Epidermal growth factor and epidermal growth factor receptor-dependent phosphorylation of a Mr = 34,000 protein substrate for pp60src

A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine.     

Identification and characterization of a novel cytoskeleton-associated pp60src substrate.

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.     

Phenothiazine binding by a homolog of calpactin, the pp60src tyrosine kinase substrate

Microvilli isolated from 13762 mammary ascites tumor cells contain a major calcium-sensitive protein (AMV-p35) that can be isolated with microvillar microfilament cores prepared by Triton X-100 extraction in the presence but not absence of calcium. AMV-p35 can be readily purified from ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid extracts of the microfilament cores by chromatography on an anion exchange column, to which it does not bind. Immunoblot analysis indicates that AMV-p35 is related to calpactin I, the pp60src tyrosine kinase substrate. In the presence of calcium, AMV-p35 binds approximately 4 mol of chlorpromazine per mole of protein in a binding process showing apparent positive cooperativity, similar to calmodulin; however, in contrast to calmodulin, AMV-p35 also binds phenothiazine in the absence of calcium.     

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.     

Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies.

Previous work in this laboratory has established that a rat liver nuclear phosphoprotein (B2:Mr 68,000, pI 6.5-8.2) is associated with actively transcribed nucleosomes, as demonstrated by its preferential release after mild treatment with micrococcal nuclease. In the present report we provide further immunological evidence ('Western Blot' analysis, solid-phase radioimmunoassay and indirect immunofluorescence) that in addition establishes the presence of this phosphoprotein in the nuclear-matrix protein fraction. This paradoxical localization suggests that this phosphoprotein may function in two separate and distinct roles within the realm of nuclear organization.     

Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate.

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.     

Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src.

Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src. A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein.     

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.     

Diurnal variation of lymphocyte subsets identified by monoclonal antibodies.

Monoclonal antibodies specific for lymphocyte subsets were used to examine circulating lymphocytes obtained at frequent intervals from healthy subjects. A diurnal rhythm was found in the total numbers of lymphocytes, T cells, inducer/helper cells, suppressor/cytotoxic cells, Ia positive cells, and B cells. The lowest levels of all subsets were seen at 0900 hours and the highest levels at 2100. In some subjects the ratio of helper to suppressor cells varied considerably during the sample period, though the ratio was relatively constant for the group as a whole.     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).     

The submembranous location of p11 and its interaction with the p36 substrate of pp60 src kinase in situ

p36, a major cytoplasmic substrate of pp60 src kinase, is present beneath the plasma membrane. It can be isolated either as a monomer or as a heterotetramer (protein I) containing two copies each of p36 and a unique p11 polypeptide. To compare the expression rules of p36 and p11 as well as their cellular distributions, monoclonal antibodies to the two porcine proteins were isolated. In tissue culture cells p11-specific antibodies decorated the same submembranous compartment previously seen with antibodies to p36 and fodrin or spectrin and followed the p36 images under all fixation/extraction conditions tested. Immunofluorescence microscopy on tissue sections showed coincident expression patterns of both proteins confirming and extending previous results with p36 antibodies. Antibodies with limited cross-species reaction have been used to trace the fate of porcine p11 and p36 injected into cultured cells. Both proteins are incorporated in the submembranous compartment, where they remain in Triton cytoskeletons prepared in the presence but not in the absence of Ca2+. The incorporation of p36 in vivo conforms with its Ca2+-dependent binding to actin, fodrin, and certain phospholipids in vitro. In contrast, the incorporation of p11 seems to depend on an in situ interaction with p36 or an exchange with endogenous p11 present on p36. The combined results indicate a strong coupling of p11 and p36 in cellular compartmentalization and tissue differentiation.     

Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex

Two related cellular proteins, p80 and p85 (cortactin), become phosphorylated on tyrosine in pp60src-transformed cells and in cells stimulated with certain growth factors. The amino-terminal half of cortactin is comprised of multiple copies of an internal, tandem 37- amino acid repeat. The carboxyl-terminal half contains a distal SH3 domain. We report that cortactin is an F-actin-binding protein. The binding to F-actin is specific and saturable. The amino-terminal repeat region appears to be both necessary and sufficient to mediate actin binding, whereas the SH3 domain had no apparent effect on the actin- binding activity. Cortactin, present in several different cell types, is enriched in cortical structures such as membrane ruffles and lamellipodia. The properties of cortactin indicate that it may be important for microfilament-membrane interactions as well as transducing signals from the cell surface to the cytoskeleton. We suggest the name cortactin, reflecting the cortical subcellular localization and its actin-binding activity.     

Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies

We identify two tissue-specific molecules in the sea urchin embryo by an immunofluorescence technique capable of co-localizing monoclonal antibodies on the same tissue section. The technique uses monovalent Fab-fluorochrome conjugates as secondary reagents to avoid cross-talk of subsequent antibody probes. Using this technique, we show that two cell surface molecules are expressed by different cell populations in the embryo. The technique is generally applicable for antibodies regardless of species or subtype specificity, and uses commercially available reagents. The technique provides sufficient amplification and resolution for analytical work, yet is rapid enough for screening procedures. As a fluorescent counterstain, use of the dye 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) in the protocol provides a distinct fluorescent background staining of the tissue without interference with the specific antibody staining.     

Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation.

F/St mice are unique in producing high levels of both ecotropic and xenotropic murine leukemia virus. The high ecotropic virus phenotype is determined by three or more V (virus-inducing) loci. A single locus for inducibility of xenotropic murine leukemia virus was mapped to chromosome 1 close to, but possibly not allelic to, Bxv-1. Although the high ecotropic virus phenotype is phenotypically dominant, the high xenotropic virus phenotype was recessive in all crosses tested. Suppression of xenotropic murine leukemia virus is governed by a single gene which is not linked to the xenotropic V locus.     

Three different conformational states of pregnancy zone protein identified by monoclonal antibodies

Human pregnancy zone protein (PZP), related to human alpha 2-macroglobulin, forms dimeric/tetrameric (360/720 kDa) species. PZP binds proteinases which cause the cleavage of internal thiol esters in the molecule. Both the binding of proteinases, i.e. chymotrypsin, (CT) to PZP, forming PZP.CT complexes, or reaction with methylamine (MA) forming PZP.MA complexes, cause transition to a new similar conformational state. Reactivity of selected monoclonal antibodies against PZP towards the three PZP derivatives demonstrated differences in the reactivity pattern. PZP and PZP.MA share one determinant, which is missing on the PZP.CT complex. PZP after transition to PZP.CT, but not to PZP.MA, presents a neodeterminant detected by one of six monoclonal antibodies. The findings demonstrate that at least three different conformational states exist for PZP and its derivatives. Access to discriminating immunochemical tools makes possible an evaluation of the relative abundance of the different complexes in vivo.     

Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.     

Immunochemical characterization of molecules identified in human thymic epithelium by anti-thymulin monoclonal antibodies

Four high molecular weight molecules (ranging from 59 to 48 kd) were evidenced in the human thymic epithelium, after one and two dimensional immunoblot analyses, using monoclonal antibodies directed against synthetic thymulin. One of these immunoreactive proteins might correspond to the intra-thymic precursor of the hormone.     

Stage-specific and common antigens of Brugia malayi identified with monoclonal antibodies

To identify parasite antigens that trigger protective, pathogenic, and allergic immune responses during filarial infections, we generated a series of monoclonal antibodies to infective larvae, adult worms, and microfilariae of Brugia malayi, a human pathogen. Quantitative and qualitative analysis of the reaction patterns of these monoclonal antibodies indicates the existence of stage-specific antigens of B. malayi, as well as of antigens shared by different stages of this parasite and by other related and unrelated helminths. These antibodies should provide invaluable tools for the analysis of host-parasite interactions in filariasis at the molecular level.