Heat-shock protein hsp90 governs the activity of pp60v-src kinase.

During or immediately after synthesis in vertebrate cells, the oncogenic protein-tyrosine kinase pp60v-src associates with the approximately 90-kDa heat-shock protein (hsp90). In this complex, pp60v-src is not functional as a kinase. When pp60v-src is subsequently found inserted into the plasma membrane, it is active as a kinase and is no longer associated with hsp90. We have taken advantage of genetic manipulations possible in Saccharomyces cerevisiae to investigate the function and specificity of the association between hsp90 and pp60v-src. Expression of pp60v-src is known to be toxic to S. cerevisiae cells. We find that this toxicity is due to a very specific effect on growth, arrest at a particular point in the cell cycle. In cells expressing v-src, a mutation that lowers the level of hsp90 expression (i) relieves cell cycle arrest and rescues growth, (ii) reduces the level of tyrosine phosphorylation mediated by pp60v-src, (iii) changes the pattern of tyrosine phosphorylation, and (iv) reduces the concentration of pp60v-src. We conclude that hsp90 does not simply suppress pp60v-src kinase activity during transit to the plasma membrane, as previously suggested, but also stabilizes the protein and affects both its activity and specificity. This function of hsp90 is highly selective for pp60v-src: the same hsp90 mutation has no effect on the activity or specificity of the exogenous pp160v-abl tyrosine kinase; similarly, it does not affect the specificity and has only a very small effect on the activity of the exogenous pp60c-src kinase.     

Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src.

Protein phosphatase 1, one of four major protein phosphatases involved in cellular regulation, was phosphorylated in vitro by pp60v-src, the transforming gene product of Rous sarcoma virus. Phosphorylation was accompanied by a loss of protein phosphatase activity. The inactivation of protein phosphatase 1 was time-dependent and the extent of inactivation correlated closely with the stoichiometry of phosphorylation. Under optimal conditions, 0.34 +/- 0.01 mol of phosphate were incorporated per mol of protein phosphatase and the activity of the enzyme was decreased by 39 +/- 2%. The inactivation required the presence of both MgATP and pp60v-src. There was no loss of activity when adenosine 5'-[beta gamma-imido]triphosphate was used in place of ATP. Phosphorylation of protein phosphatase 1 occurred exclusively on tyrosine residues and was blocked by specific antibodies to pp60v-src. During preincubation of pp60v-src at 41 degrees C, its protein kinase activity towards casein was lost rapidly. The ability of pp60v-src to phosphorylate and inactivate protein phosphatase 1 declined in parallel with the loss of casein kinase activity. Limited chymotryptic digestion of 32P-labeled protein phosphatase 1 (Mr 37,000) resulted in its quantitative conversion to a Mr 33,000 species. Conversion to this species was accompanied by the loss of 32P-labeling and by reactivation of the protein phosphatase. When various concentrations of chymotrypsin were used in the digestion, there was a close correlation between conversion to the Mr 33,000 species and the restoration of protein phosphatase activity. pp60v-src was unable to phosphorylate or inactivate a partially proteolyzed species of protein phosphatase 1 (Mr 33,000/34,000).     

Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk.

A recombinant baculovirus was constructed for the production of the serine-specific protein kinase, pp90rsk (where rsk is ribosomal S6 kinase), in insect cells. The Xenopus pp90rsk expressed in the infected cells had nearly undetectable enzyme activity in contrast to the same enzyme coproduced with the v-src oncogene product pp60v-src. The transforming gene product pp60v-src very effectively activated pp90rsk, whereas the products of c-src and the myristoylation-minus nontransforming virus NY315 were markedly less effective. Only a fraction of the total pp90rsk population was activated, and it could be partially separated from unactivated protein by ion-exchange chromatography. When compared to the unactivated form, the activated enzyme displayed about a 4000-fold increase in the capacity to phosphorylate the ribosomal protein S6. The enhanced enzymatic activity appeared to be due to phosphorylation of pp90rsk.     

Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.

The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.     

pp60v-src tyrosine kinase is expressed and active in sarcoma-free avian embryos microinjected with Rous sarcoma virus.

Early embryonic avian tissue is resistant to transformation by Rous sarcoma virus. To determine the nature of this resistance, we examined the expression and properties of the Rous sarcoma virus transforming protein pp60v-src, in infected embryonic chicken limbs in ovo. Lysates from Rous sarcoma virus-infected limbs contained the viral structural protein p19gag, as detected by immunoblot analysis, and showed pp60v-src kinase activity in vitro. Immunoblot analysis of lysates with anti-phosphotyrosine antibodies revealed a number of phosphotyrosine-containing proteins present in lysates of Rous sarcoma virus-infected embryos but not in lysates of control, uninfected embryos. Anti-phosphotyrosine immunoreactivity was observed in frozen sections in the same cell types that expressed pp60v-src and p19gag. These studies demonstrate that pp60v-src is co-expressed with viral structural determinants in infected embryonic avian tissue. Furthermore, pp60v-src is active in ovo as a tyrosine-specific phosphotransferase, despite the apparent lack of sarcoma induction. The localization pattern of the major src gene substrate p36 (calpactin I) was compared with that of p19gag by double-label immunofluorescence and found to be generally nonoverlapping. These observations are consistent with the concept that the induction of tumors in ovo requires complementation between viral determinants and host factors. These host factors, which may be critical substrates of pp60v-src, are subject to developmental regulation in the avian embryo.     

A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: positive in vivo regulation by sterol.

The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp60v-src were examined in a sterol auxotroph of Saccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v-src shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, approximately equal to 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control.     

Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src).

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp60v-src and pp60-c-src are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v-src and pp60c-src. We have therefore characterized the sites of tyrosine phosphorylation in the two proteins. Tyrosine phosphorylation of pp60v-src in infected cells occurs mainly (if not entirely) at residue 419 in the deduced amino acid sequence of the protein. Surrounding this residue is the sequence Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg. This peptide is distinguished by the fact that three out of the four amino acids that precede the phosphorylated tyrosine are acidic in nature. These results define what may prove to be a widely used site for tyrosine phosphorylation in the regulation of cellular function. The same site was phosphorylated when partially purified pp60v-src was used in a phosphotransfer reaction in vitro. The results with pp60c-src were more complex. The site of tyrosine phosphorylation in vitro appeared to be the same as that found in pp60v-src. By contrast, phosphorylation of pp60c-src in vivo apparently occurred at a different, and currently unidentified, tyrosine residue. It is therefore possible that pp60v-src and pp60c-src respond differently to regulatory influences in the intact cell.     

The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane.

Oncogenesis by avian sarcoma virus is attributable to a single viral gene (src) which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. A closely related protein, pp60proto-src, occurs in uninfected cells from a wide variety of vertebrate species and is presumed to be the product of a cellular gene that served as progenitor for src. We explored the location of these proteins within the cell by using immunoprecipitation to analyze subcellular fractions prepared from avian sarcoma virus-transformed rat and chicken cells and from uninfected rat cells. We found that both pp60src and pp60proto-src were associated with the plasma membrane as active protein kinases and could be recovered efficiently only by disrupting the membranes with nonionic detergent. Our findings, in conjunction with those of other investigators, indicate that both proteins are embedded in the membrane by means of a hydrophobic domain(s); available evidence indicates that pp60src is not exposed on the surface of the cell but is accessible at the cytoplasmic aspect of the plasma membrane. These conclusions lend credence to two current speculations. First, pp60src and pp60proto-src may have similar or even identical functions. Second, neoplastic transformation may originate from derangements in the plasma membrane or its affiliated structures.     

Stimulation of ribosomal protein S6 kinase activity by pp60v-src or by serum: dissociation from phorbol ester-stimulated activity.

Ribosomal protein S6 kinase activity was measured in lysates prepared from serum-deprived chicken embryo fibroblasts (CEF) treated for various times with phorbol 12-myristate 13-acetate (PMA). Maximal activity was observed within 15 min, and it declined to the initial level by 4 hr. Incubation of these cells with PMA 4-60 hr after the initial treatment did not result in an additional increase in S6 protein kinase activity. These results are consistent with down-regulation of the PMA receptor, protein kinase C, and the dependence of PMA-stimulated S6 kinase activity on this enzyme. Long-term pretreatment of CEF with PMA only partially attenuated the stimulation of the S6 protein kinase activity by serum or by expression of the Rous sarcoma virus transforming gene product, pp60v-src. A similar protein kinase activity also was stimulated in cells treated with cycloheximide or sodium vanadate. Pretreatment with PMA had little effect on this response. These data indicate that it is likely that there are at least two mechanisms through which S6 kinase activity can be regulated, one of which apparently utilizes protein kinase C whereas the other(s) does not. Additional experiments show PMA-stimulated glucose transport was not attenuated by long-term incubation with phorbol ester, suggesting that another mechanism, which is not dependent on the presence of protein kinase C, maintains this response after the proposed down-regulation of the PMA receptor.     

Nonmyristoylated p60v-src fails to phosphorylate proteins of 115-120 kDa in chicken embryo fibroblasts.

We have used anti-phosphotyrosine antibodies to identify a large number of tyrosine phosphoproteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Most of these proteins were found in the 100,000 X g supernatant when cells were separated into soluble and particulate fractions; however, one group of proteins, of 115-120 kDa, was found in the particulate fraction. The phosphotyrosine content of the 115- to 120-kDa proteins was greatly reduced in chicken embryo fibroblasts infected with mutants of RSV (NY314 and SD10) encoding nonmyristoylated forms of the viral src gene product that do not associate with cellular membranes. Since RSV mutants NY314 and SD10 do not transform cells, phosphorylation of this group of 115- to 120-kDa membrane proteins may be related to the process of transformation.     

Receptor synthesis and routing to the plasma membrane.

Hormones and other extracellular proteins exert their effects on the cells that they influence by interacting with receptors in the plasma membranes of these target cells. For these interactions to occur, the receptor proteins must be efficiently synthesized, processed, and transported to the plasma membrane. This review summarizes the events and organelles involved in this process.     

Hepatocyte plasma membrane antigens. I. Determination of antibodies bound to the hepatocyte plasma membrane by 125I-protein A binding assay

Anti-liver-specific membrane lipoprotein (anti-LP-1) and anti-Tamm-Horsfall glycoprotein (anti-THGP) rabbit antibodies were found to bind to Chang liver cells, a cultured human hepatocyte cell line, and PLC/PRF/5, a hepatoma cell line. The antibodies bound were determined by an immunofluorescence staining and a semiquantitative 125I-protein A binding assay. The 125I-protein A binding assay was successfully adapted to determine anti-hepatocyte plasma membrane antibodies in sera of patients with lupoid hepatitis and chronic active hepatitis. The percentage of 125I-protein A bound in 10 normal subjects were 1.5 +/- 0.4 (mean +/- standard deviation) for PLC/PRF/5 and 1.6 +/- 0.6 for Chang liver cell, while those in 2 patients with lupoid hepatitis were 7.2 +/- 0.3, 5.9 +/- 0.1, and those in 8 patients with chronic active hepatitis 3.9 +/- 1.3, 3.2 +/- 1.5, respectively. Furthermore, a blocking study revealed that LP-1 and THGP were partially involved in antigen sites recognized with anti-hepatocyte plasma membrane antibodies in sera of a patient with lupoid hepatitis. The retaining ability of antibody binding to the hepatocytes after the absorption with non-hepatocyte cells suggested the presence of antibodies specific for the hepatocyte plasma membrane in the patient's serum.     

Chitin synthetase zymogen is attached to the yeast plasma membrane.

Pretreatment of yeast protoplasts with concanavalin A, according to the method used by G. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3H]concanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found In this fraction; in the zymogen form. About 20% of the activity sedimented in a plasma-membrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a lysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation.     

pp60c-src expression in the developing rat brain.

We have studied pp60c-src expression in the striatum, hippocampus, and cerebellum of the developing rat brain. In the striatum, pp60c-src protein kinase activity peaks during embryonic development and then declines in the adult. The peak activity occurs in the striatum on embryonic day 20 (E20) when it is 18- to 20-fold higher than the activity in fibroblasts and 4- to 5-fold higher than the activity in the striatum at E15 or in the adult striatum. In the hippocampal region, pp60c-src activity reaches a maximum shortly after birth but remains high throughout life. On postnatal day 2 (P2) the activity in the hippocampus is 9- to 13-fold higher than the activity in fibroblasts and twice as high as the activity in the hippocampus at E18. In the cerebellum, the kinase activity remains constant from E20 onward and is 6- to 10-fold higher than that observed in fibroblasts. The increase in pp60c-src kinase activity observed during the development of the striatum and hippocampus is due to an increase in the amount of pp60c-src protein and to an increase in the specific activity of the kinase. The increase in specific activity in these regions coincides with the peak periods of neurogenesis and neuronal growth. In the striatum, we have found that the increase in pp60c-src activity also parallels the increase observed in culture as embryonic striatal neurons differentiate. Taken together, our results are consonant with the idea that pp60c-src is the product of a developmentally regulated gene that is important for the differentiation and/or the continuing function of neurons.     

Altered membrane association of p60v-src and a murine 63-kDa N-myristoyl protein after incorporation of an oxygen-substituted analog of myristic acid

A number of viral and cellular proteins contain covalently bound lipid. In a subset of these acyl proteins, the 14-carbon saturated fatty acid myristic acid is attached through an amide linkage to an NH2-terminal glycine residue. Myristoyl-CoA:protein N-myristoyltransferase (NMT) transfers the myristoyl moiety from myristoyl-CoA to these nascent proteins and is highly selective for fatty acid chain length. We have found that 10-(propoxy)decanoyl-CoA (11-oxymyristoyl-CoA), an analog of myristic acid with reduced hydrophobicity, acts as a substrate for NMT both in vitro and in vivo. Comparison of the in vitro kinetic properties of a number of synthetic octapeptide substrates of NMT using myristoyl-CoA or 11-oxymyristoyl-CoA indicated that there is an interaction between the acyl-CoA and peptide binding sites of this acyltransferase. Peptide catalytic efficiency with 11-oxymyristoyl-CoA was reduced relative to that with myristoyl-CoA, but the extent of the reduction varied widely among the octapeptides tested. These in vitro data accurately predicted that only a subset of myristoyl proteins synthesized in Saccharomyces cerevisiae and a murine myocyte-like cell line (BC3H1) would incorporate 11-oxy[3H]myristate. Substitution of the myristoyl moiety by the 11-oxymyristoyl moiety does not significantly affect the membrane association of most N-myristoyl proteins. However, for the tyrosine kinase p60v-src and a 63-kDa N-myristoyl protein in BC3H1 cells, analog incorporation results in marked redistribution from the membrane to the cytosolic fraction. These studies demonstrate the utility of heteroatom-containing analogs for analysis of the role of myristate in acyl protein targeting. The sequence-specific nature of analog incorporation and the protein-specific effects on membrane association suggests that these compounds may represent a useful class of antiviral and antitumor agents.     

Monoclonal antibody to a plasma membrane antigen of neurons.

Fusion of spleen cells from a mouse immunized with chicken embryo retina cells with clonal mouse myeloma cells yielded a lymphocyte hybrid cell line that produced antibody that bound to neural tissue such as retina, brain, spinal cord, and dorsal root ganglia but not to other tissues tested. The antigen was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies in chicken retina, but little or no antigen was detected on axons or dendrites, Müller cells, or retina pigment cells. The activity of antigen A2B5 is relatively stable at 100 degrees C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside, and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition at 0.2 microM) or by N-acetylneuraminic acid (half-maximal inhibition at 5000 microM) but not by other purified gangliosides tested. These results suggest that the antigen is a complex ganglioside in plasma membranes of retina neuron cell bodies but not axons or dendrites.     

Thyroglobulin inhibition of thyrotropin binding to thyroid plasma membrane.

The effect of thyroglobulin (TG) on binding of TSH to thyroid plasma membranes was studied in vitro. Human and bovine thyroid plasma membranes have specific binding sites for bovine [125I]TSH. The binding of [125I]TSH is inhibited by the addition of purified TG (100 ng/microgram/ml). The inhibitory mechanism appears to be noncompetitive when subjected to Lineweaver-Burk analysis. Incubation of TG with TSH did not show an interaction, as assessed by sucrose gradient centrifugation. Plasma membranes prepared from human thyroid tissue have specific binding sites for human TG, as shown by [125I]TG binding assay. The TG binding was not affected by adding low concentrations of unlabeled bovine TSH. In the presence of very high concentrations of TSH, TG binding was increased. Hemoglobin, beta-lactoglobin, and ovalbumin did not have an inhibitory effect on [125I]TSH and [125I] TG binding to membrane preparations. Both [125I]TSH and [125I]TG binding were inhibited by 10 mM neuraminic acid. These results suggested that 1) TG released from thyroid gland may have a regulatory effect on TSH binding to its specific receptor, and 2) there are specific binding sites for TG on the thyroid plasma membrane.     

Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.

Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.     

Transfer of synaptic vesicle antigens to the presynaptic plasma membrane during exocytosis.

We have utilized immunofluorescence techniques to look for synaptic vesicle antigens on the plasma membrane of resting and active nerve terminals. Rabbit antiserum was raised against purified cholinergic synaptic vesicles from the electric organ of Narcine brasiliensis, a marine electric ray. Antibodies to synaptic vesicles were shown to bind selectively to nerve terminals in cryostat sections of frog nerve-muscle preparations. Binding was demonstrated indirectly by using fluorescein-labeled goat anti-rabbit antibodies. Structures in cross sections that bound antiserum were identified as nerve terminals because of their size, shape, and position and because they coincided with sites that bound rhodamine-conjugated alpha-bungarotoxin and had acetylcholine esterase activity. Presumably, sectioning gave antibodies access to binding sites within the nerve terminal. However, when antibodies to synaptic vesicles were added to the bathing medium of intact neuromuscular preparations prior to sectioning, antibody binding was marginal or undetectable, suggesting that few vesicle antigens were normally accessible on the outer surface of resting nerve terminals. When intact preparations were stimulated to release their vesicular acetylcholine by the addition of 1 mM LaCl3, antibody binding to the intact nerve terminals became striking. These findings suggest that the synaptic vesicle membrane and the synaptic terminal plasma membrane differ in composition. They also provide further support for the exocytotic hypothesis of neurotransmitter release, which predicts that vesicle markers should be exposed on the outside of nerve terminals when vesicles fuse with the plasma membrane during stimulation.