Summer Undergraduate Research Program - Fred Hutchinson ...

“Best” Posters from the 2012 SURP Poster Session
Conclusions
Site-Directed Mutagenesis GST Pull-Down
Methods
Introduction
2012 Lee Hartwell Poster Award
The NPF sequence of Dab2 C-terminus is required for
binding to Dab2 PTB domain.
Clathrin-mediated endocytosis is an essential and vital cellular
function for the internalization of membrane proteins and
extracellular macromolecules.
The NPxY sequence of
cargo competes with the
NPF sequence of Dab2
C-terminus for PTB
domain binding.
The Dab2 PTB domain binding site for cargo is similar
but not identical to the binding site for Dab2 C-terminus.
These conclusions suggest the importance of the cargo/
adaptor/accessory protein complex to the endocytic process.
Results
Making it
possible for
Dab2 to do this:
Then maybe the
presence of cargo
initiates this:
If Dab2
without cargo
looks like this:
Dab2 PTB cargo-blocking mutation F166V
inhibits Dab2 PTB/C-terminus binding.
The presence of receptor inhibits the
binding of Dab2 C-terminus to Dab2 PTB
domain.
Figure 4. A point mutation
was made at the amino acid
F166, a location in the Dab2 PTB
domain that is known to inhibit
cargo binding1 . These results
demonstrate that in the presence
of the mutation F166V, binding of
Dab2 C-terminus and Dab2 PTB
domain is inhibited.
Figure 1. When the FxNPxY
sequence of LDL receptor is
incubated with Dab2 GST-PTB,
the C- terminus of Dab2 and the
PTB domain of Dab2 no longer
bind. This suggests that LDL
receptor could be a possible
competitor of Dab2 C-terminus at
the PTB domain binding site.
108
Eps
15
Previous Cooper lab data suggests that a cargo/ adaptor/
accessory protein complex is needed for endocytosis to take
place.
Dab2 is an adaptor
protein that binds to
the FxNPxY
internalization signal
sequence of LDL
receptors. The
accessory protein that
Eps 15
interacts with Dab2 is
Eps 15. It binds to the
C-terminus of Dab2
through its EH
domain.
Teckchandani, et. al, MBC. 2012.
Future Directions
Determine the binding affinity of accessory proteins to
Dab2 C-terminus in open conformation vs. closed.
Dab2 PTB cargo-blocking mutation S122T
shows no effect on Dab2 PTB/C-terminus
binding.
NPF sequence mutations in Dab2 C-terminus
decrease Dab2 C-terminus binding to Dab2
PTB domain.
Recent in vitro binding experiments showed that the end of
Dab2 C-terminus is also important for binding to Dab2 PTB
domain.
Determine the contribution of the amino acid F761 to find
a constitutively open/closed configuration of Dab2.
Produce an x-ray crystallography image of the binding
site of Dab2 C-terminus and Dab2 PTB domain.
Acknowledgements
Figure 5. A point mutation was
made at the amino acid S122, a
location in the Dab2 PTB domain
that is known to inhibit cargo
binding1 . These results demonstrate
that in the presence of the mutation
S122T, binding of Dab2 C-terminus
andDab2 PTB domain is unaffected.
Figure 2. Point mutations
were made to each amino acid
in the NPF sequence of the
Dab2 C-terminus. These results
demonstrate decreased Cterminus-PTB
domain binding
when an N mutation is made
and no C-terminus-PTB domain
binding when P and F mutations
are made.
Because of this, Dab2 C-terminus amino acids 752-766
became the area of focus. Sequence analysis revealed a
similarity between Dab2 C-terminus amino acid sequence
and LDL receptor tail sequence.
Cargo
A special thanks to Erin Mulkearns-Hubert for her unwavering
patience, assistance, and understanding. Thank you to the rest of the
members of the Cooper lab for fostering my education and teaching
me that science is about life-long learning. I would also like to thank
Jonathan Cooper for being the backbone of curiosity, NMSU as my
home institution, and the Fred Hutchinson Cancer Research Center
for this life-changing opportunity. The Fred Hutchinson Cancer
Research Center Summer Undergraduate Research Program is
supported in part by the Cancer Center Support Grant (CCSG) CURE
Supplement: 3 P30 CA015704-38S1. The FHCRC/NMSU partnership
is supported in parts by NCI grants: 5 U54 CA132381 (FHCRC)and 5
U54 CA132383 (NMSU). Additional funding provided by grant
GM066257.
+His-C-Term
?
FxNPxY
FGNPFA
NPF sequence mutations in Dab2 C-terminus
decrease Dab2 C-terminus binding to Eps 15
EH domain.
F166
P
Y
S122
Y
From this, we hypothesized that
the C-terminus of Dab2 might also
bind to the PTB domain of Dab2
in the same location where LDL
Dab2 PTB domain
shown inlaid with
amino acids N,P,
and Y of FxNPxY
sequence of LDL
receptor. This figure
has been modified1 .
Figure 3. These results
demonstrate a decreased
interaction between Dab2 Cterminus
and Eps 15 EH
domain when each of the amino
acids in the NPF sequence is
mutated. Other NPF sequences
in the C-terminus may be
responsible for residual binding.
receptor binds.
Objective
Contact
Rylie Hightower
Fred Hutchinson Cancer Research Center/New Mexico State University
E-mail: swimmer1@nmsu.edu
The main focus of this research was to map the region of
Dab2 C-terminus that interacts with Dab2 PTB domain and
Eps 15.
1. "Yun et al. Journal of Biological Chemistry, 2003."