Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

Carboxyl methylation of platelet rap1 proteins is stimulated by guanosine 5'-(3-O-thio)triphosphate

Carboxyl methylation of platelet ras-related proteins, known as rap proteins, was investigated in this study. Platelet membrane proteins of Mr 23,000 incorporated radioactivity in the presence of S-[methyl-3H]adenosylmethionine and platelet cytosol. About 97% of the radioactivity present in the Mr 23,000 proteins was liberated as volatile methanol under basic (1 M sodium hydroxide) conditions. Cycloheximide, an inhibitor of protein synthesis, inhibited incorporation of S-[methyl-3H]adenosylmethionine by 25%. These results suggest that at least 75% of the radioactivity present in the Mr 23,000 proteins is due to carboxyl methylation and not due to the incorporation of S-[methyl-3H]adenosylmethionine into proteins or due to the incorporation of base-stable methyl groups into side chains of arginine, histidine, or lysine residues. Protein methylation did not occur if membranes or cytosol alone was incubated with S-[methyl-3H]adenosylmethionine. Guanosine 5'(3-O-thio)triphosphate increased methylation of the Mr 23,000 proteins in a time- and concentration-dependent manner. Acetyl-farnesylcysteine, a synthetic substrate for carboxyl methyltransferases, completely blocked methylation of the Mr 23,000 membrane proteins. On the basis of one- and two-dimensional Western blots using rap-specific antisera, the Mr 23,000 methylated proteins were identified as rap1 proteins. The existence of the carboxyl-terminal CAAX motif in rap1 proteins, similar to the CAAX motif present in p21ras as well as in the yeast mating factors, leads us to suggest that methylation of rap1 proteins possibly occurs at the alpha-carboxyl-terminal cysteine.     

Carboxyl methylation of cytosolic proteins in intact human erythrocytes. Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase

Intact human erythrocytes incubated with L-[methyl-3H]methionine incorporated radioactivity into base-labile linkages with membrane and cytosolic proteins which are characteristic of protein methyl esters. Kinetic analysis of the methylation reactions in intact cells shows that individual erythrocytes contain approximately 38,000 and 115,000 protein methyl esters with biological half-lives of 150 min or less in the membrane and cytosolic protein fractions, respectively. Fractionation of the methylated cytosolic species by gel filtration chromatography at pH 6.5 followed by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 reveals that many different cytosolic proteins serve as methyl acceptors and that the degree of modification varies widely for individual proteins. For example, hemoglobin is modified to the extent of 3 methyl groups/10(6) polypeptide chains, while carbonic anhydrase contains 1 methyl group/approximately 16,500 polypeptide chains at steady state. Aspartic acid beta-[3H]methyl ester (Asp beta-[3H]Me) can be isolated from carboxypeptidase Y digests of cytosol proteins. By synthesizing and separating diastereomeric L-Leu-L-Asp beta Me and L-Leu-D-Asp beta Me dipeptides, we show that all of the Asp beta-[3H]Me recovered from cytosolic proteins is in the D-stereoconfiguration. Based on these data and on previous observations that erythrocytes contain a single methyltransferase which also methylates red cell membrane proteins at D-aspartyl residues both in vivo (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464) and in vitro (O'Connor, C. M., and Clarke, S. (1983) J. Biol. Chem. 258, 8485-8492), we propose that protein carboxyl methylation is part of a generalized mechanism for metabolizing damaged proteins. The infrequent and spontaneous occurrence of D-aspartyl residues in proteins adequately explains the broad substrate specificity and limited stoichiometries of protein carboxyl methylation reactions.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.

A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.     

Membrane protein carboxyl methylation increases with human erythrocyte age. Evidence for an increase in the number of methylatable sites

The level of carboxyl methylation of membrane proteins has been measured in intact human erythrocyte populations of different ages separated by density gradient centrifugation. Age separation was confirmed by measurement of cytosolic pyruvate kinase specific activity in each fraction. When cells of different ages were incubated with L-[methyl-3H]methionine, the steady state level of 3H radioactivity covalently bound to membrane proteins is observed to be at least 3-fold higher in older erythrocytes. Because the specific radioactivity of the methyl group donor S-adenosyl-L-[methyl-3H]methionine was identical in all age fractions, this represents an increase in the extent of modification of membrane proteins by carboxyl methylation. Of the three major methylated erythrocyte membrane proteins, this increase in carboxyl methylation with age is 4 to 7-fold for bands 2.1 and 3, while the increase in band 4.1 is 3 to 4-fold. This increase in the steady state level of methylation with age cannot be explained by changes in either the intrinsic rate of methyl transfer or by changes in the rate constant of methyl turnover. We, therefore, propose that the age-dependent change in carboxyl methylation is due to an increase in the number of available acceptor sites as the erythrocyte ages in vivo. Since methylation of acidic residues on erythrocyte membrane proteins has been detected exclusively on D-aspartic acid residues (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464), these results are consistent with an accumulation of D-aspartic acid in membrane protein due to spontaneous racemization a the cell ages. The relationship of these observations to possible functions of erythrocyte membrane protein carboxyl methylation is discussed.     

S-adenosylmethionine-dependent protein methylation in mammalian cytosol via tyrphostin modification by catechol-O-methyltransferase

It has previously been shown that incubation of mammalian cell cytosolic extracts with the protein kinase inhibitor tyrphostin A25 results in enhanced transfer of methyl groups from S-adenosyl-[methyl-3H]methionine to proteins. These findings were interpreted as demonstrating tyrphostin stimulation of a novel type of protein carboxyl methyltransferase. We find here, however, that tyrphostin A25 addition to mouse heart cytosol incubated with S-adenosyl-[methyl-3H]methionine or S-adenosyl-[methyl-14C]methionine stimulates the labeling of small molecules in addition to proteins. Base treatment of both protein and small molecule fractions releases volatile radioactivity, suggesting labile ester-like linkages of the labeled methyl group. Production of both the base-volatile product and labeled protein occurs with tyrphostins A25, A47, and A51, but not with thirteen other tyrphostin family members. These active tyrphostins all contain a catechol moiety and are good substrates for recombinant and endogenous catechol-O-methyltransferase. Inhibition of catechol-O-methyltransferase activity with tyrphostin AG1288 prevents both base-volatile product formation and protein labeling from methyl-labeled S-adenosylmethionine in heart, kidney, and liver, but not in testes or brain extracts. These results suggest that the incorporation of methyl groups into protein follows a complex pathway initiated by the methylation of select tyrphostins by endogenous catechol-O-methyltransferase. We suggest that the methylated tyrphostins are further modified in the cell extract and covalently attached to cellular proteins. The presence of endogenous catechols in cells suggests that similar reactions can also occur in vivo.     

In vivo methylation of prokaryotic elongation factor Tu.

In Salmonella typhimurium and Escherichia coli, elongation factor Tu (EF-Tu) is methylated as shown by its incorporation of labeled methyl residues from [methyl-3H]methionine. Analysis of the nature of the methyl-containing residues by protein hydrolysis, followed by paper chromatography and high voltage electrophoresis showed that both mono- and dimethyllysine are present. Eighty per cent of the EF-Tu molecules are methylated if methylation occurs at a unique lysine residue. The EF-Tu fraction which is not methylated is still able to accept methyl groups, as shown by methylation of approximately 10% of the EF-Tu after addition of chloramphenicol (D-(-)-threo-2,2-dichloro-N-[beta-hydroxy-alpha-(hydroxymethyl)-o-nitrophenethyl] acetamide) to inhibit further protein synthesis. There is no evidence of turnover of the methyl residues. We attempted to separate the methylated from the nonmethylated form of EF-Tu by isoelectric focusing on polyacrylamide gel, but were unable to do so.     

Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid-Turnover Pool Determine Methyl Accepting Capacity

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.     

Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).     

Identification of highly methylated arginine residues in an endogenous 20-kDa polypeptide in cancer cells.

Enzymatic methylation of endogenous proteins in several cancer cell lines was investigated to understand a possible relationship between protein-arginine methylation and cellular proliferation. Cytosolic extracts prepared from several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S-adenosyl-L-[methyl-3H]methionine revealed an intensely [methyl-3H]-labeled 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purified histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-kDa polypeptide, indicating that it is unlikely to be any of the histone subclasses. The [methyl-3H]group in the 20-kDa polypeptide was stable at pH 10-11 (37 degrees C for 30 min) and methylation was not stimulated by GTPgammaS (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N(G)-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenous 20-kDa arginine-methylation is a cellular proliferation-related posttranslational modification reaction.     

Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.     

Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae

Eukaryotic elongation factor 1A (eEF1A) is an abundant cytosolic protein in Saccharomyces cerevisiae and is well conserved amongst species. This protein undergoes multiple posttranslational modifications, including the N-methylation of four side chain lysine residues. However, the enzyme(s) responsible for catalyzing these modifications have remained elusive. Here we show by intact protein mass spectrometry that deletion of either of two genes coding for putative methyltransferases results in a loss in mass of eEF1A. Deletion of the YHL039W gene, a member of the SET domain subfamily including cytochrome c and ribosomal protein lysine methyltransferases, results in an eEF1A mass loss corresponding to a single methyl group. Deletion in the YIL064W/SEE1 gene, encoding a well conserved seven beta strand methyltransferase sequence, has been shown previously to affect vesicle transport; in this work we show that deletion results in the loss of two methyl group equivalents from eEF1A. We find that deletion of thirty-five other putative and established SET domain and seven beta strand methyltransferases has no effect on the mass of eEF1A. Finally, we show that wild type extracts, but not YIL064W/SEE1 mutant extracts, can catalyze the S-adenosylmethionine-dependent in vitro methylation of hypomethylated eEF1A. We suggest that YHL039W (now designated EFM1 for elongation factor methyltransferase 1) and YIL064W/SEE1 encode distinct eEF1A methyltransferases that respectively monomethylate and dimethylate this protein at lysine residues.     

Identification of a site for carboxyl methylation in human alpha-globin

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.     

Isolation of glutamic acid methyl ester from an Escherichia coli membrane protein involved in chemotaxis.

We have isolated glutamic acid 5-methyl ester from an Escherichia coli protein that is involved in chemotaxis. The bacteria were first incubated with [methyl-3H]methionine under conditions which are known to result in methylation of the protein. The protein, isolated by gel electrophoresis, was then digested by successive treatment with three proteolytic enzymes. One of the products was [methyl-3H]glutamic acid 5-methyl ester, identified by comparison with an authentic sample in the following studies: (a) chromatography on an automatic amino acid analyzer, (b) chromatography on paper in two solvent systems, (c) chromatography on paper of the N-acetyl derivatives, and (d) stability of the ester bond to various pH conditions. No aspartic acid 4-methyl ester was found in the enzymatic digest. Treatment of the methylated protein with alkali released the radioactivity as [3H]methanol, which was identified by gas chromatography and by preparation of the 3,5-dinitrobenzoate.     

Relationship among methylation,isoprenylation, and GTP binding in 21-to 23-kDa proteins of neuroblastoma

1. Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [alpha-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate. 2. A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [alpha-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP. 3. The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (greater than 70%). 4. Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (greater than 80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated. 5. Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding. 6. Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.     

Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5'-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.     

Evidence for the carboxyl methylation of nuclear lamin-B in the pancreatic beta cell

Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin-B) undergo posttranslational modifications (e.g., isoprenylation and methylation) at their C-terminal cysteine. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Herein, we examined whether nuclear lamin-B undergoes carboxyl methylation in islet beta cells. A 65- to 70-kDa protein was carboxyl methylated in intact rat islets and clonal beta (HIT or INS) cells or in homogenates which could be immunoprecipitated using lamin-B antiserum. Incubation of purified HIT cell-nuclear fraction with [(3)H]S-adenosyl methionine yielded a single carboxyl methylated protein peak (ca. 65-70 kDa); this protein was immunologically identified as lamin-B. Several methylation inhibitors, including acetyl farnesyl cysteine, a competitive inhibitor of protein prenyl cysteine methylation, inhibited the carboxyl methylation of lamin-B, indicating that the carboxyl-methylated amino acid is cysteine. These findings, together with our recent observations demonstrating that inhibition of protein isoprenylation causes apoptotic death of the pancreatic beta cell, raise an interesting possibility that inhibition of C-terminal cysteine modifications of lamin-B might result in disruption of nuclear assembly, leading to further propagation of apoptotic signals, including DNA fragmentation and chromatin condensation.