Characterization of Ha-ras, N-ras, Ki-Ras4A, and Ki-Ras4B as in vitro substrates for farnesyl protein transferase and geranylgeranyl protein transferase type I.

Ras proteins are small GTP-binding proteins which are critical for cell signaling and proliferation. Four Ras isoforms exist: Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B. The carboxyl termini of all four isoforms are post-translationally modified by farnesyl protein transferase (FPT). Prenylation is required for oncogenic Ras to transform cells. Recently, it was reported that Ki-Ras4B is also an in vitro substrate for the related enzyme geranylgeranyl protein transferase-1 (GGPT-1) (James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 6221-6226). In the current studies, we compared the four isoforms of Ras as substrates for FPT and GGPT-1. The affinity of FPT for Ki-Ras4B (Km = 30 nM) is 10-20-fold higher than that for the other Ras isoforms. Consistent with this, when the different Ras isoforms are tested at equimolar concentrations, it requires 10-20-fold higher levels of CAAX-competitive compounds to inhibit Ki-Ras4B farnesylation. Additionally, we found that, as reported for Ki-Ras4B, N-Ras and Ki-Ras4A are also in vitro substrates for GGPT-1. Of the Ras isoforms, N-Ras is the highest affinity substrate for GGPT-1 and is similar in affinity to a standard GGPT-1 substrate terminating in leucine. However, the catalytic efficiencies of these geranylgeranylation reactions are between 15- and 140-fold lower than the corresponding farnesylation reactions, largely reflecting differences in affinity. Carboxyl-terminal peptides account for many of the properties of the Ras proteins. One interesting exception is that, unlike the full-length N-Ras protein, a carboxyl-terminal N-Ras peptide is not a GGPT-1 substrate, raising the possibility that upstream sequences in this protein may play a role in its recognition by GGPT-1. Studies with various carboxyl-terminal peptides from Ki-Ras4B suggest that both the carboxyl-terminal methionine and the upstream polylysine region are important determinants for geranylgeranylation. Furthermore, it was found that full-length Ki-Ras4B, but not other Ras isoforms, can be geranylgeranylated in vitro by FPT. These findings suggest that the different distribution of Ras isoforms and the ability of cells to alternatively process these proteins may explain in part the resistance of some cell lines to FPT inhibitors.

Phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase C beta 1 and is essential for its activation.

Earlier studies of a site-specific mutant of protein kinase C beta 1 (PKC beta 1) altered at Thr635 and Thr642 indicated that these phosphorylation sites are critical for enzymatic function (Zhang, J., Wang, L., Petrin, J., Bishop, W. R., and Bond, R. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,6130-6134). To determine the contribution of the individual threonines, we report here on two site-specific mutants in which either Thr635 or Thr642 was changed to alanine. When transiently overexpressed in Cos cells wild-type PKC beta 1 exists in two forms: a Triton-insoluble form with high electrophoretic mobility and a slower migrating Triton-soluble form. Mutation at Thr642 (but not Thr635) results in production of only the fast-migrating form. [35S]Methionine pulse-chase labeling indicates that wild-type PKC beta 1 is synthesized as the fast-migrating form and is subsequently converted to the slow-migrating form. 32P labeling shows that only the slow-migrating form is a phosphoprotein. Mutation of Thr642 abolishes this phosphorylation. Finally, the Thr642 mutant PKC beta 1 lacks enzymatic activity and, when expressed in NIH 3T3 cells, reduces phorbol ester-induced c-fos promoter activity. These results indicate that Thr642 phosphorylation is an early event in the processing of newly synthesized PKC beta 1 and is required for enzymatic function. These results support a role for a PKC kinase in PKC processing and activation.

Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

The autophosphorylation sites of the beta 2 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A.J., Paladini, R.D. & Koshland, D.E. (1990) Science 294, 408-411]. To define the role of autophosphorylation we constructed three site-directed mutants of PKC beta 1 isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC beta 1 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a beta isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA. Our results indicate that (i) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (ii) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subcellular localization in COS cells.

Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase.

In the adult rat brain, the gene for glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is expressed predominantly as a 3.7-kilobase transcript. Earlier data showed that embryonic brain expresses an RNA transcript distinct from the adult form; however, the exact structure of this form was not elucidated. Here, transcripts expressed in the embryonic but not the adult brain were cloned and analyzed. These transcripts include an exon not expressed in the adult inserted into coding sequence. The embryonic exon contains a stop codon that is in-frame with the coding sequence. The exon is found in genomic DNA within the GAD gene where it is flanked by introns with conventional splice sites. On the basis of these structural data, we propose the hypothesis that, early in brain development, transcripts encoding a truncated form of GAD are expressed. The deduced protein cannot function as a decarboxylase because the stop codon in the embryonic exon occurs upstream of the binding site for pyridoxal phosphate, an essential cofactor. Thus, alternative splicing plays a crucial role in the pathway leading to the development of functional GABAergic neurons. The central nervous system-derived cell lines B65 and C6 express a mixture of the adult and embryonic forms of GAD mRNA. They therefore are useful clonal models of central nervous system cells in the early phases of differentiation.

Characterization of a cDNA coding for rat glutamic acid decarboxylase.

cDNA clones have been isolated for rat glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) and 3216 bp of the sequence have been determined. This sequence extends the previously reported feline GAD cDNA sequence both in the 5' (67 bp) and 3' (887 bp) directions and contains the polyadenylation signal and tail. The cDNA codes for a 67 kDa mol. wt. protein beginning from the putative initiator methionine found in the feline sequence. Extensive homology to feline GAD was identified at the amino acid level (97% identity) within the coding region. This interspecies homology is high compared to other neurotransmitter synthesizing enzymes and suggests selective pressure to maintain the primary sequence throughout the full length of the protein. Homology is found 5' to the putative initiator methionine. Extensive stretches of homology are also found in the 3' non-coding region. These conserved non-coding regions may play a role in GAD mRNA regulation. The rat cDNA sequence will facilitate investigations into the structure and regulation of the GAD gene.

Pattern of expression of glutamic acid decarboxylase mRNA in the developing rat brain.

The time and pattern of appearance of glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) mRNA during the development of the rat brain were analyzed. RNA transfer blot analysis of poly(A)+ RNA from whole brain shows that a 3.7-kilobase transcript is the most abundant form of the message from embryonic day 15 (E15) through adulthood. By E15 this form is present at about 50% of its adult abundance relative to other poly(A)+ mRNA species. At birth the abundance is approximately the same as in the adult. In contrast, the enzyme activity level is only 8% of the adult level at birth and takes 3 weeks to reach adult levels. There are qualitative changes in GAD mRNA during development. Several large (7-9 kilobases) transcripts with strong homology to GAD are enriched in early developmental stages but are barely detectable in the adult. A nuclease protection assay shows a developmentally regulated heterogeneity in a coding portion of the mRNA.

Degradation of aspartate transcarbamylase in Bacillus subtilis is deficient in rel mutants but is not mediated by guanosine polyphosphates.

Degradation of aspartate transcarbamylase in growing and starved Bacillus subtilis was deficient in relA and relC mutants, but these effects were not correlated with differences in the intracellular level of guanosine polyphosphates.

Nutritional regulation of degradation of aspartate transcarbamylase and of bulk protein in exponentially growing Bacillus subtilis cells.

The rate of degradation of aspartate transcarbamylase in exponentially growing Bacillus subtilis cells was determined by measurement of enzyme activity after the addition of uridine to repress further enzyme synthesis and by specific immunoprecipitation of the enzyme from cells grown in the presence of [3H]leucine. Aspartate transcarbamylase was degraded with a half-life of about 1.5 h in cells growing on a glucose-salts medium with NH4+ ions as the sole source of nitrogen. Replacement of NH4+ in this medium with a combination of the amino acids aspartate, glutamate, isoleucine, proline, and threonine reduced the degradation rate to an undetectable level. Various other amino acids and amino acid mixtures had smaller effects on the rate of degradation. The carbon source also influenced the degradation rate, but to a smaller extent than the nitrogen source. The effects of these nutritional variables on the rate of bulk protein turnover in growing cells were generally similar to their effects on degradation of aspartate transcarbamylase. Since the degradation of aspartate transcarbamylase has been shown to be 10 to 20 times faster than bulk protein turnover, the results suggest that a substantial portion of protein turnover in growing cells represents regulable, rapid degradation of a number of normal proteins, of which aspartate transcarbamylase is an example.