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.

Calpain-10 (CAPN10) is the first type 2 diabetes susceptibility gene to be identified through a genome scan, with polymorphisms being associated with altered CAPN10 expression. Functional data have been hitherto elusive, but we report here a corresponding increase between CAPN10 expression level and regulated insulin secretion. Pancreatic beta-cell secretory granule exocytosis is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor protein complex of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin 1, and vesicle-associated membrane protein 2. We report, for the first time, direct binding of a calpain-10 isoform with members of this complex. Furthermore, SNAP-25 undergoes a Ca2+-dependent partial proteolysis during exocytosis, with calpain protease inhibitor similarly suppressing both insulin secretion and SNAP-25 proteolysis. Based upon these findings, we postulate that an isoform of calpain-10 is a Ca2+-sensor that functions to trigger exocytosis in pancreatic beta-cells.

No abstract

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.