Two protein tyrosine phosphatases, Ptp2 and Ptp3, modulate the subcellular localization of the Hog1 MAP kinase in yeast.

The MAP kinase Hog1 transiently accumulates in the nucleus upon activation. Although Hog1 nuclear export correlates with its dephosphorylation, we find that dephosphorylation is not necessary for export. Unexpectedly, a strain lacking the nuclear protein tyrosine phosphatase, Ptp2, showed decreased Hog1 nuclear retention, while a strain lacking the cytoplasmic Ptp3 showed prolonged Hog1 nuclear accumulation, consistent with Ptp2 being a nuclear tether for Hog1 and Ptp3 being a cytoplasmic anchor. In support of this result PTP2 overexpression sequestered Hog1 in the nucleus while PTP3 overexpression restricted Hog1 to the cytoplasm. Thus, Ptp2 and Ptp3 regulate Hog1 localization by binding Hog1.

Differential regulation of the cell wall integrity mitogen-activated protein kinase pathway in budding yeast by the protein tyrosine phosphatases Ptp2 and Ptp3.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity and protein tyrosine phosphatases (PTPs) in yeasts. In Saccharomyces cerevisiae, two PTPs, Ptp2 and Ptp3, inactivate the MAPKs, Hog1 and Fus3, with different specificities. To further examine the functions and substrate specificities of Ptp2 and Ptp3, we tested whether they could inactivate a third MAPK, Mpk1, in the cell wall integrity pathway. In vivo and in vitro evidence indicates that both PTPs inactivate Mpk1, but Ptp2 is the more effective negative regulator. Multicopy expression of PTP2, but not PTP3, suppressed growth defects due to the MEK kinase mutation, BCK1-20, and the MEK mutation, MKK1-386, that hyperactivate this pathway. In addition, deletion of PTP2, but not PTP3, exacerbated growth defects due to MKK1-386. Other evidence supported a role for Ptp3 in this pathway. Expression of MKK1-386 was lethal in the ptp2Delta ptp3Delta strain but not in either single PTP deletion strain. In addition, the ptp2Delta ptp3Delta strain showed higher levels of heat stress-induced Mpk1-phosphotyrosine than the wild-type strain or strains lacking either PTP. The PTPs also showed differences in vitro. Ptp2 was more efficient than Ptp3 at binding and dephosphorylating Mpk1. Another factor that may contribute to the greater effectiveness of Ptp2 is its subcellular localization. Ptp2 is predominantly nuclear whereas Ptp3 is cytoplasmic, suggesting that active Mpk1 is present in the nucleus. Last, PTP2 but not PTP3 transcript increased in response to heat shock in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1.

A proteolytic pathway that recognizes ubiquitin as a degradation signal.

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.

A yeast protein similar to bacterial two-component regulators.

Many bacterial signaling pathways involve a two-component design. In these pathways, a sensor kinase, when activated by a signal, phosphorylates its own histidine, which then serves as a phosphoryl donor to an aspartate in a response regulator protein. The Sln1 protein of the yeast Saccharomyces cerevisiae has sequence similarities to both the histidine kinase and the response regulator proteins of bacteria. A missense mutation in SLN1 is lethal in the absence but not in the presence of the N-end rule pathway, a ubiquitin-dependent proteolytic system. The finding of SLN1 demonstrates that a mode of signal transduction similar to the bacterial two-component design operates in eukaryotes as well.

A gene encoding a putative tyrosine phosphatase suppresses lethality of an N-end rule-dependent mutant.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, mutational inactivation of the N-end rule pathway is neither lethal nor phenotypically conspicuous. We have used a "synthetic lethal" screen to isolate a mutant that requires the N-end rule pathway for viability. An extragenic suppressor of this mutation was cloned and found to encode a 750-residue protein with strong sequence similarities to protein phosphotyrosine phosphatases. This heat-inducible gene was named PTP2. Null ptp2 mutants grow slowly, are hypersensitive to heat, and are viable in either the presence or absence of the N-end rule pathway. We discuss possible connections between dephosphorylation of phosphotyrosine in proteins and the N-end rule pathway of protein degradation.

Multiple sites of methyl esterification of calmodulin in intact human erythrocytes.

Aspartyl and asparaginyl residues are susceptible to spontaneous chemical degradation reactions that result in the formation of isomerized and racemized aspartyl residues. At least a subset of these abnormal residues are recognized by a widely distributed protein D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77) that can participate in their conversion to normal L-aspartyl residues. We have used this methyltransferase as a probe to identify modified aspartyl and asparaginyl residues in peptides and proteins. In purified calmodulin from bovine brain, major sites of methylation were found to originate from the Asp-2 residue near the amino terminus and the Asp-78 residue in the alpha-helix that connects the two globular calcium-binding domains. When purified calmodulin was incubated at physiological temperature and pH in the absence of calcium, additional methylation sites were found in three of the four calcium-binding sites. In this work we have analyzed the methyl esterification of human calmodulin catalyzed by this enzyme in intact erythrocytes. On the basis of results from peptide mapping studies, Asp-2, Asp-78/80, and residues in calcium-binding domains III and IV appear to be methylated. Methylation of sites in the calcium-binding regions appears to reflect the low concentration of free calcium in human erythrocytes. We also found that calmodulin isolated from erythrocytes and methylated in vitro contains major methylation sites at Asp-2 and Asp-78/80 but not in the calcium-binding sites. Comparison of the number of available methylation sites of calmodulin in intact cells and in material aged in vitro supports the hypothesis that repair processes can occur in erythrocytes.

The gamma subunit of brain G-proteins is methyl esterified at a C-terminal cysteine.

The gamma polypeptide of brain G-proteins is carboxyl methylated when the purified beta gamma subunit complex is reconstituted with S-adenosyl-[3H-methyl]-L-methionine and a methyltransferase present in detergent-stripped brain membranes. By chromatographic analysis of the 3H-amino acid generated by exhaustive proteolysis and performic acid oxidation of the 3H-methylated beta gamma complex, we show that this modification occurs on the alpha-carboxyl group of a C-terminal cysteine residue. Our result suggests that brain G-protein may undergo multiple covalent modification steps, including proteolytic removal of the three terminal amino acids from the predicted common C-terminal Cys-Xaa-Xaa-Xaa sequence, and the methyl esterification of the resulting terminal cysteine residue. This modification is likely to be associated with lipidation at the sulfhydryl group of the same cysteine, which would explain the tight membrane binding property of the brain beta gamma complex.

The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terminal cysteine.

Retinal rod cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE), a key regulatory enzyme involved in visual excitation, is one of several outer segment membrane proteins that are carboxyl methylated in the presence of the methyl donor S-adenosyl-L-[3H-methyl]methionine. By chromatographic analyses of the 3H-methyl amino acid generated by exhaustive proteolysis of purified PDE, followed by performic acid oxidation of the digest, we have shown that this modification occurs at a C-terminal cysteine residue of the alpha subunit of this enzyme. When PDE is subjected to limited proteolysis with trypsin, a 3H-methylated fragment of 1000 daltons or less is rapidly removed prior to the degradation of its inhibitory gamma subunit. This small fragment remains membrane bound, whereas the bulk of the enzyme is released, indicating that a domain responsible for anchoring PDE to the membrane is located near the C terminus. Based on the C-terminal amino acid sequence of Cys-Cys-Val-Gln predicted from the alpha cDNA sequence, we conclude that PDE undergoes posttranslational modifications, including the proteolytic removal of two or three terminal amino acids, and methyl esterification of the alpha-carboxyl group of the terminal cysteine residue. We speculate that the sulfhydryl group of the methylated cysteine is also lipidated to mediate membrane binding. These modifications may play an important role in delivering the nascent PDE chains to the membrane and in correctly positioning the PDE molecule in the rod disks for phototransduction.

Enzymatic methylation of 23-29-kDa bovine retinal rod outer segment membrane proteins. Evidence for methyl ester formation at carboxyl-terminal cysteinyl residues.

A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77). To determine the site(s) of methylation, 3H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins (Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647).

Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin.

We have previously shown that the D-aspartyl/L-isoaspartyl protein carboxyl methyltransferase recognizes two major sites in affinity-purified preparations of bovine brain calmodulin that arise from spontaneous degradation reactions. These sites are derived from aspartyl residues at positions 2 and 78, which are located in apparently flexible regions of calmodulin. We postulated that this flexibility was an important factor in the nonenzymatic formation and enzymatic recognition of D-aspartyl and/or L-isoaspartyl residues. Because removal of Ca2+ ions from this protein may also lead to increased flexibility in the four Ca2+ binding regions, we have now characterized the sites of methylation that occur when calmodulin is incubated in buffers with or without the calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid (EGTA). Calmodulin was treated at pH 7.4 for 13 days at 37 degrees C under these conditions and was then methylated with erythrocyte D-aspartyl/L-isoaspartyl methyltransferase isozyme I and S-adenosyl-L-[methyl-3H]methionine. The 3H-methylated calmodulin product was purified by reverse-phase HPLC and digested with various proteases including trypsin, chymotrypsin, endoproteinase Lys-C, clostripain, and Staphylococcus aureus V8 protease, and the resulting peptides were separated by reverse-phase HPLC. Peptides containing Asp-2 and Asp-78, as well as calcium binding sites II, III, and IV, were found to be associated with radiolabel under these conditions. When calmodulin was incubated under the same conditions in the presence of calcium, methylation at residues in the Ca2+ binding regions was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation.

We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.

Two major isozymes of the protein D-aspartyl/L-isoaspartyl methyltransferase from human erythrocytes.

We have been able to separate protein carboxyl methyltransferase activity from human erythrocyte cytosol into two major fractions by DEAE-cellulose chromatography. These isozymes, designated I and II, are characterized by their isoelectric points of approximately 6.6 and 5.5 as determined by isoelectric focusing in polyacrylamide gels. The ratio of the isozymes (II/I) was found to range from 0.52 to 1.2 in blood samples from 14 individuals. We did not detect differences in this ratio between males and females. We also found no differences between freshly drawn and outdated blood samples. Both isozymes catalyzed the methylation of proteins such as ovalbumin as well as synthetic L-isoaspartyl-containing peptides.

Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase.

Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoaspartyl and/or D-aspartyl residues but not of normal L-aspartyl residues. In order to identify the origin of these unusual residues which occur in subpopulations of a variety of cellular proteins, we studied the in vitro methylation by the erythrocyte enzyme of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. Methylated glucagon was digested with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg C, and the labeled fragments were separated by high-performance liquid chromatography and identified. In separate experiments, methyl acceptor sites were determined by digesting glucagon first with proteases and then assaying purified glucagon fragments for methyl acceptor activity. Using both approaches, we found that the major site of methylation, accounting for about 62% of the total, was at the position of Asp-9. Chemical analysis of fragments containing this residue indicated that this site represents an L-isoaspartyl residue. A second site of methylation, representing about 23% of the total, was detected at the position of Asn-28 and was also shown to represent an L-isoaspartyl residue. Methyl acceptor sites were not detected at the positions of Asp-15 or Asp-21. Preincubation of glucagon under basic conditions (0.1 M NH4OH, 3 h, 37 degrees C) increased methylation at the Asn-28 site by 4-8-fold while methylation at the Asp-9 site remained unchanged. These results suggest that methylation sites can originate from both aspartyl and asparaginyl residues and that these sites may be distinguished by the effect of base treatment.