Biomedical subjects

Lukas Bürkle

Publications and source records attributed to Lukas Bürkle.

7 recordsLinked to original sources

PubMed2006

Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana.

The signal of the plant hormone cytokinin is perceived by membrane-located sensor histidine kinases and transduced by other members of the plant two-component system. In Arabidopsis thaliana, 28 two-component system proteins (phosphotransmitters and response regulators) act downstream of three receptors, transmitting the signal from the membrane to the nucleus and modulating the cellular response. Although the principal signaling mechanism has been elucidated, redundancy in the system has made it difficult to understand which of the many components interact to control the downstream biological processes. Here, we present a large-scale interaction study comprising most members of the Arabidopsis cytokinin signaling pathway. Using the yeast two-hybrid system, we detected 42 new interactions, of which more than 90% were confirmed by in vitro coaffinity purification. There are distinct patterns of interaction between protein families, but only a few interactions between proteins of the same family. An interaction map of this signaling pathway shows the Arabidopsis histidine phosphotransfer proteins as hubs, which interact with members from all other protein families, mostly in a redundant fashion. Domain-mapping experiments revealed the interaction domains of the proteins of this pathway. Analyses of Arabidopsis histidine phosphotransfer protein 5 mutant proteins showed that the presence of the canonical phospho-accepting histidine residue is not required for the interactions. Interaction of A-type response regulators with Arabidopsis histidine phosphotransfer proteins but not with B-type response regulators suggests that their known activity in feedback regulation may be realized by interfering at the level of Arabidopsis histidine phosphotransfer protein-mediated signaling. This study contributes to our understanding of the protein interactions of the cytokinin-signaling system and provides a framework for further functional studies in planta.

Arabidopsis↗

PubMed2005

Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.

Various modifications of the conventional yeast two-hybrid system have played an essential role in confirming or detecting protein-protein interactions among nuclear and cytoplasmic proteins. These approaches have permitted the identification of novel interaction partners, as well as provided hints as to their function. However, membrane proteins, such as receptor tyrosine kinases, G protein-coupled receptors, membrane-bound phosphatases, and transporters, which represent important classes of signaling molecules, are difficult to study using classical protein interaction assays because of their hydrophobic nature. Here, we describe a genetic system that allows the identification of integral membrane-interacting proteins. This so-called "split-ubiquitin membrane-based yeast two-hybrid assay" involves fusing the halves of ubiquitin to two interacting proteins, at least one of which is membrane bound. Upon interaction of these two proteins, the halves of ubiquitin are brought together, and the transcription factor that is fused to a membrane protein of interest is cleaved and released. The free transcription factor then enters the nucleus and activates transcription of reporter genes. We also describe how this technology is used to screen complementary DNA libraries to identify novel binding partners of a membrane protein of interest.

Amino Acid Substitution↗

PubMed2005

In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system.

In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between different vectors has not yet been studied in great detail. Here we describe the construction of four cDNA libraries from different tissues of Arabidopsis thaliana, the shuttling of the libraries into expression vectors, and evaluation of this method as well as its suitability for downstream applications. Libraries were constructed from seedlings, hormone treated seedlings, flowers, developing seeds and primary leaves in the "entry vector" of the Gateway cloning system. After initial characterization of the libraries, they were shuttled into an expression vector (a yeast two-hybrid prey vector). To monitor for a size bias generally assumed to be inherent to in vitro recombination methods, the libraries were characterized before and after the transfer into the expression vector. However no significant difference could be detected. The functionality of the in vitro recombination system for the shuttling of entire libraries was then further tested by protein-protein interaction screens. The results of the library characterization and of the yeast two-hybrid screens and their implications for large-scale proteomic approaches are discussed.

Arabidopsis↗

PubMed2005

Immediate-early and delayed cytokinin response genes of Arabidopsis thaliana identified by genome-wide expression profiling reveal novel cytokinin-sensitive processes and suggest cytokinin action through transcriptional cascades.

Cytokinins are hormones that regulate many developmental and physiological processes in plants. Recent work has revealed that the cytokinin signal is transduced by two-component systems to the nucleus where target genes are activated. Most of the rapid transcriptional responses are unknown. We measured immediate-early and delayed cytokinin responses through genome-wide expression profiling with the Affymetrix ATH1 full genome array (Affymetrix Inc., Santa Clara, CA, USA). Fifteen minutes after cytokinin treatment of 5-day-old Arabidopsis seedlings, 71 genes were upregulated and 11 genes were downregulated. Immediate-early cytokinin response genes include a high portion of transcriptional regulators, among them six transcription factors that had previously not been linked to cytokinin. Five plastid transcripts were rapidly regulated as well, indicating a rapid transfer of the signal to plastids or direct perception of the cytokinin signal by plastids. After 2 h of cytokinin treatment genes coding for transcriptional regulators, signaling proteins, developmental and hormonal regulators, primary and secondary metabolism, energy generation and stress reactions were over-represented. A significant number of the responding genes are known to regulate light (PHYA, PSK1, CIP8, PAT1, APRR), auxin (Aux/IAA), ethylene (ETR2, EIN3, ERFs/EREBPs), gibberellin (GAI, RGA1, GA20 oxidase), nitrate (NTR2, NIA) and sugar (STP1, SUS1) dependent processes, indicating intense crosstalk with environmental cues, other hormones and metabolites. Analysis of cytokinin-deficient 35S:AtCKX1 transgenic seedlings has revealed additional, long-lasting cytokinin-sensitive changes of transcript abundance. Comparative overlay-analysis with the software tool mapman identified previously unknown cytokinin-sensitive metabolic genes, for example in the metabolism of trehalose-6-phosphate. Taken together, we present a genome-wide view of changes in cytokinin-responsive transcript abundance of genes that might be functionally relevant for the many biological processes that are governed by cytokinins.

Arabidopsis↗

PubMed2004

Identification of barley CK2alpha targets by using the protein microarray technology.

We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha. As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E. coli expression vector. By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters. Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones. All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases. Based on these results we selected 768 clones expressing different barley proteins for protein purification. The purified proteins were robotically arrayed onto FAST slides. The generated protein microarrays were incubated with an expression library-derived barley CK2alpha in the presence of [gamma-33P]ATP, and signals were detected by X-ray film or phosphor imager. We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2alpha as high mobility group proteins and calreticulin.

Casein Kinase II↗

PubMed2003

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis.

Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.

Adenine↗

PubMed2002

Large-scale plant proteomics.

Large-scale and high throughput approaches increasingly play an essential role in the study of biological systems, which are per se highly complex. Therefore, they need to be examined by these extensive methods to receive information about the large genomic and proteomic networks. In plant biology, this purpose has a strong support through the accessability of the complete genome sequence of the model plant Arabidopsis thaliana. This brief review intends to focus on the basics and the state-of-the-art of these high-throughput technologies and their application to plant proteomics. It describes protein microarrays, the use of antibodies, 2-DE and MS methods and the yeast two hybrid system, which are emerging as the major technologies for plant proteomics.

Arabidopsis↗