Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4

Abstract

In T lymphocytes, the Src-family protein tyrosine kinase p56lck (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.

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56lck

“…In the latter case, the absence of Hsp90 activity could result in misfolded Lck that is unable to associate with membranes and is targeted for degradation. To discriminate between these two possibilities, we investigated the effect of Hsp90 inhibitors on the biosynthesis and membrane binding of UD-GFP, a construct composed of the unique domain of Lck fused to the N terminus of GFP (Bijlmakers et al, 1997). The N-terminal unique domain is important for the membrane binding and subcellular distribution of Lck (Kwong and Lublin, 1995;Yurchak and Sefton, 1995;Bijlmakers et al, 1997;Zlatkine et al, 1997).…”

“…To discriminate between these two possibilities, we investigated the effect of Hsp90 inhibitors on the biosynthesis and membrane binding of UD-GFP, a construct composed of the unique domain of Lck fused to the N terminus of GFP (Bijlmakers et al, 1997). The N-terminal unique domain is important for the membrane binding and subcellular distribution of Lck (Kwong and Lublin, 1995;Yurchak and Sefton, 1995;Bijlmakers et al, 1997;Zlatkine et al, 1997). At the extreme N terminus, myristic and palmitic acids are attached, modifications that are crucial for the interaction with membranes (Resh, 1994).…”

“…The final PCR product was checked by sequencing. LckY505F and Lck⌬SH2 were transfected into NIH-3T3 fibroblasts by electroporation as described previously (Bijlmakers et al, 1997).…”

“…The murine T-cell line LSTRA was grown in this medium supplemented with 50 M 2-mercaptoethanol. HeLa cells stably transfected with Lck or the unique domain of Lck attached to green fluorescent protein (UD-GFP) (Bijlmakers et al, 1997) were grown in DMEM, 4% FCS, pen/strep, and 1 mg/ml G418. NIH-3T3 cells stably transfected with Lck (Bijlmakers et al, 1997), LckY505F, Lck⌬SH2, or c-Src (Pelchen-Matthews et al, 1992 were grown in DMEM, 10% FCS, and pen/strep.…”

“…Subsequently, the completed protein becomes membrane associated and is palmitoylated on either one or both of two cysteine residues close to its N terminus (Paige et al, 1993;Shenoy-Scaria et al, 1993). Failure to undergo either of these acylations generates a cytosolic protein that cannot couple to CD4 or CD8 (Turner et al, 1990;Kwong and Lublin, 1995;Yurchak and Sefton, 1995;Bijlmakers et al, 1997;Zlatkine et al, 1997).…”