Role of the SH3 domain of Lck in its trafficking and localization in T-cells

Su Sien Ong, Purdue University

Abstract

T cell development and activation are governed by signaling through the T cell receptor (TCR). Lck, a member of the Src-family of non-receptor protein tyrosine kinases, plays a pivotal role in normal T cell development and activation. The SH3 domain of Lck is a protein-protein binding domain that interacts with proline-rich sequences with the consensus sequence PxxP. A crippling point mutation within the SH3 domain of Lck (W97A) resulted in a mutant form of Lck that exhibited a distinct subcellular localization. I showed that while WTLck-GFP localized primarily to the plasma membrane and the Golgi, W97ALck-GFP showed decreased Golgi localization. This indicates that the SH3 domain of Lck influences its trafficking and localization in T cells. Using GST pulldown assays coupled with mass spectrometry, I identified several proteins that bound specifically to WTLck-SH3 and not to W97ALck-SH3. One of these proteins was dynamin2 (Dyn2). Dyn2 plays a pivotal role in receptor endocytosis and the regulation of actin dynamics. I demonstrated that Lck and Dyn2 interact at the Golgi. I further showed that the Golgi recruitment and accumulation of Dyn2 is dependent on the SH3 domain of Lck. This Golgi accumulation of Dyn2 subsequently led to Golgi fragmentation and vesiculation. Since Golgi fragmentation is pivotal in mediating Golgi positioning, it follows that this process positively regulates MTOC and Golgi polarization to the immune synapse (IS). Accordingly, I demonstrated that the W97ALck expressing cells exhibited a defect in MTOC polarization to the IS. Taken together, I propose that Lck-Dyn2 interaction serves to mediate stable IS formation through MTOC and Golgi polarization. This is the first report of a novel role for Lck as an adaptor protein in mediating TCR signaling.

Degree

Ph.D.

Advisors

Harrison, Purdue University.

Subject Area

Molecular biology

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