Open Collections

The interactions of LCK in T cells and its regulation by CD45

University of British Columbia

Lck is a Src-family tyrosine kinase whose activity is essential for T cell activation and development. The phosphorylation of two key tyrosine residues in Lck, Tyr 505 and Tyr 394, regulate its activation by inducing profound changes in conformation. In vitro, CD45, a leukocyte-specific tyrosine phosphatase, preferentially dephosphorylated Tyr 394, as opposed to Tyr 505, and this specificity depended on the Lck non-catalytic domains and not the sequence specificity of the phosphatase catalytic pocket. The Lck non-catalytic domains enhanced the rate of CD45-mediated Tyr 394 dephosphorylation, and inhibited, but did not block catalysis at Tyr 505. A direct, specific, non-catalytic interaction was observed between the second phosphatase domain of CD45 (D2) and subdomain X(SD10) of Lck. This interaction was also shown to influence CD45 substrate specificity as the replacement of Erkl SD10 with that of Lck resulted in the conversion of Erkl into a more efficient CD45 substrate. In T cells, the majority of CD45 is constitutively associated with a smaller protein aptly named CD45-associated protein (CD45AP). An interaction between Lck and CD45AP was determined to occur independently of CD45 in T cells. A direct CD45AP:Lck interaction was confirmed in vitro, and was shown to antagonize the ability of CD45 to interact with Lck. Moreover, the presence of CD45AP significantly inhibited CD45-mediated Lck dephosphorylation at Tyr 394, but not Tyr 505, suggesting a role for CD45AP in sustaining Lck activity in the cell. CD44 is a widely expressed transmembrane adhesion molecule that requires the activity of Src-family kinases in T cells to induce cytoskeletal changes associated with cell adhesion. Here, Src-family kinases Lck and Fyn were both shown to associate with CD44 in T cells. In vitro, the CD44 cytoplasmic domain bound to Lck directly in a zinc dependent manner, but did not bind to Fyn. The treatment of T cells with cation chelator 1,10-phenanthroline both disrupted the CD44:Lck association and blocked CD44-induced cytoskeletal changes. This suggests that the zinc-dependent Lck association with CD44 plays a key role in mediating CD44-induced cell signaling events required for cell adhesion.

Thesis/Dissertation

application/pdf

2009-11-17

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/15133

Microbiology and Immunology

University of British Columbia

UBCV

DSpace