A single point mutation has pleiotropic effects on pp60v-src function.

The Rous sarcoma virus mutant tsLA29 encodes a pp60v-src molecule that is temperature sensitive for both tyrosine kinase activity and its ability to locate at the cell periphery. The defect in localization appears to be due to a perturbation in events following complex dissociation, since the mutant enzyme shows a rapidly reversible association with the cytoskeleton when shifted between permissive and restrictive temperatures. Although tsLA29 pp60v-src differs from the wild type at three amino acid residues, studies with chimeric proteins show that only one of the mutations, an alanine-for-proline substitution at residue 507, accounts for all the temperature-sensitive characteristics. Moreover, a single second site mutation, at residue 427, can restore the wild phenotype. Cells infected with a chimeric virus encoding only the alanine substitution at position 507 have a conspicuously fusiform morphology, suggesting that this mutation also has subtle effects on pp60v-src function that are apparently compensated for by the other mutations in native tsLA29.     

Features of the pp60v-src carboxyl terminus that are required for transformation.

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins.     

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.     

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.     

Deletions and insertions within an amino-terminal domain of pp60v-src inactivate transformation and modulate membrane stability.

We previously showed (V. W. Raymond and J. T. Parsons, Virology 160:400-410, 1987) that variants of the Prague A strain of Rous sarcoma virus containing large deletions impinging on a region of the src gene encoding amino acid residues 143 to 169 were defective for transformation of chicken cells in culture. Here we report that introduction of small (tri-and tetrapeptide) deletions into a region of pp60v-src containing amino acid residues 155 to 175 was found to inactivate transformation. In addition, insertion of four, but not one, amino acid residues at position 161 also inhibited transformation. Biochemical analysis of the src proteins encoded by individual transformation-defective variants revealed that the structural alterations introduced into this domain had only marginal effects upon src tyrosine-specific protein kinase activity. However, the src proteins encoded by defective variants exhibited a significantly shorter half-life within the cell, although these proteins efficiently and rapidly associated with cellular membranes. Our results suggest that the structural domain encompassing residues 155 to 177 may influence the stability of pp60src in the cellular membrane, possibly via the interaction of src with a cellular membrane component(s) or substrate(s).     

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.     

Biochemical properties of p60v-src mutants that induce different cell transformation parameters.

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.     

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.     

Identification of the domain in ErbB2 that restricts ligand-induced degradation

Ligand-induced receptor degradation is an important process for down-regulation of plasma membrane receptors. While epidermal growth factor receptor (EGFR) is rapidly internalised and degraded upon ligand stimulation, ErbB2, the closest member to EGFR in ErbB receptor family, is resistant in ligand-induced degradation. To understand the molecular mechanisms underlying the impairment in ligand-induced degradation of ErbB2, we attempted to determine structural factor in ErbB2 that restricts the degradation. By analysis of ligand-induced degradation of EGFR/ErbB2 chimeras, we have identified a region between amino acid residues F1030 and L1075 in ErbB2 as the domain that restricts the ligand-induced degradation. We designated this domain as the Blocking ErbB2 Degradation or the BED domain. Replacement of the BED domain in an EGFR/ErbB2 chimera with the corresponding region of EGFR changed this chimera from a non-degradable to a degradable receptor, indicating that the BED domain is the factor restricting the ligand-induced degradation of ErbB2. In addition, we found that a non-degradable EGFR/ErbB2 chimera was not defective in tyrosine phosphorylation, ubiquitination and interaction with c-Cbl, rather, was defective in ligand-induced internalisation, suggesting that the endocytosis defect is the cause restricting the degradation of ErbB2, and that c-Cbl-catalysed mono-ubiquitination is not involved in the impairment in ligand-induced degradation of ErbB2.     

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 pp60^v-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 pp60^v-src preparation at 41°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 pp60^v-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 pp60^v-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 pp60^v-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.     

Modulation of Werner syndrome protein function by a single mutation in the conserved RecQ domain

Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures.     

In vitro synthesis of pp60v-src: myristylation in a cell-free system.

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.     

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association.     

Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain

We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.     

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

The tyrosine protein kinase activities of pp60c-src and pp60v-src were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp60c-src was about 10-fold lower than that of pp60v-src for exogenous substrate phosphorylation but was only 1.1- to 2-fold lower than that of pp60v-src for autophosphorylation. Six glycolytic enzymes, including three not previously identified as substrates for pp60src phosphorylation, were phosphorylated by both pp60c-src and pp60v-src. Levels of pp60c-src fourfold higher than the amount of pp60v-src in src-plasmid-transformed cells did not detectably alter the level of phosphotyrosine in cellular proteins, but increasing the expression of pp60c-src another twofold (which induces cells to form foci in monolayer culture (P.J. Johnson, P.M. Coussens, A.V. Danko, and D. Shalloway, Mol. Cell. Biol. 5:1073-1083, 1985) resulted in a threefold increase in the level of cellular protein phosphotyrosine. Immunoprecipitation and analysis of the alkali-stable phosphoproteins by two-dimensional electrophoresis showed that, in contrast to pp60v-src-transformed cells, pp36 and enolase are only weakly phosphorylated in these high-level pp60c-src overexpresser cells. Even allowing for the in vitro differences in specific activities of phosphorylation, these results suggest that the pp60c-src tyrosine protein phosphorylating activity may be restricted relative to that of pp60v-src by additional in vivo mechanisms.     

A novel growth-inducible gene that encodes a protein with a conserved cold-shock domain.

We have isolated a cDNA that encodes a novel member of the Y-box binding protein family, termed as RYB-a (Rat Y-box Binding protein-a). RYB-a is a 31 kDa protein that contains a conserved cold-shock domain and an amino acid alignment similar to those of charge zipper proteins. Expression of RYB-a mRNA was highly abundant in the skeletal muscle, spleen, and fetal liver. The expression is very low in new-born and adult livers, suggesting its expression is under developmental regulation. In addition, the expression of RYB-a mRNA was induced in the liver during regeneration and by stimulation of quiescent fibroblast cells with serum. Induction in the fibroblasts was inhibited by treating the cell with a specific tyrosine kinase inhibitor, genistein or by detachment of cell-adhesion. Since both treatments are known to inhibit G1 cells to enter S phase, RYB-a gene is thought to be a member of growth-inducible genes.     

Sequence and functional differences between Schmidt-Ruppin D and Schmidt-Ruppin A strains of pp60v-src.

We show that Schmidt-Ruppin D pp60v-src kinase activity is reduced by a mutation previously shown to be associated with Schmidt-Ruppin A pp60v-src temperature sensitivity and that its reduced transforming activity is associated with a conformational change in the SH3 region. The evolutionary relationship of seven v-src strains was studied by using parsimony analysis.     

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.     

A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains.

WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (betaB5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (betaB7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second beta-sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.