Abstract
The gene sap/shd1a, which encodes a 128-residue SH2 domain protein, is frequently deleted or mutated in the X-linked lymphoproliferative syndrome (XLP). The SAP SH2 domain differs from others in the same class in that it is not only capable of binding to a phosphotyrosine-containing peptide, it can also associate with an SH3 domain using a distinct surface. This novel mode of ligand-binding is initially discovered in the SLAM-SAP-Fyn complex that plays a critical role in T cell and natural killer cell activation. To identify additional binding partners for SAP, we screened a panel of 12 SH3 domains derived from regulatory proteins and identified NCK1 as a novel target of SAP in T cells. NMR analysis demonstrated that the NCK1 and Fyn SH3 domains possessed comparable affinities for SAP and engaged the same set of residues on the surface of the SAP SH2 domain. Depletion of SAP by siRNA caused a significant decrease in NCK1 tyrosine phosphorylation as well as the phosphorylation of other T cell receptor (TCR) downstream proteins such as LAT and SLP-76. Moreover, SAP was shown to regulate T cell proliferation through the MAP-kinase Erk. Taken together, our work identifies NCK1 as a novel physiological partner for SAP and a direct regulator of TCR signaling and T cell proliferation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / metabolism
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Amino Acid Sequence
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Cell Line
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Extracellular Signal-Regulated MAP Kinases / metabolism
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Fluorescent Antibody Technique, Direct
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Gene Knockdown Techniques
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Humans
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Intracellular Signaling Peptides and Proteins / metabolism*
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Jurkat Cells
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Large Neutral Amino Acid-Transporter 1 / metabolism
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Lymphoproliferative Disorders / genetics
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Lymphoproliferative Disorders / metabolism
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Magnetic Resonance Spectroscopy
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Oncogene Proteins / chemistry
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Oncogene Proteins / metabolism*
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Phosphoproteins / metabolism
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Phosphorylation
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Proto-Oncogene Proteins c-fyn / chemistry
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Proto-Oncogene Proteins c-fyn / metabolism
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RNA, Small Interfering / genetics
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Signal Transduction
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Signaling Lymphocytic Activation Molecule Associated Protein
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T-Lymphocytes / immunology*
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src Homology Domains / physiology*
Substances
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Adaptor Proteins, Signal Transducing
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Intracellular Signaling Peptides and Proteins
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Large Neutral Amino Acid-Transporter 1
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Nck protein
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Oncogene Proteins
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Phosphoproteins
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RNA, Small Interfering
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SH2D1A protein, human
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SLP-76 signal Transducing adaptor proteins
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Signaling Lymphocytic Activation Molecule Associated Protein
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FYN protein, human
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Proto-Oncogene Proteins c-fyn
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Extracellular Signal-Regulated MAP Kinases