The Lck SH3 domain negatively regulates localization to lipid rafts through an interaction with c-Cbl.
Lck is a member of the Src family of protein-tyrosine kinases and is essential for T cell development and function. Lck is localized to the inner surface of the plasma membrane and partitions into lipid rafts via dual acylation on its N terminus. We have tested the role of Lck binding domains in regulating Lck localization to lipid rafts. A form of Lck containing a point mutation inactivating the SH3 domain (W97ALck) was preferentially localized to lipid rafts compared with wild type or SH2 domain-inactive (R154K) Lck when expressed in Lck-deficient J.CaM1 cells. W97ALck incorporated more of the radioiodinated version of palmitic acid, 16-[(125)I]iodohexadecanoic acid. Overexpression of c-Cbl, a ligand of the Lck SH3 domain, depleted Lck from lipid rafts in Jurkat cells. Additionally, Lck localization to lipid rafts was enhanced in c-Cbl-deficient T cells. The association of Lck with c-Cbl in vivo required a functional SH3 domain. These results suggest a model whereby the SH3 domain negatively regulates basal localization of Lck to lipid rafts via association with c-Cbl.
Hawash
Ibrahim Y
IY
Department of Biology, Purdue University, West Lafayette, Indiana 47907, USA.
Kesavan
Kamala P
KP
Magee
Anthony I
AI
Geahlen
Robert L
RL
Harrison
Marietta L
ML
D000818:Animals; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry* / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D014162:Transfection; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D049452:Green Fluorescent Proteins; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); C067419:CBL protein, human