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Systems Biochemistry of Signal Transduction

     Group Leader: Gary Davidson

     tel.: +49 721 608 26103
     fax: +49 721 608 23354
     email: gary davidsonPsc5∂kit edu

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Outline of research

A huge number of protein components that function within signal pathways have been identified, and epistasis analysis has ordered most of them with respect to where they function in pathways. In contrast, information on the biochemical regulation of these components that ensues upon pathway activation is relatively incomplete. This lack of knowledge limits our understanding of how signaling is regulated.

All cellular signaling depends on interconnected signaling proteins that relay information along molecular pathways by means of sequential protein modification. For example, once a cell surface receptor is activated a "chain-reaction" of protein modifications is initiated that propagates a flow of information, the exact directionality and specificity of which is dictated by the repertoire of signaling components, as well as their potential modifiers, present.

In order to gain a better understanding of the biochemical regulation of signaling, we focus primarily on post-translational modification (PTM) of signaling pathway components. We use our experience of working in the field of developmental signaling pathways and employ "modification screening" approaches to identify novel modifiers of known pathway components (Davidson et al., 2005; Davidson et. al.,2008).

We use mostly standard protein detection methods as a read-out in our screening platforms, such as SDS-PAGE and western blot (see below for schematic representation of modification screen and equipment used). Our unique position within the Forschungszentrum Karlsruhe (FZK) however allows close collaboration with experts in nanotechnology and microfuidics and this may allow the development of new, high-throughput technologies for PTM screening. The biological relevance of candidate modifiers is then studied using a combination of mammalian and Drosophila cell culture as well as Xenopus embryos.

Schematic example of screening platform to identify novel modifiers

1. cDNA/RNAi libraries

cDNA expression libraries as well as RNAi libraries are used for gain-of- function and loss-of-function screening approaches, respectively. In the diagram above a pooled cDNA library is represented, where each well of the 96-well plate contains 96 independent cDNA expression plasmids pooled and ready for transfection.

2. Transfection of library clones with epitope tagged "modification targets", encoding signaling pathway components of choice.

The signaling pathway "targets" we select for modification screens are generally epitope tagged to allow highly specific and sensitive detection by antibodies (myc and V5 are good tags for this purpose). Several different pathway components are cotransfected with the cDNA library pools, allowing more modification information to be gained per screen and to provide internal controls that allow one to distinguish between specific and non-specific modifications.

3. SDS-PAGE / Western Blot screening

The read-out of choice for a relatively unbiased identification of protein modifications is SDS-PAGE followed by Western blot identification of signaling components using tag specific or, when available, signaling component specific antibodies. This method will identify any modification that alters the migration properties of the proteins in some way as well as the relative levels. Screening is typically performed in 96-well format and specialized pipettes allow single-step loading of 12 samples from one row of a 96-well plate to a mini SDS-PADE gel. The 8 gels from one 96-well plate of cell of cell lysates are run simultaneously and transferred to nitrocellulose. Automated Western blot is then performed using a BioLane HTI instrument (pictured below).

BioLane HTI instrument

One or two 96-well plates are easily screened in one day and ready for WB developing the next morning. Any modification of a particular protein band seen is then noted and re-screened in grid format using pooled rows and columns from the original 96-well plate of individual cDNA clones. The potential modifier is thus identified.

Previous Work

Our previous work on expression based modification screening identified Casein Kinase 1 gamma (CK1g) as a modifier of the Wnt signaling pathway co-receptor, low density lipoprotein receptor related protein 6 (LRP6). We demonstrated that CK1g is a kinase that specifically phosphorylates multiple, conserved residues within the cytoplasmic domain of LRP6 and, in doing so, acts as an essential, positive regulator of Wnt/b-catenin signaling by promoting LRP6-Axin association (Davidson et. al., 2005). A recent and related project using a kinome-wide Drosophila RNAi screen identified another LRP6 kinase (Davidson et. al., 2008).

Future Directions

In order to increase screening throughput we plan to use “dot-blot” analysis of protein lysates from cells after cDNA/RNAi library screening, as outlined above. The use of nitrocellulose coated microscope slides for spotting of lysates (equivalent to protein chip technology) is a method we are currently investigating. To simplify screening complexity we plan to use bioinformatic based approaches to prepare "sub-libraries" in order to screen for specific types of PTM.

Highly motivated PhD and Bachelor students are encouraged to apply for a position in our lab.

 

 

Updated: February, 2015