The action of v-src on gap junctional permeability is modulated by pH

The product of the viral src gene (v-src) is the protein tyrosine kinase pp60v-src. Among the known consequences of pp60v-src activity is the reduction in permeability of gap junctions, an effect that is counteracted by the calcium antagonist TMB-8 (8-N,N-[diethylamino]octyl-3,4,5-trimethoxybenzoate). We show here that a decrease in intracellular pH (pHi) also counteracts the v-src effect: junctional permeability of cells containing active v-src kinase rose with decreasing pHi in the range 7.15 to 6.75, whereas junctional permeability of cells containing inactive v-src kinase or no v-src at all was insensitive to pH in that range. Low pH also counteracted the known action of diacylglycerol on junction, but only when pp60v-src kinase was inactive. Immunoblots of whole-cell lysates using an antibody against phosphotyrosine show that phosphorylation on tyrosine of at least one cellular protein, specific for pp60v-src kinase activity, was reduced by low pH but not by TMB-8. These results suggest that TMB-8 does not inhibit v-src action on junctional permeability by interfering with tyrosine phosphorylation of a protein crucial for closure of gap junction channels, but that the inhibition by low pH may be via this mechanism.     

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5'')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.     

The pp60v-src tyrosine kinase desensitizes epidermal growth factor binding to 3T3 fibroblasts by two distinct protein kinase C-independent mechanisms

Protein kinase C (Ca2+/phospholipid-dependent enzyme) is known to phosphorylate the epidermal growth factor receptor and reduce its affinity for epidermal growth factor. Transformation of 3T3 fibroblasts by the oncogenic tyrosine kinase pp60v-src is accompanied by an elevation of cellular diacylglycerol and partial activation of protein kinase C (Wolfman, A., Wingrove, T. G., Blackshear, P. J., and Macara, I. G. (1987) J. Biol. Chem. 262, 16546-16552). We therefore asked whether pp60v-src can down-modulate the epidermal growth factor receptor. We report that within 15 min of activating temperature-sensitive pp60v-src, binding of 125I-labeled epidermal growth factor to 3T3 cells falls at least 50%. Two distinct processes control the down-modulation by pp60v-src. The first is rapid and transient, while the second requires protein synthesis and persists long after inactivation of pp60v-src. Surprisingly, both mechanisms seem to be protein kinase C-independent. Both operate by decreasing the affinity of the epidermal growth factor receptor for its ligand.     

Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference

The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.     

Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus.

The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.     

Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations

Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.     

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles.

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.     

Expression of pp60v-src alters the ionic permeability of the plasma membrane in rat cells

The transmembrane potential of Rous sarcoma virus (RSV)-infected Rat-1 cells, expressing the pp60v-src protein kinase, is markedly less negative (by approximately 30 mV) than that of their normal counterparts. By contrast, the membrane potential of Rat-1 cells infected with Kirsten sarcoma virus is virtually unaltered. The RSV-induced membrane depolarization is shown to be due to a severalfold increase in the cation permeability ratio (PNa/PK) of the plasma membrane. When cells infected with a temperature-sensitive mutant of RSV (ts LA29), encoding a src protein with heat-labile kinase activity, are shifted from the nonpermissive to the permissive temperature, a rapid and sustained membrane depolarization is observed. Conversely, thermal inactivation of the ts LA29 pp60v-src kinase activity rapidly restores the membrane potential to near normal levels. Addition of epidermal growth factor, platelet-derived growth factor, or insulin to uninfected cells fails to cause a detectable change in membrane potential. We conclude that, unlike growth factor receptor tyrosine kinases, pp60v-src can induce, either directly or indirectly, a major change in the membrane permeability to monovalent cations.     

Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane.

We have investigated the characteristics of platelet-activating factor (PAF)-stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets.     

Serine/threonine-specific protein kinase activity associated with viral pp60src protein

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352-360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate.     

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.     

Studies in pig heart tissue on various 60,000 Da phosphoproteins

Pig heart tissue have been shown to contain 3 different 60,000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60v-src since none of them is immunoprecipitated by sera that precipitate pp60v-src. The possibility that the three 60,000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c-src and/or c-src related genes is discussed.     

Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos.

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.     

Ecto-protein kinase activities in normal and transformed cells

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.     

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.