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J E Buss

Publications and source records attributed to J E Buss.

At least 37 records · Page 2Linked to original sources
PubMed1992

Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity.

We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val12]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (approximately 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.

3T3 Cells↗
PubMed1992

Structure and biological effects of lipid modifications on proteins.

Both the prevalence of lipid modifications of proteins and their importance for protein function and cellular localization have been widely observed. The advances made during the past year in defining the enzymology of lipid addition and in understanding the biological consequences of these modifications on protein function are discussed.

Acyltransferases↗
PubMed1992

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

3T3 Cells↗
PubMed1992

Prenoids and palmitate: lipids that control the biological activity of Ras proteins.

Ras proteins can be modified by two types of lipids--an isoprenoid and the fatty acid palmitate. These lipids help the otherwise cytoplasmic Ras protein to interact with the plasma membrane of a cell. The biological consequences of this association between Ras and membranes are dramatic, and can alter a cell's behavior from normal growth into malignancy. The scope and limits of our knowledge of the steps, structures and enzymes involved in this molecular transformation from soluble inactivity to membrane-bound potency are offered below. The prospects of regulating Ras function by controlling its intracellular location provides a tantalizing opportunity to translate research into a novel therapeutic reality.

Amino Acid Sequence↗
PubMed1991

Isoprenoid modification of rab proteins terminating in CC or CXC motifs.

Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b. Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid). Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins.

Amino Acid Sequence↗
PubMed1991

The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

Animals↗
PubMed1990

Functional analysis of protein N-myristoylation: metabolic labeling studies using three oxygen-substituted analogs of myristic acid and cultured mammalian cells provide evidence for protein-sequence-specific incorporation and analog-specific redistribution.

Covalent attachment of myristic acid (C14:0) to the NH2-terminal glycine residue of a number of cellular, viral, and oncogene-encoded proteins is essential for full expression of their biological function. Substitution of oxygen for methylene groups in this fatty acid does not produce a significant change in chain length or stereochemistry but does result in a reduction in hydrophobicity. These heteroatom-containing analogs serve as alternative substrates for mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97) and offer the opportunity to explore structure/function relationships of myristate in N-myristoyl proteins. We have synthesized three tritiated analogs of myristate with oxygen substituted for methylene groups at C6, C11, and C13. Metabolic labeling studies were performed with these compounds and (i) a murine myocyte cell line (BC3H1), (ii) a rat fibroblast cell that produces p60v-src (3Xsrc), or (iii) NIH 3T3 cells that have been engineered to express a fusion protein consisting of an 11-residue myristoylation signal from the Rasheed sarcoma virus (RaSV) gag protein linked to c-Ha-ras with a Cys----Ser-186 mutation. This latter mutation prevents isoprenylation and palmitoylation of ras. Two-dimensional gel electrophoresis of membrane and soluble fractions prepared from cell lysates revealed different patterns of incorporation of the analogs into cellular N-myristoyl proteins (i.e., protein-sequence-specific incorporation). In addition, proteins were identified that underwent redistribution from membrane to soluble fractions after incorporating one but not another analog (analog-specific redistribution). Comparable studies using the model RaSV-ras chimeric protein also demonstrated analog-specific differences in incorporation, varying from approximately 25% of the total RaSV-ras chimeric protein with 5-octyloxypentanoate to greater than 50% with 12-methoxydodecanoate. Modification by this latter compound was so extensive that the amount of membrane-associated N-myristoylated protein was decreased. Incorporation of each of the analogs caused a dramatic redistribution to the soluble fraction, comparable to that seen when myristoylation was completely blocked by mutating the protein's site of myristate attachment (glycine) to an alanine residue. The demonstration that these analogs differ in the extent to which they are incorporated and in their ability to cause redistribution of any single protein suggests that they may also have sufficient selectivity to be of potential therapeutic value.

Animals↗
PubMed1990

Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.

Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.

Animals↗
PubMed1989

Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal.

The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.

Animals↗
PubMed1989

Activation of cellular p21ras by myristoylation.

p21ras is palmitoylated on a cysteine residue near the C-terminus. Changing Cys-186 to Ser in oncogenic forms produces a non-palmitoylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. To examine whether palmitate acts in a general way to increase ras protein hydrophobicity, or is involved in more specific interactions between p21ras and membranes, we constructed genes that encode non-palmitoylated ras proteins containing myristic acid at their N-termini. Myristoylated, activated ras, without palmitate (61Leu/186Ser) exhibited both efficient membrane association and full transforming activity. Unexpectedly, we found that myristoylated forms of normal cellular ras were also potently transforming. Myristoylated c-ras retained the high GTP binding and GTPase characteristic of the cellular protein and, moreover, bound predominantly GDP in vivo. This implied that it continued to interact with GAP (GTPase-activating protein). While the membrane binding induced by myristate permitted transformation, only palmitate produced a normal (non-transforming) association of ras with membranes and must therefore regulate ras function by some unique property that myristate does not mimic. Myristoylation thus represents a novel mechanism by which the ras proto-oncogene protein can become transforming.

Acylation↗
PubMed1989

p21ras is modified by a farnesyl isoprenoid.

Association of oncogenic ras proteins with cellular membranes appears to be a crucial step in transformation, ras is synthesized as a cytosolic precursor, which is processed to a mature form that localizes to the plasma membrane. This processing involves, in part, a conserved sequence, Cys-Ali-Ali-Xaa (in which Ali is an amino acid with an aliphatic side chain and Xaa is any amino acid), at the COOH terminus of ras proteins. Yeast a-factor mating hormone precursor also possesses a COOH-terminal Cys-Ali-Ali-Xaa sequence. However, while the COOH-terminal cysteine has been implicated as a site of palmitoylation of ras proteins, in mature a-type mating factor this residue is modified by an isoprenoid, a farnesyl moiety. We asked whether the Cys-Ali-Ali-Xaa sequence signaled different modifications for the yeast peptides (farnesylation) than for ras proteins (palmitoylation) or whether ras proteins were similar to the mating factors and contained a previously undiscovered isoprenoid. We report here that the processing of ras proteins involves addition of a farnesyl moiety, apparently at the COOH-terminal cysteine analogous to the cysteine modified in the yeast peptides, and that farnesylation may be important for membrane association and transforming activity of ras proteins.

Amino Acid Sequence↗
PubMed1988

A 22 kDa ras-related G-protein is the substrate for an ADP-ribosyltransferase from Clostridium botulinum.

A ribosyltransferase from C. botulinum type D ADP-ribosylated a protein of 22 kDa (p22) in human astrocytoma (1321N1) cells. ADP-ribosylation of membrane-bound p22 was potentiated by 2 mM MgCl2 or guanine nucleotides but was much reduced in the presence of 10 mM Mg2+ plus GTP gamma S. p22 was immunoprecipitated by a monoclonal antibody (142-24E05) raised against a peptide sequence common to the ras gene family but not by other ras or G-protein antibodies. p22 was also ADP-ribosylated in Drosophila but was not detected in Dictyostelium. These data suggest that the 22 kDa botulinum toxin substrate is a GTP-binding protein and a member of the ras protein family.

Adenosine Diphosphate Ribose↗
PubMed1988

The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras.

We have used oligonucleotide-directed mutagenesis to replace the N-terminal amino acids of p21v-ras with residues which mimic the amino terminus of p60v-src. p21v-ras protein possessing only the first five amino acids of p60src was not myristylated, while substitution of residue 6 (serine) produced a protein p21(GSSKS) which incorporated [3H]myristic acid that was stable to hydroxylamine, sensitive to inhibitors of protein synthesis, and found in both the normally nonacylated precursor and mature forms of p21(GSSKS). This defines the minimum framework of the p60v-src myristylation signal (glycine 2 and serine 6) and identifies serine 6 as a crucial part of that signal for myristylation of a protein in vivo.

Amino Acid Sequence↗
PubMed1987

Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins.

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

Animals↗
PubMed1986

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation.

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.

Actins↗
PubMed1986

The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity.

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.

Animals↗