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.

To study the acylation of p21N‐ras with palmitic acid we have used cells which express the human N‐ras gene to high levels under control of the steroid‐inducible MMTV–LTR promoter. Addition of [3H]palmitate to these cells resulted in detectable incorporation of label into p21N‐ras within 5 min, which continued linearly for 30‐60 min. Inhibition of protein synthesis for up to 24 h before addition of [3H]palmitate had no effect on acylation of p21N‐ras, suggesting that this can occur as a late post‐translational event. Acylated p21N‐ras with a high SDS–PAGE mobility is found only in the membrane fraction, whereas approximately 50% of the [35S]methionine‐labelled p21N‐ras is cytoplasmic and has a lower mobility. Conversion of the acylated high mobility form to a deacylated form of slightly lower mobility can be achieved with neutral hydroxylamine, which is known to cleave thioesters. This treatment also results in partial removal of p21N‐ras from the membranes. A remarkably high rate of turnover of the palmitate moiety can be demonstrated by pulse–chase studies (t1/2 approximately 20 min in serum‐containing medium) which cannot be attributed to protein degradation. The data suggest an active acylation–deacylation cycle for p21N‐ras, which may be involved in its proposed function as a signal transducing protein.

Nonuniformity in the spatial patterning of gap junctions between heart muscle cells is now recognized as an important determinant of electromechanical function in working myocardium. Breakdown of the normal geometry of electrical intercellular connectivity in diseased myocardium correlates with reentry, arrhythmia, and conduction disturbance. The developmental mechanism(s) that determines this precise spatial order in gap junction organization in normal myocardium is at present unknown. To examine this question, we have used immunoelectron and immunoconfocal microscopy to analyze the spatial distributions of gap junctional (connexin43), desmosomal (desmoplakin), and adherens junctional (N-cadherin) components during maturation of rodent and canine left ventricular myocardium. In rats, a striking divergence in the distribution of gap junctions and cell adhesion junctions emerged within the first 20 days of postnatal life. It was found that although gap junctions initially demonstrated dispersed distributions across myocyte cell membranes, desmosomes and adherens junctions showed more rapid polarization toward cell termini (ie, nascent intercalated disks) after birth. Over subsequent postnatal development (20 to 90 postnatal days), gap junctions became progressively concentrated in these cell adhesion junction-rich zones of membrane. Quantitative analyses of this process in a series of rats aged 15 embryonic and 1, 5, 10, 20, 40, 70, and 90 postnatal days indicated that significantly higher levels (P < .01) of N-cadherin and desmoplakin than of connexin43 were immunolocalized to cell termini by as early as postnatal day 5. Although all three junctions types showed increasing polarization to myocyte termini with development, variation between junctions remained significant (P < .05) at all times points between 5 and 70 postnatal days. Only at 90 postnatal days, when the animals were nearly full grown, did the proportions of gap junction, desmosome, and adherens junction at intercalated disks become statistically similar (P > .05). Examination of myocardium from 1- and 3-month-old canines revealed that related differential changes to the spatiotemporal distribution of intercellular junctions occurred during postnatal maturation of the dog heart, suggesting that the process was not rodent specific. It is concluded that this progressive change in the organization and pattern of association between gap junctions and cell adhesion junctions is likely to be an important factor in maturation of electromechanical function within the mammalian heart.

We have recently identified a novel N-terminal cysteine-containing motif which specifies the palmitoylation of several G-protein alpha-subunits [Parenti, Viganó, Newman, Milligan and Magee (1993) Biochem. J. 291, 349-353]. A related motif occurs at the N-terminus of members of the Src family of protein tyrosine kinases except for Src itself and Blk. We have investigated whether the Src, Fyn, Yes and Lck gene products are palmitoylated. Src was not labelled with [3H]palmitate when endogenously expressed in COS cells. In contrast, endogenous Yes immunoprecipitated from COS cells was palmitoylated. Fyn was palmitoylated in insect cells infected with a recombinant baculovirus and the palmitoylation was independent of protein synthesis, suggesting a dynamic turnover of this lipid. Fatty acid analysis indicated that most of the label was incorporated as palmitate. Lck was palmitoylated when expressed by transfection in COS cells. All of these protein tyrosine kinases were also detectably myristoylated in each of the systems tested. Experiments performed with mutants of Lck expressed by transfection in COS cells indicated that cysteines at positions 3 and 5 were both palmitoylation sites and that myristoylation was required for palmitoylation. To confirm that palmitoylation was occurring on cysteines in the N-terminal region of Fyn, site-directed mutagenesis was used to replace the cysteines at positions 3 and 6 with alanine. The resulting protein was not palmitoylated but was still myristoylated when expressed in COS cells. A glycine to alanine mutant at position 2 was also not palmitoylated, showing that myristoylation is a prerequisite for palmitoylation. Our data indicate that Src family members containing the N-terminal cysteine motif are indeed palmitoylated. By analogy with Ras, it is possible that palmitoylation may play an important role in the localization and function of Src family protein tyrosine kinases.