1. TCR Stimulation Induces the
Recruitment of Effector
Molecules
When the TCR is stimulated by an antigen,
proteins inside the T-Cell are tyrosine
phosphorylated. This induces molecular
interactions that drive signal transduction,
leading to T-Cell activation. The proteins
SLP76 and ADAP are critical for T-Cell
signaling.
T-Cells Become Activated
Following Stimulation of the
T-Cell Antigen Receptor (TCR)
The surface of each T-Cell displays
thousands of identical T-Cell Antigen
Receptors (TCR). The TCR is stimulated by a
histocompatibility molecule in complex with
an antigen, which is found on the surface of
an antigen presenting cell. An antigen is a
substance foreign to the body that evokes
an immune response. Each TCR is specific
for a particular antigen and every T-Cell has
a unique antigen specificity. Those that
recognize an antigen will become activated
and work to clear the infection.
A Mutagenesis Approach to Understanding Molecular Interactions Between
Adaptor Proteins in T-Cells
Henry Chen, Nathan P. Coussens, Lawrence E. Samelson
From the Laboratory of Cellular and Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892
The SH2 Domain is Required
for SLP76 Clustering and
T-Cell Signaling
SLP76 has 3 domains including an SH2
domain at the C-terminus. An SH2 domain is
a part of a protein that helps it bind to other
proteins by recognizing specific
phosphotyrosine residues. In imaging
studies of stimulated T-Cells with
fluorescent SLP76 chimeras, the protein is
observed in structures called microclusters,
which contain many protein complexes.
When the SH2 domain is mutated, SLP76
microclusters and T-Cell signaling are
impaired. This indicates that the SH2 domain
plays an important role in T-Cell activation.
ADAP may facilitate the clustering of SLP76
through multipoint binding.
Introduction
T-Cells belong to a group of white blood
cells called lymphocytes. The main job of a
T-Cell is to fight infections. There are many
different kinds of T-Cells (helper, cytotoxic,
natural killer) that act in different ways to
identify, directly attack, and destroy
infectious agents. T-Cells are produced in
the bone marrow and mature in the thymus.
WT SH2 Mutant
Introduction of the Y771F
Mutation Into Cyan Fluorescent
Protein Tagged ADAP
Chimeras
The four constructs shown above had been
generated previously.
I made four additional constructs by adding
the Y771F mutation to each of the existing
ADAP constructs. In this process, the ADAP
DNA sequence was changed to code for
phenylalanine instead of tyrosine.
Phenylalanine, unlike tyrosine, cannot be
phosphorylated and bind to an SH2 domain.
This means that ADAP will no longer be able
to bind SLP76 at those specific sites.
GAGAGATCTATGATGATAT
GAGAGATCTTTGATGATAT
The hydroxyl group of tyrosine is
phosphorylated, which permits binding to an
SH2 domain. The OH is missing on the
phenylalanine.
Tyrosine
Phenylalanine
Transfection of T-Cells with
ADAP Constructs
The ADAP DNA constructs were transfected
into Jurkat T-Cells. The cells were
transfected with DNA encoding either WT
ADAP or the triple mutation (Y591F-Y651F-
Y771F). Since the ADAP protein is fused to a
cyan-fluorescent protein (CFP) tag which
fluoresces blue light, the cells that are
transfected will appear blue in confocal
microscope images.
Results
The transfected ADAP proteins were
captured by immunoprecipitation. This
image shows that only the cells transfected
with ADAP DNA express a protein
recognized by an antibody to CFP. The
molecular weight of this protein
corresponds to that of the ADAP-CFP fusion
protein. Unfortunately, the transfection
efficiency was low and we could only
immunoprecipitate small amounts of the
ADAP protein. At that level, it was not
possible to detect any associated SLP76
protein.
Future Directions
We will optimize the immunoprecipitation
assay to detect SLP76 with wild-type ADAP.
We will then repeat the assay with the
different ADAP mutants. Transfecting more
cells may allow us to detect SLP76
associating with ADAP.
3D Reconstruction
Bunnell, S.C. et al. Mol. Cell. Biol. 2006 26:7155-7166.
SLP76 Associates with ADAP
Phosphotyrosines 595, 651 and
771
SLP76 associates with ADAP during T-Cell
activation. We hypothesize that the three
binding sites promote SLP76 microcluster
formation by multipoint binding.
ANTIGENANTIGEN
-CN- Tyr
112
Tyr
128
Tyr
145
P P P
Pro R,K
SLP76
533 AA SH2 Domain
Experimental Approach
Characterize interactions between SLP76
and constructs of ADAP with different
combinations of mutations to the tyrosine
residues 595, 651 and 771.
Existing
Constructs 595 651 771
Missing
Constructs
595 651 771
595 651 771 595 651 771
Examine Interactions Between
SLP76 and ADAP by
Immunoprecipitation
The T-Cells were lysed and ADAP was
captured by antibodies. In stimulated cells,
SLP76 binds ADAP so the proteins co-
precipitate.
http://molecularsciences.org
ADAP
SLP
Detection of SLP76 and ADAP by
Western Blotting
The proteins were separated on a gel and
transferred onto a nitrocellulose membrane.
The membrane was then incubated with
antibodies that bind to the protein of
interest. The antibodies have enzymes that
generate light which can be detected by film.
http://www.virology.ws
EGFP WT SH2
EGFP-SLP76
2D Image