GPIb–IX with the 14‐3‐3 isoforms, questioning the importance of this domain. Conclusions: Our study suggests that, to inhibit GPIb‐induced integrin αIIbβ3 activation, a more appropriate strategy than inhibiting individual 14‐3‐3 isoforms would be to target the 14‐3‐3‐binding motif on GPIb or, alternatively, the conserved 14‐3‐3 I‐helix.

Objective-The identification of platelet-reactive proteins exclusively present in atherosclerotic plaques could provide interesting targets for effective and safe antithrombotic strategies. In this context, we explored platelet adhesion and activation to tenascin-C (TN-C), a matrix protein preferentially found within atheroma. Methods and Results-We show that platelets efficiently adhere to TN-C under both static and flow conditions.Videomicroscopy revealed a unique behavior under flow, with platelets exhibiting stationary adhesion to TN-C; in contrast, platelets rolled over von Willebrand factor and detached from fibrinogen. Platelet interaction with TN-C was predominantly supported by integrin ␣ 2 ␤ 1 under static conditions, whereas under high shear, it was dependent on both the ␣ 2 ␤ 1 integrin and the glycoprotein Ib-IX complex. Integrin ␣ IIb ␤ 3 appeared to play a secondary role but only at low shear rates. The glycoprotein Ib-IX-dependent interaction was indirect, relying on von Willebrand factor, and increased as a function of wall shear rate. Von Willebrand factor bound directly to TN-C, as shown by ELISA and coimmunoprecipitation, suggesting that it acts as a bridge between TN-C and platelets. The adhesion of platelets to TN-C triggered their activation, as demonstrated by a shape change and increases in intracellular calcium level. Key Words: blood flow Ⅲ platelets Ⅲ receptors Ⅲ thrombosis Ⅲ tenascin-C T he central role of platelets in arterial thrombosis renders them attractive targets for antithrombotic drugs. Clopidogrel and integrin ␣ IIb ␤ 3 blockers, 2 antiplatelet agents widely used in clinical practice, have greatly reduced cardiovascular-associated death; however, because they directly target the hemostatic function of platelets, their use is linked to an increased bleeding risk. The development of more selective antithrombotic drugs causing minimal perturbation of hemostasis could pave the way to new and more effective strategies in the treatment of ischemic events. This task is challenged by the fact that the main cellular and molecular events implicated in thrombosis are also those regulating hemostasis. Nevertheless, differences exist between these 2 conditions, such as the rheological microenvironment and the nature of the vascular material exposed to platelets. Thrombosis occurs in advanced atherosclerotic arteries that present abnormal luminal narrowing or stenosis inducing local disturbance of blood flow. 1 In addition, the marked inflammation and active tissue remodeling found in atheroma profoundly modify the composition of the subendothelium. Atherosclerotic plaques are rich in highly reactive platelet-adhesive materials, such as collagens or lipids, and overexpress extracellular matrix proteins that are essentially absent from the healthy vessel wall. The identification of platelet-adhesive proteins preferentially expressed in diseased vessels could provide novel targets for more selective antithrombotic therapies. Conclusion-ThisIn this context, tenascin-C (TN-C) appears to be a...