Three distinct forms of atrial natriuretic factor receptors: kidney tubular epithelium cells and vascular smooth muscle cells contain different types of receptors

Three distinct ANF receptor subtypes have been identified and characterized from cultured canine kidney tubular (MDCK) cells and rat thoracic aortic smooth muscle (RTASM) cells. These three ANF receptor subtypes include; (1) a disulfide-linked 140 kDa protein found in RTASM cells which was reduced by sulfhydryl reagent dithiothreitol (DTT) to a 70 kDa band, (2) a disulfide-unlinked 120 kDa protein, specific to MDCK cells whose Mr was not reduced by DTT and (3) a 68-70 kDa protein prevalent in both RTASM and MDCK cells whose Mr was not reduced by DTT. The non-reducible 68-70 kDa and the reducible 140 kDa proteins showed strong affinities to the full-length ANF (99-126) and truncated ANF (103-123) peptides, however, non-reducible 120 kDa protein showed strong affinity only to the full length ANF (99-126) but negligible or very weak affinity to truncated ANF (103-123). These findings suggest that distinct ANF receptor subtypes are present in renal and vascular cells which might be linked to diverse physiological functions of ANF such as natriuresis and diuresis in kidney and vasorelaxation in vascular smooth muscle cells.     

Alternative splicing gives rise to two forms of the p68 Ca2(+)-binding protein

The p68 Ca2+ and phospholipid binding protein of the lipocortin/calpactin family appears to exist as two forms. These may be resolved into a closely-spaced polypeptide doublet by SDS-PAGE. The cloning and sequencing of p68 revealed an apparent 18 nucleotide alternative splice sequence, which could account for this observation. We show here that an antiserum directed against a synthetic peptide corresponding to the region containing the splice sequence, recognises only the upper band of the p68 doublet by both immunoprecipitation and Western blotting. These results are consistent with alternative splicing being responsible for the generation of the two forms of p68.     

The acidic peptide-specific type II protein kinases from the nucleus and the cytosol of porcine liver are distinct

The second messenger-independent acidic peptide-specific protein kinase II (casein kinase II) from the cytosol of porcine liver has been purified to apparent homogeneity by using DEAE-cellulose, hydroxyl apatite, and phosphocellulose chromatography. The native enzyme has an apparent Mr of 150,000. After sodium dodecyl sulfate-gel electrophoresis a band of Mr = 39,000 and a slightly diffuse band of Mr = 27,000 were found indicating an alpha 2 beta 2 structure of this protein kinase. A thorough comparison with the corresponding enzyme from the nucleus was performed. The two enzymes differ in the subunit composition, as the nuclear enzyme is composed of subunits with a Mr of 95,000; they further differ in the heparin sensitivity and binding to blue dextran-Sepharose. Distinct differences in their nucleotide binding sites were found upon mapping with ATP analogs, although both enzymes utilize ATP as well as GTP. On the other hand, both enzymes phosphorylate identical sites in the casein variants beta A2 and alpha S1B at comparable rates. These results demonstrate for the first time the existence of distinct nucleus and cytoplasm specific type II "casein kinases".     

Transforming growth factor-beta inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes.

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-beta (TGF-beta). In this paper, we show that TGF-beta inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-beta-treated chondrocytes show reduced 125I-IL-1 binding that returns to a normal level when TGF-beta is removed from the culture medium. The inhibitory effect of TGF-beta is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor-ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of Mr = 116 and 80 kDa and a minor band of Mr = 100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-beta treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-beta to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.     

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.     

Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the alpha subunit of the vertebrate enzyme.

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds

Herbimycin A, an antibiotic which reverses Rous sarcoma virus transformation, inhibited irreversibly the auto- and trans-phosphorylation activities of p60v-src in in vitro immune complex kinase assays. The addition of a sulfhydryl compound such as dithiothreitol, 2-mercaptoethanol, glutathione (reduced form) or cysteine abolished the ability of herbimycin A to inactivate p60v-src kinase as well as the ability to reverse transformed cell morphology, whereas the addition of oxidized glutathione, cystine or methionine showed no effect. The sulfhydryl alkylating reagent N-ethylmaleimide also, although less effectively, inactivated p60v-src kinase activity in vitro. These results suggest the likelihood that sulfhydryl groups of p60v-src are involved in the inactivation of v-src tyrosine kinase activity by herbimycin A.     

Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier

Two anion-transporting systems, i.e., the dicarboxylate carrier and the 2-oxoglutarate carrier, have been purified from rat liver mitochondria and functionally identified. The dicarboxylate carrier has been isolated in active form by hydroxyapatite chromatography after partial removal of the solubilizing detergent Triton X-114 from the mitochondrial extract. The SDS gel electrophoresis of this preparation consists mainly of one protein band with an apparent Mr of 28,000, identified as the dicarboxylate carrier. Complete purification of the 28 kDa protein in inactive form has been achieved by sequential chromatography on hydroxyapatite and Celite followed by SDS extraction of the retained protein. The 2-oxoglutarate carrier has been purified by hydroxyapatite chromatography after extensive removal of Triton X-114 from the detergent extract. SDS gel electrophoresis of the purified fraction shows a single band with an apparent Mr of 32,500. When reconstituted into liposomes, the functional properties of the two isolated carrier proteins resemble closely those of the dicarboxylate and the 2-oxoglutarate transport systems characterized in mitochondria.     

Biochemical and structural properties of a Hodgkin's disease-related membrane protein

The HeFi-1 mAb recognizes a membrane protein on Hodgkin's disease cells and on a limited number of other human cells that are either tumorigenically transformed or virally activated. Herein biochemical and structural analyses of the HeFi-1 reactive membrane protein (HRMP) were done to identify its potential importance in cellular transformation in the Hodgkin's disease cell line L428, in the T cell lymphoma line HuT 78, and in several EBV-transformed lymphoblastoid cell lines. Immunoprecipitation studies demonstrated that the mature form of the HRMP had an apparent Mr of 120 kDa in tumor cells and 116 kDa in the EBV-transformed cell lines and that it was phosphorylated at both serine and tyrosine residues in all cell lines tested. The precursor to the HRMP is an 86-kDa core protein that, after processing by high mannose N-linked glycosylation, migrates with an apparent Mr of 90 kDa. This protein is then further processed to the mature 120-kDa HRMP in part by O-linked glycosylation, the addition of sialic acid residues, and by the conversion of N-linked oligosaccharides from the high mannose to the complex type. Detectable amounts of the 90-kDa molecule can be found in the membrane and, although this protein can be phosphorylated in vitro, it is not phosphorylated in intact cells. The combined results of this study suggest that the HRMP is involved in cellular metabolism and show that an unusual amount of post-translational processing of the 90-kDa precursor results in the formation, and perhaps phosphorylation, of the mature 120-kDa HRMP.     

Serine 123 Phosphorylation Modulates p21 Protein Stability and Activity by Suppressing Ubiquitin-independent Proteasomal Degradation

The cyclin-dependent kinase inhibitor p21^Waf1/Cip1 is a major regulator of the cell cycle and plays an important role in many cellular processes, including differentiation, stress response, apoptosis, and tumorigenesis. We previously cloned the gene encoding dog p21 and found that unlike its human ortholog, dog p21 is expressed as two isoforms, one high molecular mass band of 23 kDa and one low molecular mass band of 19 kDa. In the current study, we found that the high molecular mass band is phosphorylated, whereas the low molecular mass band is hypophosphorylated. Moreover, by generating multiple mutants of dog p21, we found that serine 123 and proline 124, which form a consensus site for proline-directed phosphorylation, are required for expression of the high molecular mass p21 isoform through phosphorylation at serine 123. Most importantly, we showed that serine 123 phosphorylation inhibits ubiquitin-independent proteasomal degradation of p21 protein and subsequently, prolongs p21 protein half-life and enhances the ability of p21 to suppress cell proliferation. Taken together, these data reveal that serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation.     

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.     

Functional role of GTPase-activating protein in cell transformation by pp60v-src.

Morphological transformation of NIH 3T3 cells was observed following coexpression of a portion of the ras GTPase-activating protein (GAP) comprising the amino terminus (GAP-N) and a mutant of v-src (MDSRC) lacking the membrane-localizing sequence. Cells expressing either of these genes alone remained nontransformed. Coexpression of GAP-N with MDSRC did not alter the subcellular localization, kinase activity, or pattern of cellular substrates phosphorylated by the MDSRC product. In contrast to SHC, phospholipase C-gamma 1, and the p85 alpha phosphatidylinositol 3'-kinase subunit, the endogenous GAP product (p120GAP) was highly tyrosine-phosphorylated only in cells transformed by wild-type v-src. Furthermore, for transformation induced by wild-type v-src as well as by coexpression of MDSRC and GAP-N, a strict correlation was observed between cell transformation, elevated tyrosine phosphorylation of p62, p190, and a novel protein of 150 kDa, and complex formation between these proteins and p120GAP. As with cells transformed by wild-type v-src, the MDSRC plus GAP-N transformants remained dependent on endogenous Ras. The results suggest that tyrosine phosphorylation and complex formation involving p120GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.     

Vitellogenin diversity in tropical lizards (Anolis pulchellus): identification and partial characterization.

1. Three size-classes of vitellogenin polypeptides were detected by electrophoretic and radiolabeling techniques in 32Pi-labeled plasma of vitellogenic female lizards but not in male animals. Based on their apparent Mr, the polypeptides were designated as VTG-226-201K, VTG-169-153K and VTG-116-123K. 2. Structural differences were found between VTG-169-153K and VTG-116K by partial proteolysis with S. aureus V8 protease and radiolabeling techniques. 3. Autoradiography of a 3-10% native gradient gel revealed three different species of VTG in 32Pi-labeled plasma of stimulated males: VTG-I (Mr = 850,000), VTG-II (Mr = 750,000) and VTG-III (Mr = 610,000). By 2D PAGE, it was shown that VTG-169K and VTG-116K are components of VTG-I, while VTG-153K and VTG-116K comprise VTG-III. These results suggest an oligomeric structure for native VTG. 4. Amino acid analysis, 32Pi incorporation, electrophoretical behavior and Mr estimation demonstrated homology between VTG-116K and the S1-lipovitellin from the lizard egg. 5. These results strongly indicate an unusual multiplicity of VTG forms in tropical lizards when compared to other egg-laying vertebrates.     

Use of antibodies in the analysis of connexin 43 turnover and phosphorylation

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.     

Dlmo基因编码一个32kDa的蛋白

为了获取研究Dlmo（果蝇LMO基因的简称）的功能信息,使用耦联网织红细胞体外翻译系统将Dlmo基因进行体外转录翻译得到32kDa的蛋白产物。该蛋白产物与抗人类LMO2蛋白的多抗抗体发生免疫沉淀,并得到了32kDa的阳性带。为了证实Dlmo基因在体外和体内翻译能得到同一大小蛋白,从2～4小时果蝇胚盘中分别抽提核和胞浆提取物进行Western印迹分析,免疫血清可以识别胞浆提取物中的32kDa蛋白,而在核提取物中未曾见到,证实体外和体内翻译产物相同。免疫组织化学的分析在0～4小时胚盘外周胞浆中有Dlmo基因的阳性染色信号,结果证实,Dlmo与人类LMO不同,其表达产物是一个胞浆蛋白。 Abstract：In order to gain some insight into a possible function of Dlmo gene,we used the TNT coupled reticulocyte lysate systems as an in vitro translation system to detect the Drosoplila protein. We found a 32kDa protein product.To demonstrate that the DLMO protein has the same size in vivo as in vitro we studied nuclear and cytoplasmic extracts from 2-4h embryos in Western blots. The immuno serum recognizes a 32kDa protein in the cytoplasm that is not present in the nucleus.The products were immune precipitated with the polyclonal anti-LMO2 antibody raised against the human protein and seen a positive band of 32kDa.Using immuno histochemical analysis was seen a positive staining in the basal cytoplasm of blastoderm embryos at 4 hours. This result confirms that the DLMO is a cytoplasmprotein, not like human LMO protein.     

Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.     

Purification and Characterization of p68/70, Regeneration-Associated Proteins from Goldfish Brain

Abstract: Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu²⁺-charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5 2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high-resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a hetero-dimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydro-phobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton 114. Analysis of hydrolytic products indicated p68/70 to be a glyco-protein, containing 11% carbohydrate.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.