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J F Hancock

Publications and source records attributed to J F Hancock.

44 records · Page 3Linked to original sources
PubMed1990

Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy.

The intrinsic fluorescence properties of the oncogene protein p21N-ras,p21H-ras and one of its transforming mutants, p21N-ras (Val12), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect.

Circular Dichroism↗
PubMed1989

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.

Cysteine↗
PubMed1989

Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis.

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.

Amino Acid Sequence↗
PubMed1989

Targeting of oncoproteins to membranes by fatty acylation.

Post-translational modification of proteins with hydrophobic lipid-derived substituents is increasingly becoming recognized as a major route for targeting proteins to membranes. Glycosylphosphatidylinositol (GPI) anchors are found at the C terminus of a wide range of cell surface proteins, and may endow the cell with the ability to release them in a controlled fashion via specific phospholipases. We have concentrated on the direct attachment (acylation) of long-chain fatty acids (myristate, C14:0 and palmitate, C16:0) to proteins associated with the cytoplasmic face of cellular membranes. Two such proteins, the products of the src and ras oncogenes, require acylation respectively with myristate and palmitate for their membrane association and biological activity, including transformation. N-terminal myristoylation of p60src seems to be a cotranslational stable modification. However, our recent results show that post-translational modification of p21ras is a complex cascade of events involving proteolysis, methylation and thioesterification of palmitate. This last acylation event is dynamic in vivo and may regulate ras function. Enzymological studies of these modification events are in progress. A better understanding of acylation may provide targets for future pharmacological intervention.

Acylation↗
PubMed1988

The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product.

About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins and control cell proliferation by regulating signal transduction pathways at the plasma membrane. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site, strongly implicating GAP as the biological target for regulation by p21.

Animals↗
PubMed1988

Mutant but not normal p21 ras elevates inositol phospholipid breakdown in two different cell systems.

Expression of oncogenic mutant p21 ras either in stably transformed NIH3T3 fibroblasts or transiently in COS-1 cells elevates the basal rate of inositol phosphate production. Additional mutations in the effector region or the carboxy terminal region which abolish transforming capacity on NIH3T3 cells block the effect of oncogenic mutant p21 ras on basal rates. Overexpression of normal (Gly12) p21 ras has no such effect on this second messenger signalling system. These differences between overexpressed normal p21 ras and oncogenic mutant p21 ras strongly suggest that the increased basal rate of inositol phosphate production is a direct consequence of constitutively activated p21 ras activity.

Cell Line↗
PubMed1987

Haemorrhagic cystitis in bone marrow transplantation patients: possible increased risk associated with prior busulphan therapy.

Following bone marrow transplantation employing conditioning including 'high-dose' cyclophosphamide, 65 patients were studied for the subsequent development of symptomatic haemorrhagic cystitis. There was no protection from the urothelial toxicity of cyclophosphamide metabolites afforded by the concurrent administration of 2-mercaptoethane sodium sulphonate (mesna) if timing errors in administration were made. Other factors which might increase the risk of haemorrhagic cystitis due to cyclophosphamide administration include the prior administration of busulphan to patients with chronic granulocytic leukaemia, in whom the incidence of haemorrhagic cystitis was 36% compared with 4% in all other patients. We have also investigated the use of intravesical prostaglandin E2 as a treatment for haemorrhagic cystitis in eight patients, two of whom appeared to obtain major benefit.

Adolescent↗