Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.

Recent progress in the structural identification of human melanoma antigens recognized by autologous cytotoxic T cells has led to the recognition of a new melanocyte differentiation antigen, Melan-A(MART-1). To determine the properties of the Melan-A gene product, Melan-A recombinant protein was produced in Escherichia coli and used to generate mouse monoclonal antibodies (mAbs). Two prototype mAbs, A103 and A355, were selected for detailed study. Immunoblotting results with A103 showed a 20-22-kDa doublet In Melan-A mRNA positive melanoma cell lines and no reactivity with Melan-A mRNA-negative cell lines. A355, in addition to the 20-22-kDa doublet, recognized several other protein species in Melan-A mRNA-positive cell lines. Immunocytochemical assays on cultured melanoma cells showed specific and uniform cytoplasmic staining in Melan-A mRNA-positive cell lines. Immunohistochemical analysis of normal human tissues with both mAbs showed staining of adult melanocytes and no reactivity with the other normal tissues tested. Analysis of 21 melanoma specimens showed homogenous staining of tumor cell cytoplasm in 16 of 17 Melan-A mRNA-positive cases and no reactivity with the three Melan-A mRNA-negative cases.

A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain.

Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.

The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.

Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.

Organ-specific and Agamous-regulated expression and glycosylation of a pollen tube growth-promoting protein.

Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin-binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death.

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells.

A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two-hybrid system in the yeast Saccharomyces cerevisiae. Sam68 (Src-associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein. 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein. Upon poliovirus infection, Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs. Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs. These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication.

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.

Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition.

Cytotoxic lymphocytes are characterized by their inclusion of cytoplasmic granules that fuse with the plasma membrane following target cell recognition. We previously identified a cytotoxic granule membrane protein designated p15-TIA-1 that is immunochemically related to an RNA-recognition motif (RRM)-type RNA-binding protein designated p40-TIA-1. Although it was suggested that p15-TIA-1 might be derived from p40-T1A-1 by proteolysis, N-terminal amino acid sequencing of p15-TIA-1 immunoaffinity purified from a natural killer (NK) cell line by using monoclonal antibody (mAb) 2G9 revealed that p15-T1A-1 is identical to the deduced amino acid sequence of NKG7 and GIG-1, cDNAs isolated from NK cells and granulocyte-colony-stimulating factor-treated mononuclear cells, respectively. Epitope mapping revealed that mAb 2G9 recognizes the C terminus of p15-T1A-1 and p40-T1A-1. The deduced amino acid sequence of p15-T1A-1/NKG7/GIG-1 predicts that the protein possesses four transmembrane domains, and immuno-electron microscopy localizes the endogenous protein to the membranes of cytotoxic granules in NK cells. Given its subcellular localization, we propose to rename-this protein GMP-17, for granule membrane protein of 17 kDa. Immunofluorescence microscopy of freshly isolated NK cells confirms this granular localization. Target cell-induced NK cell degranulation results in translocation of GMP-17 from granules to the plasma membrane, suggesting a possible role for GMP-17 in regulating the effector function of lymphocytes and neutrophils.