120802_NC_My Poster
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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. GAGAGATCT A T GATGATAT GAGAGATCT T T GATGATAT The hydroxyl group of tyrosine is phosphorylated, which permits binding to an SH2 domain. The OH is missing on the phenylalanine. Tyrosine Phenylalani ne 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 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. ANTIGEN -C N- 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 Construct s 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
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Transcript of 120802_NC_My Poster
TCR Stimulation Induces the Recruitment of Effector
MoleculesWhen 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.
IntroductionT-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.
ResultsThe 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 DirectionsWe 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.
ANTIGEN
-CN- Tyr112
Tyr128
Tyr145
P P P
Pro R,KSLP76533 AA SH2 Domain
Experimental ApproachCharacterize interactions between SLP76 and constructs of ADAP with different combinations of mutations to the tyrosine residues 595, 651 and 771.
ExistingConstructs 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
