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Role of the SH3 Domain in the Trafficking and Localization of Lck in T Lymphocytes
Author(s) -
Ong Su Sien,
Tao Andy W.,
Geahlen Robert L.,
Harrison Marietta L.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a926
Subject(s) - subcellular localization , microbiology and biotechnology , sh3 domain , proto oncogene tyrosine protein kinase src , signal transduction , protein subcellular localization prediction , kinase , tyrosine kinase , protein domain , chemistry , biology , gene , biochemistry , cytoplasm
Lck, a member of the Src family of non‐receptor protein tyrosine kinase, plays a pivotal role in normal T cell development and activation. The SH3 domain of Lck is a protein‐protein interacting domain that binds proline‐rich sequences with the consensus sequence PxxP. A point mutation in the SH3 domain of Lck (W97A) leads to the failure to support signaling downstream of Ras, while retaining its ability to support proximal signaling events. This mutant also shows a distinct subcellular localization. Wild‐type Lck is localized at the membrane, with a significant intracellular localization, while W97A Lck is predominantly localized at the membrane. The goal of this project is to determine if the difference in the subcellular localization mediated by the SH3 domain of Lck plays a role in supporting the signaling events downstream of Ras. The specific aims of this project are to determine 1) the subcellular localization of WT Lck and W97A Lck; 2) the mechanism underlying the aberrant trafficking and localization of W97A Lck; 3) if the altered trafficking is responsible for the inability of the W97A mutant to support signaling to the ERK pathway. In order to study the trafficking and localization of Lck, we employed Lck‐GFP constructs. Microscopy and biochemical separations will be employed to study the localization of WT Lck and W97A Lck. GST pulldown assays coupled with mass spectrometry technology was used to determine the binding partners of the SH3 domain of Lck. Preliminary studies using mass spectrometry identified over ten cellular proteins that specifically associated with the SH3 domain of WT Lck. Supported by NIH Grant GM048099.