Renilla luciferase-yellow fluorescence protein chimerae as indicators for real-time monitoring of camp and camp-induced phosphorylation in living cells based on bioluminescence resonance energy transfer (BRET)

Ziel

Intracellular monitoring offers the great advantage of imaging and studying signalling pathways in real time. Two genetically encoded indicators for camp and camp phosphorylation will be developed based on bioluminescence energy transfer (BRET). A) A number of chimeras will be prepared by fusion of a donor protein, Refill Lucifer's (Relic) and an acceptor protein, the Yellow Fluorescence Protein (EYFP) to either end of protein kinas A (PKA) catalytic and regulatory subunits. The camp will disrupt the energy transfer by dissociating the subunits. The chimera with the best BRET characteristics will be used for transfixion and in vivo camp monitoring. B) A second sensor will be composed by the Relic and EYFP joined by the kinas inducible domain (KID) which contains a phosphorylation site for PKA. We expect that the phosphorylation-dependent conformant ional change of KID will affect the BRET between Relic and EYFP. These BRET changes will be studied in vitro and in vivo and will permit an intracellular view of phosphorylation dynamics in real time. The proposed indicators may find applications in intracellular screening for PKA inhibitors and as leader molecules for the des gin of chimerical indicators containing phosphate acceptor sites for other kinesis. The above BRET sensors are expected to offer several advantages and wider cellular applications over the Fluorescence Energy transfer (FRET) sensors reported so far since they do not require excitation illumination.

Wissenschaftliches Gebiet

Programm/Programme

FP5-LIFE QUALITY - Specific Programme for research, technological development and demonstration on "Quality of life and management of living resources", 1998-2002

Thema/Themen

1.1.1.-3. - Key action The "Cell factory"

Aufforderung zur Vorschlagseinreichung

Data not available

Finanzierungsplan

RGI - Research grants (individual fellowships)

Koordinator

University of Patras

EU-Beitrag

Keine Daten

Adresse

Karatheodori
26500 PATRAS
Griechenland

Gesamtkosten