Regulation of the calcium/NF-AT T cell activation pathway by the D2 domain of CD45.
CD45 contains tandem repeated protein tyrosine phosphatase (PTP) domains and is essential for the initiation of the earliest activation events resulting from Ag ligation of the TCR. The second PTP domain (D2) contains four CK2 phosphorylation sites in a unique 19-aa insert, which are targets of CK2 phosphorylation. This study was designed to evaluate the roles of these Ser residues in T cell activation. Transient transfection of the CD45- T cell line, J45.01, with CD45 cDNA incorporating four Ser to Ala (S/A) mutations in the 19-aa insert did not affect the magnitude of NF-AT activation resulting from TCR ligation. However, the basal level of NF-AT activity in unstimulated cells expressing the CD45 S/A mutation was elevated 9- to 10-fold. Increased basal NF-AT was dependent on extracellular Ca2+ stores as judged by EGTA treatment. In additional experiments, isolation of stable clones derived from transfection of the CD45 S/A mutant into CD45- H45.01 cells showed sustained calcium flux after TCR engagement. The sustained calcium flux returned to baseline levels after addition of EGTA, suggesting that the expression of the CD45 S/A mutant may have prevented deactivation of plasma membrane calcium channels. Consideration of both transient and stable transfection systems suggests that in addition to being essential for initial events in T cell triggering, the intact CD45 D2, 19-aa insert is necessary for regulation of TCR-mediated calcium signaling pathways.
Wang
Y
Y
Department of Microbiology, Michigan State University, East Lansing, MI 48824, USA.
Liang
L
L
Esselman
W J
WJ
D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics / Q000276:immunology*; D002999:Clone Cells / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D008213:Lymphocyte Activation / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D017434:Protein Structure, Tertiary / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D017493:Leukocyte Common Antigens; D002118:Calcium