Regulation of the subcellular localization of Lck to lipid rafts

Lck is a member of the Src family of protein tyrosine kinases and is highly expressed in T lymphocytes. Lck is a membrane-associated protein and plays an essential role in T cell antigen receptor (TCR) signaling. Effective TCR signaling is dependent on the localization of Lck to hydrophobic microdomains within the plasma membrane known as lipid rafts. Palmitate moieties covalently bound to cysteines 3 and 5 in the extreme N-terminus of Lck are implicated in targeting Lck to rafts. We hypothesized that (1) palmitoylation provides Lck with the necessary hydrophobicity to associate with rafts and (2) Lck binding to other proteins plays a role in the regulation of Lck raft localization. To test the first hypothesis, we utilized a novel heteroatom-substituted analog of palmitic acid, 13-oxypalmitate (13-OP) and examined the effects of 13-OP treatment on Lck subcellular localization and function. To test the second hypothesis we utilized genetic, biochemical and microscopy approaches and examined the localization of various Lck mutants to rafts. In both studies the palmitoylation of Lck was directly analyzed using the radioactive analog of palmitic acid, [125I]-ω-iodopalmitic acid. These studies led to: (1) the demonstration that 13-OP modified Lck and inhibited the localization of Lck to rafts and inhibited TCR signaling, and (2) the identification of the Lck SH3 domain as a negative regulator of Lck raft localization though an interaction with c-Cbl. The findings provide important insights on the mechanisms regulating Lek raft localization and provide useful tools for studying the role of rafts in T cell function