CD45, also known as Leukocyte-common antigen (LCA), is a type-1 receptor-like protein tyrosine phosphatase (PTPase), which contains two PTPase domains, the membrane proximal domain (DI) and the membrane distal domain (D2). It is uniquely expressed on the surface of all leukocytes and their hematopoietic precursors and plays an important role in immunoreceptor signaling. In this study, I identified a novel CD45-interacting protein, Fyb, through screening human leukocyte cDNA library with a modified yeast two-hybrid system. Fyb is an adaptor protein mainly involved in the regulation of T cell receptor-induced integrin clustering and adhesion. It contains several regions with potential to mediate protein-protein interactions and has been shown to bind to the Src family kinase Fyn, the adapter protein SLP-76, Ena/VASP proteins, the adapter protein SKAP55, and SKAP55-HOM in T cell receptor signaling. I also demonstrated that the interaction of Fyb with CD45 was through involves the C-terminal region of Fyb, where several tyrosine residues, such as Tyr 594 and Tyr624, are critical for its interaction with other proteins in signaling. The interaction of Fyb with CD45 is phosphotyrosine-dependent since Src kinase is required for the interaction. Mutation analysis revealed that the tyrosine residue 624 of Fyb is the pivotal tyrosine residue involved in its association with CD45, whereas tyrosin 594 was not found to be required for binding. By mutation of CD45, it was demonstrated that the phosphatase catalytic D1 domain of CD45 was involved in the interaction with Fyb. More significantly, I found that only the substrate-trapping mutant (Asp to Val), but not the wild type CD45 interacted with Fyb, suggesting that Fyb is a potential CD45 substrate. Furthermore, I demonstrated the association of the C-terminal fragment of Fyb with CD45 in mammalian cells. However, I was not able to determine the interaction of the wild type Fyb with CD45 in 293 cells.
Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.98530 |
Date | January 2005 |
Creators | Zhang, Linhua, 1968- |
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 | Master of Science (Division of Experimental Medicine.) |
Rights | © Linhua Zhang, 2005 |
Relation | alephsysno: 002328802, proquestno: AAIMR24838, Theses scanned by UMI/ProQuest. |
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