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High resolution imaging of ERK pathway protein distribution for the analysis of the dynamics of compartmentalisation of signalling processes

Our experiments have involved the detection of hERK1 (human ERK1) localization, cytoplasmic-nuclear translocation, and dimerization in live cells using high-resolution bio-imaging methods. Specific fluorescent tagging of hERK1 in vivo has been achieved through the use of eGFP (enhanced green fluorescent protein ) fusion proteins. This eGFP-hERK1 has been shown to respond to stimulation in the same way as wild type hERK1, indicating that the presence of the GFP tag does not inhibit its function. A fusion protein with photoactivatable GFP (paGFP) has also been generated. EGFP-hERK14, a mutant of hERK1 in which the four amino acids necessary for homodimer formation are deleted, has also been characterized. The creation of these constructs and their successful transfection into ERK1-knock-out cells (provided by WP3) allowed us to study the real-time dynamics of ERK in the native environment of the cell; these investigations are still in progress. We have monitored real-time detection of eGFP-hERK1 translocation to the nucleus upon activation and compared this with the quiescent state, the kinetics of EGFP-hERK14 and the effects of nuclear export inhibitors (leptomycin B). Detection and characterization of translocation to and from the nucleus is essential for interpreting the role of ERK in signal transduction.

We determined hERK1 translocation rates under different conditions using various FRAP/FLIP protocols in which the cytoplasm, nucleus or parts of the nucleus were bleached. We determined that the GFP-hERK1 was in continuous exchange between cytoplasm and nucleus and mobile within the nucleus. Dimerization of ERK has been proposed to be a requirement for nuclear translocation. However, GFP-hERK1Δ4 also accumulated in the nucleus upon stimulation with an apparent rate similar to wild type. This result was consistent with parallel real-time homoFRET measurements (a technique which detects interactions between like fluorophores) indicating that the wild type GFP-hERK1 does not dimerize before moving to the nucleus. Comparison of the rates determined with those predicted by different models is currently underway. Studies to detect interactions with other signalling cascade partners can now be designed using FRET with eGFP-ERK1 as a donor or acceptor.
In addition, new derivatives of the bisarsenylated FlAsH probe have been developed in order to facilitate biochemical identification and fractionation of the intracellular partners of ERK1. The generation of a hERK1 construct containing a new and improved FlAsH tag (a hexapeptide motif including 4 cysteines) at the C-terminus has shown positive labeling results with a reduction in the background signal, which was a problem present earlier in the project.

Deliverable achieved:
2.2 Spatio-temporal description of (lack of) homo-association of ERK1

Milestones achieved:
M14: Constuction and expression of GFP-ERK1.


Tom JOVIN, ( )
Tel.: +49-551-2011381
Fax: +49-551-2011467
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