Single- and dual-parameter FRET kinase probes based on pleckstrin.

Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe.

Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B.

Endoplasmic reticulum-localized protein-tyrosine phosphatase PTP1B terminates growth factor signal transduction by dephosphorylation of receptor tyrosine kinases (RTKs). But how PTP1B allows for RTK signaling in the cytoplasm is unclear. In order to test whether PTP1B activity is spatially regulated, we developed a method based on Förster resonant energy transfer for imaging enzyme-substrate (ES) intermediates in live cells.

Cellular uptake of PNA--terpyridine conjugates and its enhancement by Zn2+ ions.

Conjugation of usually impermeable peptide nucleic acids (PNA) to the chelator 2,2':6',2' '-terpyridine strongly promotes cellular and nuclear uptake by cultured HeLa cells. Cellular accumulation is further enhanced in the presence of extracellular Zn2+.

A dual parameter FRET probe for measuring PKC and PKA activity in living cells.

Cell function is regulated by complex and often interdependent networks of signaling molecules. To accurately describe these networks, it is important to monitor multiple signals in parallel. To this end, we have developed a genetically encoded, FRET-based probe that independently monitors both protein kinase A (PKA) and protein kinase C (PKC) activity in vivo.

Multiparameter imaging for the analysis of intracellular signaling.

In biological experimentation and especially in drug discovery there is a trend towards more complex test systems. Cell-based assays are replacing conventional binding or enzyme assays more and more. This development is strongly driven by novel fluorescent probes that give insight into cellular processes.

Genetically encoded FRET probe for PKC activity based on pleckstrin.

We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo.