Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.     

Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein.

To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.     

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.     

Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Neural tissues express high levels of the cellular src gene product pp60c-src.

Chicken embryo tissues were examined for the expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. Three assays, including a solid-phase radioimmunoassay, a competitive radioimmunoprecipitation assay, and an immune complex protein kinase assay, were employed. Elevated levels of pp60c-src were detected in lysates from several neural tissues, including brain, retina, and spinal ganglia. Other tissues contained 8- to 10-fold-lower levels of pp60c-src, levels comparable to those found in chicken embryo fibroblasts. Expression of pp60c-src in brain tissues was also shown to vary with the developmental stage of the embryo.     

Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries.

Many proteins involved in intracellular signal transduction contain a small, 50-60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein-protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P-X-X-P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage-displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.     

Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.     

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.