Anthony I. Magee

All ras proteins are polyisoprenylated but only some are palmitoylated.

The C-terminal CAAX motif of the yeast mating factors is modified by proteolysis to remove the three terminal amino acids (-AAX) leaving a C-terminal cysteine residue that is polyisoprenylated and carboxyl-methylated. Here we show that all ras proteins are polyisoprenylated on their C-terminal cysteine (Cys186). Mutational analysis shows palmitoylation does not take place on Cys186 as previously thought but on cysteine residues contained in the hypervariable domain of some ras proteins. The major expressed form of c-K-ras (exon 4B) does not have a cysteine residue immediately upstream of Cys186 and is not palmitoylated. Polyisoprenylated but nonpalmitoylated H-ras proteins are biologically active and associate weakly with cell membranes. Palmitoylation increases the avidity of this binding and enhances their transforming activity. Polyisoprenylation is essential for biological activity as inhibiting the biosynthesis of polyisoprenoids abolishes membrane association of p21ras.

S‐acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes

LCK is a non‐receptor protein tyrosine kinase required for signal transduction via the T‐cell antigen receptor (TCR). LCK N‐terminus is S‐acylated on Cys3 and Cys5, in addition to its myristoylation on Gly2. Here the role of S‐acylation in LCK function was examined. Transient transfection of COS‐18 cells, which express a CD8‐ζ chimera on their surface, revealed that LCK mutants that were singly S‐acylated were able to target to the plasma membrane and to phosphorylate CD8‐ζ. A non‐S‐acylated LCK mutant did not target to the plasma membrane and failed to phosphorylate CD8‐ζ, although it was catalytically active. Fusion of non‐S‐acylated LCK to a transmembrane protein, CD16:7, allowed its plasma membrane targeting and also phosphorylation of CD8‐ζ when expressed in COS‐18 cells. Thus S‐acylation targets LCK to the plasma membrane where it can interact with the TCR. When expressed in LCK‐negative JCam‐1.6 T cells, delocalized, non‐S‐acylated LCK was completely non‐functional. Singly S‐acylated LCK mutants, which were expressed in part at the plasma membrane, efficiently reconstituted the induced association of phospho‐ζ with ZAP‐70 and intracellular Ca2+ fluxes triggered by the TCR. Induction of the late signalling proteins, CD69 and NFAT, was also reconstituted, although at reduced levels. The transmembrane LCK chimera also supported the induction of tyrosine phosphorylation and Ca2+ flux by the TCR in JCam‐1.6 cells. However, induction of ERK MAP kinase was reduced and the chimera was incapable of reconstituting induced CD69 or NFAT expression. These data indicate that LCK must be attached to the plasma membrane via dual acylation of its N‐terminus to function properly in TCR signalling.

THE DYNAMIC ROLE OF PALMITOYLATION IN SIGNAL TRANSDUCTION

Guanine nucleotide binding protein (G protein)-linked receptors, the alpha-subunits of heterotrimeric G proteins and members of the Src family of nonreceptor tyrosine kinases are among many polypeptides that are posttranslationally modified by the addition of palmitate, a long-chain fatty acid. Attachment of palmitate to these proteins is dynamic and may be regulated by their activation. The presence of palmitate appears to play a key role in the membrane localization of either the entire polypeptide or parts of it, and may regulate the interactions of these polypeptides with other proteins.

Dynamic fatty acylation of p21N-ras.

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.

Posttranslational processing of the ras superfamily of small GTP-binding proteins.

III. C-Terminal processing of ras proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 A. Elucidation of the processing pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 B. Sequence of processing events and importance for membrane binding . . . . . . . . . . . . . . . 83 C. Functional importance of membrane binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 D. Mechanism and specificity of membrane attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system

We have identified a protein-acyltransferase activity in membranes from mouse fibroblasts which transfers palmitate from palmitoyl-CoA to p21N-ras. Specificity of acylation has been confirmed by linkage analysis using hydroxylamine and by peptide mapping of in vivo and in vitro acylated p21N-ras. The acylation was temperature- and time-dependent, and prevented by prior boiling of membranes, consistent with an enzymatic process. The activity was detected in membranes, but not cytosol, and co-fractionated on Percoll gradients with Golgi markers. Cytosolic p21N-ras from mouse fibroblasts, which is C-terminally modified at its CAAX sequence, was a better substrate for the enzyme that recombinant bacterially expressed, unmodified p21N-ras. The activity could be solubilised in non-ionic detergents, making it amenable to purification.

Cloning and Expression Throughout Mouse Development of mfat1, a Homologue of the Drosophila Tumour Suppressor Gene fat

We present the entire sequence of the mouse Fat orthologue (mFat1), a protein of 4,588 amino acids with 34 cadherin repeats, 27 potential N‐glycosylation sites, five EGF repeats and a laminin A G‐motif in its extracellular domain. A single transmembrane region is followed by a cytoplasmic domain containing putative catenin‐binding sequences. mFat1 shows high homology to human FAT and lesser homology to Drosophila Fat. The sequence of this giant cadherin suggests that it is unlikely to have a homophilic adhesive function, but may mediate heterophilic adhesion or play a signalling role. Expression analysis shows that the mfat1 gene is expressed early in pre‐implantation mouse development, at the compact eight cell stage. Whole‐mount and section in situ analyses show that transcripts are widely expressed throughout post‐implantation development, most notably in the limb buds, branchial arches, forming somites, and in particular in the proliferating ventricular zones in the brain, being down‐regulated as cells cease dividing. RT‐PCR detects widespread expression in the adult suggesting a role in proliferation and differentiation of many tissues and cell types. Dev Den;217:233–240.

The Lck SH3 Domain Negatively Regulates Localization to Lipid Rafts through an Interaction with c-Cbl

Lck is a member of the Src family of protein-tyrosine kinases and is essential for T cell development and function. Lck is localized to the inner surface of the plasma membrane and partitions into lipid rafts via dual acylation on its N terminus. We have tested the role of Lck binding domains in regulating Lck localization to lipid rafts. A form of Lck containing a point mutation inactivating the SH3 domain (W97ALck) was preferentially localized to lipid rafts compared with wild type or SH2 domain-inactive (R154K) Lck when expressed in Lck-deficient J.CaM1 cells. W97ALck incorporated more of the radioiodinated version of palmitic acid, 16-[125I]iodohexadecanoic acid. Overexpression of c-Cbl, a ligand of the Lck SH3 domain, depleted Lck from lipid rafts in Jurkat cells. Additionally, Lck localization to lipid rafts was enhanced in c-Cbl-deficient T cells. The association of Lck with c-Cbl in vivo required a functional SH3 domain. These results suggest a model whereby the SH3 domain negatively regulates basal localization of Lck to lipid rafts via association with c-Cbl.

The human gene (DSG2) coding for HDGC, a second member of the desmoglein subfamily of the desmosomal cadherins, is, like DSG1 coding for desmoglein DGI, assigned to chromosome 18

Desmoglein is a transmembrane glycoprotein of the cadherin superfamily present in the desmosomal junction in vertebrate epithelial cells. At least two variants of desmoglein are differentially expressed in human tissues: DGI, a characteristic desmosomal protein; and HDGC, which is, for example, expressed in the simple epithelium of the colon. Using a PCR assay, we were able to assign DSG2, the gene coding for desmoglein HDGC, to chromosome 18, the same chromosomal localization to which we have previously assigned DSG1 coding for desmoglein DGI.

Keratinization is associated with the expression of a new protein related to the desmosomal cadherins DGII/III

Amino acid sequencing of a 48/46 kDa glycoprotein from human plantar callus, recognised by antisera raised against the desmosomal cadherins DGII/III has revealed N‐terminal homology to the DNA‐derived sequence of human and bovine DGII/III. However, a tryptic fragment has homology only with a bovine clone. We propose that there are two classes of DGII/III‐like molecule, that represented by the bovine cDNA clone and the 48/46 kDa protein, a monoclonal antibody against which stains mainly the suprabasal layers of human epidermis, and that represented by the human cDNA clone, identified by a monoclonal antibody which stains uniformly the living layers of the epidermis.