Intrinsic signals in the unique domain target p56(lck) to the plasma membrane independently of CD4.
In T lymphocytes, the Src-family protein tyrosine kinase p56(lck) (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell-specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles. A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.
Bijlmakers
M J
MJ
Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom.
Isobe-Nakamura
M
M
Ruddock
L J
LJ
Marsh
M
M
D016475:3T3 Cells / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015704:CD4 Antigens / Q000502:physiology*; D016827:CD8 Antigens / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D003545:Cysteine / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008854:Microscopy, Electron; D019308:Palmitic Acid / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology; D017434:Protein Structure, Tertiary; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D019061:src-Family Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D015704:CD4 Antigens; D016827:CD8 Antigens; D021382:Protein Sorting Signals; D019308:Palmitic Acid; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D003545:Cysteine