No abstract

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)2–green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B–induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.

No abstract

No abstract

We have studied the post‐translational processing of p21ras proteins. The primary translation product pro‐p21 is cytosolic and is rapidly converted to a cytosolic form (c‐p21) of higher mobility on SDS‐PAGE. c‐p21 is converted in turn to the membrane‐bound mature palmitoylated form (m‐p21) of slightly higher mobility. These processing steps are accompanied by increases in isoelectric point and in hydrophobicity as judged by Triton X‐114 partitioning. Although the increases in electrophoretic mobility and hydrophobicity precede acylation we show that mutation of Cys186, which has been shown to block acylation, also abolishes the pro‐p21 to c‐p21 conversion. Thus the Cys186 residue is involved in the processing steps prior to acylation. We have identified two processing events which contribute to the pro‐p21 conversion. Site‐directed mutagenesis to insert tryptophan, which is not present in the wild type, followed by metabolic labelling with [3H]tryptophan has allowed us to map a proteolytic processing event which removes the three C‐terminal residues. In addition, both the c‐p21 and m‐p21 forms are carboxyl‐methylated. Approximately one methyl group is incorporated per molecule of p21 at steady state, which can partially account for the increase in isoelectric point. Unlike palmitate, methyl group turnover is not observed.