Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogenactivated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.
Microarray technology plays an increasing role in proteomic research. We give an overview about recent developments in this technology focusing on molecular interaction studies using protein and antibody microarrays. We report about technical aspects in the development of protein microarrays and describe different surfaces and detection modes. Furthermore, we review the applications of protein microarrays in different molecular interaction studies including interactions of proteins with antibodies, proteins, DNA, small molecules and enzymes. Advantages and limitations of the microarray-based methods with other in vitro methods have been compared. We present the increasing applications of protein and antibody microarrays in basic research, diagnostics, drug discovery, and in vitro-risk assessment of nutrients.
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Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.
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