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.     

The 34 kd pp60src substrate is located at the inner face of the plasma membrane

The subcellular localization of the 34 kd protein substrate of the pp60src kinase was investigated by immunofluorescence microscopy and subcellular fractionation. When permeabilized fibroblasts were stained with a monoclonal anti-34 kd protein antibody, a diffuse reticular pattern was observed. The 34 kd protein was not exposed on the outside surface of the cell. Double immunofluorescence staining experiments established that the 34 kd protein distribution was similar to that of the membrane-associated protein alpha-spectrin. The 34 kd protein was found in cell sections to be concentrated along the cell edges. Taken together, these results suggested that the 34 kd pp60src substrate was associated with the inside surface of the plasma membrane. This conclusion was supported by subcellular fractionation experiments in which the 34 kd protein was observed to fractionate with the plasma membrane. These localization studies support further the hypothesis that many of the primary effects of the pp60src kinase occur at the plasma membrane.     

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.     

Demonstration of avian sarcoma virus-coded pp60src in vivo and the anti-pp60src immune response in chickens.

The avian sarcoma virus (ASV)-coded transforming protein pp60src was originally detected in vitro in ASV-transformed avian and mammalian cells in experiments involving mammalian antisera to ASV-induced tumors. It is demonstrated here that pp60src is also expressed in vivo in ASV tumors of chickens. Furthermore, the existence of the endogenous pp60src in all chicken cells does not impair the immune response to exogenous pp60src in the chicken. Whereas chicken antibodies can bind to pp60src, they do not serve as substrates for the protein kinase activity of this transforming protein.     

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.     

Detection of a tyrosine kinase in human sera and blood cells by pp60src antiserum

Using sera of Rous sarcoma virus-tumor bearing rabbits (TBR-sera) as a tool to detect pp60src kinase in immunoprecipitates, we report here that about 10% of our TBR-sera revealed tyrosine kinase activity in human serum, plasma and in soluble extracts of human blood cells. The activity found in serum represents 5-12% of the total kinase activity in blood. Most of the enzyme activity was detected in lymphocytes and polymorphonuclear cells rather than in platelets and erythrocytes. We also demonstrate that the tyrosine kinase in serum is inhibited by quercetin, a potent inhibitor of the viral pp60src protein kinase.     

Cyclic AMP treatment of Rous sarcoma virus-transformed Chinese hamster ovary cells increases phosphorylation of pp60src and increases pp60src kinase activity

Treatment of growing Rous sarcoma virus-transformed Chinese hamster ovary cells with the cyclic AMP analog 8-bromo-cyclic adenosine 3',5'-monophosphate (8-bromo-cyclic AMP) stimulates the incorporation of 32Pi into the viral transforming protein pp60src. Based on one-dimensional and two-dimensional peptide analysis and phosphoamino acid analysis, the increase is on a single phosphoserine residue at the NH2 terminus of the protein. The phosphate incorporation increases during the first 4 h of treatment. The pp60src kinase activity in extracts of cells treated with 8-bromo-cyclic AMP was stimulated about 2- to 3-fold. This stimulation of kinase activity increased during the first 3 h of treatment with 1 mM 8-bromo-cAMP and the activity was increased in both the soluble and particulate fraction of the cells. These results suggest that cyclic AMP can modulate the activity of pp60src in transformed cells.     

Sequence homologies between p36, the substrate of pp60src tyrosine kinase and a 67 kDa protein isolated from bovine aorta

A 67 kDa actin-binding protein was isolated from bovine aorta. Partial amino acid sequence determination of two large thermolysin peptides were used to compare 67 kDa bovine aorta protein and p36 the substrate of pp60src tyrosine kinase. Sequence analysis shows that 67 kDa bovine aorta protein shares common domains with p36 and possesses the consensus aminoacid sequences of mammalian Ca2+-dependent membrane-binding protein and p36/gelsolin.     

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.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

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.-).