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Cell growth regulation is essential for the survival
of multicellular and unicellular organisms. Anomalies in the pathways
that control cell growth and proliferation lead to cancers tumors
and cell death. 14-3-3 proteins a family of ubiquitously expressed
regulatory molecules have the ability to bind and modulate the function
of signaling proteins involved in pathways regulating cell growth
and proliferation. Because of their binding capabilities 14-3-3
proteins are believed to play roles as scaffolding entities in a
number of regulatory processes including mitogenic signal transduction
apoptotic death and cell cycle control. We sought to better understand
the mechanisms by which 14-3-3 binds to hExo1 in hopes of unraveling
another key component of 14-3-3 regulation. hExo1 is an exonuclease
involved in DNA repair and is known to play a major role in DNA
replication and apoptosis. We implemented the yeast two hybrid system
to monitor the interaction of 7 isoforms of 14-3-3 and 8 mutants
of the protein with hExo1. Results from the yeast two hybrid assay
suggest that hExo1 binds to 14-3-3 in the amphipatic groove in a
similar manner as other 14-3-3 ligands. Different color intensities
were seen in the plate assay which suggests the possibility of isoform
specific interactions. To explore the possibility of this type of
interaction we performed a more quantitative analysis of 14-3-3
and hExo1 interaction by employing a liquid two hybrid screen. 14-3-3
isoforms and their mutants exhibit reproducible binding patterns
with hExo1 when using the yeast two hybrid system but quantitative
liquid screens have not produced precise data linked to the strength
of these interactions. These studies may prove vital in the understanding
of neurodegenerative diseases and cancers.
Signal transduction pathways are key components of cell cycle control
and are the major mechanisms by which cell growth proliferation
and apoptosis are governed. Alterations in these pathways have been
shown to lead to or produce disease states such as cancers and neurodegenerative
diseases. An understanding of these pathways and the factors that
influence them are essential for drug discoveries and novel treatments
for these disease states. Our laboratory focuses on a group of proteins
that have been found to modulate the activity of many regulatory
proteins involved in signaling pathways. 14-3-3 proteins are a family
of ubiquitously expressed proteins whose ligands are involved in
highly regulated cellular processes. 14-3-3 proteins form dimers
consisting of 9 alpha helices. These helices form an amphipathic
groove which has been shown to be responsible for phophorylation
dependent binding of 14-3-3 with its ligands. 14-3-3 has been found
to bind to proteins of major cell growth signaling pathways involved
in cell death (ASK-1 Bad and FKHRL1) cell proliferation (Raf-1 MT)
cell cycle checkpoint (p53 Cdc25) and cell survival (IGFR-1). 14-3-3
modulates the function of proteins via three mechanisms; It may
change the localization (FKHRL-1) prevent the proteins interaction
with other moeties (Bad) or alter enzymatic activity (Cdc25). We
are interested in the interaction of 14-3-3 with hExo1. A screening
of 14-3-3 ligands through various techniques identifies hExo1 as
a potential candidate for 14-3-3 binding and modulation. hExo1 an
exonuclease is known for its involvement in DNA repair and plays
a crucial role in limiting mutations that may lead to cancers and
other anomalies in cell growth and proliferation or apoptosis. We
imployed the yeast two hybrid system to better understand the dynamics
of any interaction between hExo1 and 14-3-3 proteins.
We used the yeast two hybrid system to observe the interaction
between hExo1 and 7 isoforms of 14-3-3 and its mutants. The yeast
two hybrid system is a qualitative analysis of protein-protein interactions.
Our protein of interest 14-3-3 was fused with the activation domain
of GAL4 transcription factor and exo1 was fused to the DNA binding
domain of GAL4. We fused 7 isoforms of 14-3-3 and its mutants (K49E
R56E R5660E R60E L172D L216E L220 V176D) with alterations in the
conserved amphipathic groove of 14-3-3. If interaction occurs E-galactosidase
is activated by the GAL4 transcription factor and cleaves X-GAL
yielding a blue color in the yeast colonies on galactose XGAL plates.
We performed a more quantitative test a liquid two hybrid assay
which works similarly to the two hybrid screen but it can be quantitated
using a spectrophotometer. The substrate is CPRG instead of XGAL.
hExo1 interacts with all 14-3-3 isoforms. 14-3-3 Beta exhibits
the most interaction in the two hybrid screen. Other isoforms produce
varying degrees of interaction. L216 exhibits the most interaction.
R60E and R5660E display interactions similar to positive control.
K49E and R56E exhibit greatly reduced interaction. Quantitative
liquid assay does not produce precise results.
hExo1 interacts with all 14-3-3 isoforms. Assays of 14-3-3 isoforms
with hExo1 display varied interaction suggesting that hExo1 interaction
may be isoform specific. We attempted to perform a more quantitative
liquid assay to verify these results. The appearance of varied interaction
may also be due to varied protein expression of yeast colonies.
These data may prove essential in unraveling the mechanisms of 14-3-3
isoform specific binding. Assays of 14-3-3 mutants with hExo1 show
that binding/interactions occur similarly to other 14-3-3 ligands.
In mutants where alterations are farther from the essential phosphoserine
49 residue greater interactions occur. Further studies with a more
quantitative approach are needed to compare interactions. The liquid
two hybrid screens conditions may not be appropriate for monitoring
interactions under our constructs so we will attempt to optimize
those conditions while insuring sufficient protein expression. Collaborative
studies are concentrating on the functional significance of hExo1/14-3-3
interactions. Our studies will attempt to elucidate the mechanisms
governing its binding
Thank You Fu Lab Members, Cathy Quinone,s SURE 2003 Staff, participants,
Isabella Finkelstein and MARC *USTAR Program. This research was
supported by Howard Hughes Medical Institute Grant No. 52003727.
My laboratory studies a group of proteins that bind to a number
of different proteins. The name of the proteins that we study are
14-3-3 proteins. The proteins that they bind to are protein that
determine whether a cell grows or dies. These proteins are important
because if they are not working properly organisms like us develop
cancers and tumors. My project attempted to find out if 14-3-3 proteins
bind to a group of proteins that it has never been shown to bind
to. These proteins termed exonucleases are also important for stopping
the development of cancers. We found that our 14-3-3 protein do
in fact bind to the other proteins.
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