Visualizing CREB family transcription factor activation in living cells

Many transcription factors integrate a variety of cellular stimuli
to produce a transcriptional response. There is increasing evidence
that the timing and kinetics of activation are crucial in
determining specificity and strength of gene expression, however so
far only few tools are available to address these questions in live
cells and these have severe drawbacks, like very low signal
strength, complicated handling, irreversibility and lack of good
targeting properties.The Ca 2+- and cyclic adenosine monophosphat
responsive element-binding protein (CREB) and the related ATF-1 and
CREM are stimulus inducible transcription factors that link certain
forms of cellular activity to changes in gene expression and are
involved in differentiation, cancer, survival and neuronal
plasticity. Using fluorescence resonance energy transfer (FRET) we
here develop genetically encoded indicators that enable imaging
activation of CREB family transcription factors due to
phosphorylation of the critical serine 133 and subsequent
recruitment of a coactivator in single live cells. The indicator
for CREB activation due to phosphorylation (ICAP) consitsts of the
kinase inducible domain (KID) of CREB fused together with the KIX
of CREB binding protein (CBP) via a flexible linker, sandwiched
between a cyan and a yellow variant of the green fluorescent
protein. The specificity and reliability of ICAP as a measure for
CREB activation was demonstrated first in the cuvette and then in
the nucleus and mitochondria of HeLa cells. After that, we analyzed
the properties of ICAP in primary hippocampal neurons, where we
characterize different signaling pathways with distinct kinetics
that lead to CREB activation. Furthermore, combining the imaging of
CREB activation with calcium imaging we see a summation of CREB
activation in neurons that can be achieved by appropriately timed
depolarizing stimuli and occurs even when individual stimulations
are separated by hours. Finally, sensors for the activation of
ATF-1, CREM and the recruitment of P300, were introduced and
preliminarily characterized. On the whole, these array of
biosensors complement the toolbox for the investigation of the
activation of the CREB family of transcription factors in living
cells and organisms.