A pivotal role of cysteine 3 of Lck tyrosine kinase for localization to glycolipid-enriched microdomains and T cell activation.
Lck, a Src family protein tyrosine kinase (PTKs), is post-translationally modified by palmitoylation, a process thought to regulate the biological function, membrane affinity and glycolipid-enriched microdomain (GEM) localization of this molecule. To examine the importance of palmitoylation sites Cys3 and Cys5 in Lck, one or both of these residues was mutated to serine to create mutants S3, S5, and S3,5, respectively. Immunofluorescence and confocal microscopy of COS-7 cells transfected with these constructs showed that while S5 and S3 localized to the plasma membrane, S3,5 was localized to the cytoplasm, suggesting that palmitoylation at at least one site is essential for membrane localization. Sucrose gradient based fractionation of these mutants expressed in COS-7 cells showed that while S5 localized to GEMs in similar fashion to the wild type, GEM localization of S3 was severely inhibited. Expression of these mutants in Lck-negative JCaM1 cells showed that although S5 reconstituted activation of nuclear factor NFAT as per the wild type, S3 expression failed to do so. These results suggest that Cys3 of Lck plays a more important role than Cys5 in GEM localization and T cell activation. Additionally, it was found that the degree of T cell function recovery is positively correlated with the degree of Lck expression in GEMs.
Kosugi
A
A
School of Allied Health Sciences, Faculty of Medicine, Osaka University, 1-7, Yamada-oka, Suita, 565-0871, Osaka, Japan. kosugi@sahs.med.osaka-u.ac.jp
Hayashi
F
F
Liddicoat
D R
DR
Yasuda
K
K
Saitoh
S
S
Hamaoka
T
T
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006017:Glycolipids / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000276:immunology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D013347:Subcellular Fractions; D013601:T-Lymphocytes / Q000276:immunology*
D006017:Glycolipids; D011948:Receptors, Antigen, T-Cell; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D003545:Cysteine