T cell receptor (TCR) engagement triggers a series of biochemical and signaling cascades including protein tyrosine kinase (PTK) activation, tyrosine phosphorylation of adapter proteins and multiple protein-protein interactions. CD45 plays a critical role in regulating TCR-mediated signaling. Here, we report evidence of in vivo interaction between CD45 and the Src kinase-associated phosphoprotein (SKAP55), which acts as its substrate for dephosphorylation. Further, we demonstrate and confirm by mutational analysis that a critical residue of SKAP55, Tyr-232, mediated the association with CD45. In Jurkat cells, SKAP55 induced tyrosine phosphorylation by anti-CD3 stimulation. Biochemical analysis revealed that adapter protein SKAP55 formed homodimers through its SH3 domain and SK region. The amount of SKAP55 homodimer was enhanced in T cell activation induced by anti-CD3 stimulation. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase was dependent on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused MAP kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T-cells. Mutational analysis revealed that Tyr-271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate MAP kinase activation. / Intriguingly, TCR engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts, while Fyn kinase, which was observed in lipid rafts in resting T cells, was found in increased amounts upon TCR activation. The association between SKAP55 and Fyn kinase was also found in lipid rafts, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation. Overexpression of SKAP55 in these cells induced transcriptional activation of the IL-2 promoter, while total suppression of the IL-2 promoter activity was observed for the SKAP55-Y232F mutant. Furthermore, overexpression of SKAP55-Y232F also caused the tyrosine-hyperphosphorylation of Fyn with a decreased kinase activity. Thus, SKAP55 is an essential adaptor which couples CD45 with the Src family kinase Fyn for dephosphorylation, and acts thereby as a key component for positive regulation in TCR signaling.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84451 |
| Date | January 2002 |
| Creators | Wu, Liangtang, 1964- |
| Contributors | Shen, Shi-Hsiang (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001975152, proquestno: AAINQ88604, Theses scanned by UMI/ProQuest. |
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