research in the Parker lab at Caltech focuses on the molecular mechanisms cells
use to respond to physiological stress such as heat shock.
We are interested in understanding how heat for example is transduced
into a massive transcriptional activation response that results in the synthesis
of heat shock proteins. It is
understood that the heat shock proteins serve as a molecular chaperone and allow
the cell to survive the denaturing conditions imposed by the elevated growth
temperatures. It is not understood
how the heat signal is transduced into a transcriptional response.
In Drosophila and mammals the transcription factor
responsible for activating transcription of the heat shock genes is the heat
shock transcription factor (HSF). In
normally growing cells the HSF exists as an inactive monomer that cannot bind
DNA or activate transcription. When
cells are subjected to heat shock the factor is actively transported into the
nucleus trimerizes (giving it high affinity DNA binding properties) and
undergoes further modifications which allow it to activate transcription.
Recent work from the lab has demonstrated that the domain that regulates monomer
to trimer transition also controls nuclear entry.
When this domain is deleted from the protein it spontaneously trimerizes
with out heat shock and thus acquires DNA binding capabilities but cannot enter
the nucleus. During early stages of Drosophila development the heat shock
response cannot be induced. It is reasoned that the adverse effects on cell
cycle and cell growth brought about by Hsp70 induction must outweigh the
beneficial aspects of Hsp70 induction in the early embryo.
Although the Drosophila heat shock transcription factor (dHSF) is
abundant in the early embryo it does not enter the nucleus in response to heat
shock. In older embryos and
in cultured cells the factor is localized within the nucleus in an apparent
trimeric structure that binds DNA with high affinity.
The domain responsible for nuclear localization upon stress resides
between residues 390 and 420 of the dHSF. Using
that domain as bait in a yeast two-hybrid system we now report the
identification and cloning of a nuclear transport protein Drosophila
Biochemical methods demonstrate that the dKap-a3protein
binds tightly to the NLS. Furthermore
the dKap-a3 protein does not associate with NLSs that contain point mutations
which are not transported in vivo. Nuclear
docking studies also demonstrate specific nuclear targeting of the NLS substrate
Previous studies from other laboratories have demonstrated that
early Drosophila embryos are refractory to heat shock as a result of dHSF
nuclear exclusion. We demonstrate
that the early embryo is deficient in dKap-a3 protein until cycle 12.
From cycle 13 onward the transport factor is present and the dHSF is
localized within the nucleus thus allowing the embryo to respond to heat shock.