Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae.

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.

Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.

Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1.

Decay-accelerating factor (DAF or CD55) is a 70-kDa glycosyl-phosphatidylinositol (GPI)-anchored protein that protects cells from complement-mediated lysis by either preventing the formation of or dissociating C3 convertases. Cross-linking of DAF on human peripheral T cells by polyclonal antibodies has previously been reported to lead to lymphocyte proliferation. Two mAb, both mapping to the third short consensus repeat region of DAF, were able to trigger proliferation of human peripheral T cells. To determine the role of the GPI anchor in cell activation, we transfected EL-4 murine thymoma cells with cDNA encoding either DAF or a transmembrane form of DAF (DAF-TM). The DAF-transfected cells were able to transduce late activation events as evidenced by IL-2 production, whereas DAF-TM transfected cells were unable to do so. The GPI-anchored DAF was able to transduce early activation events leading to the tyrosine phosphorylation of a 40-kDa protein and several proteins in the 85-95 kDa range--an event absent in DAF-TM-transfected cells. Furthermore, anti-DAF immunoprecipitates of DAF-transfected cells contain tyrosine kinase activity leading to the phosphorylation of 40-, 56-60-, and 85-kDa proteins, whereas anti-DAF immunoprecipitates of DAF-TM-transfected cells did not have an associated kinase activity. Both p56lck and p59fyn were associated with DAF in DAF-transfected EL-4 cells. In HeLa cells transfected with fyn, DAF associated with p59fyn. This complex of DAF with src family protein tyrosine kinases requires the GPI anchor and suggests a pathway for signaling through GPI-anchored membrane proteins.