A. Kosugi et al., A pivotal role of cysteine 3 of Lck tyrosine kinase for localization to glycolipid-enriched microdomains and T cell activation, IMMUNOL LET, 76(2), 2001, pp. 133-138
Lek, a Src family protein tyrosine kinase (PTKs), is post-translationally m
odified by palmitoylation, a process thought to regulate the biological fun
ction, membrane affinity and glycolipid-enriched microdomain (GEM) localiza
tion of this molecule. To examine the importance o palmitoylation sites Cys
3 and Cys5 in Lck, one or both of these residues was mutated to serine to c
reate mutants S3, S5, and S3,5, respectively. Immunofluorescence and confoc
al microscopy of COS-7 cells transfected with these constructs showed that
while S5 and 53 localized to the plasma membrane, S3,5 was localized to the
cytoplasm, suggesting that palmitoylation at at least one site is essentia
l for membrane localization. Sucrose gradient based fractionation of these
mutants expressed in COS-7 cells slowed that while S5 localized to GEMs in
similar fashion to the wild type, GEM localization of 53 was severely inhib
ited. Expression of these mutants in Lck-negative JCaM1 cells showed that a
lthough Si reconstituted activation of nuclear factor. NFAT as per the wild
type, S3 expression failed to do so. These results suggest that Cys3 of Lc
k plays a more important role than Cys5 in GEM localization and T cell acti
vation. Additionally, it was found that the degree of T cell function recov
ery is positively correlated willi the degree of Lck expression in GEMs. (C
) 2001 Elsevier Science B.V. All rights reserved.