CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.
T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.
Niklinska
B B
BB
Laboratory of Immune Cell Biology, National Cancer Institute, Bethesda, Maryland 20892.
Hou
D
D
June
C
C
Weissman
A M
AM
Ashwell
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D066298:In Vitro Techniques; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009226:Myristates / Q000378:metabolism; D019000:Phosphotyrosine; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000502:physiology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D017931:DNA Primers; D008565:Membrane Proteins; D009226:Myristates; D011948:Receptors, Antigen, T-Cell; D019000:Phosphotyrosine; D014443:Tyrosine; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases; D002118:Calcium