pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.     

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.     

Developmental expression of two forms of pp60c-src in mouse brain.

The expression during mouse development of two forms of protein-tyrosine kinase pp60c-src, pp60 and pp60+, was investigated. At embryonic day 9 (E9), only pp60 was detected in whole-brain lysates. A trace of pp60+ was first seen at E10. In E18 embryos and in adults, pp60+ was the predominant form of pp60c-src in forebrain and midbrain lysates.     

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.     

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.     

pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.     

pp60c-src is developmentally regulated in the neural retina

We have localized normal cellular pp60c-src in the developing chick neural retina by immunocytochemical staining using antisera raised against bacterially expressed pp60v-src, the src gene product of Rous sarcoma virus. pp60c-src was expressed in developing retinal neurons at the onset of differentiation. Expression of pp60c-src persisted in mature neuronal cells that were postmitotic, fully differentiated, and functional. pp60c-src immunoreactivity was localized within processes and cell bodies of ganglion neurons, processes of rods and cones, and in some but not all neurons of the inner nuclear layer. Protein kinase assays and Western transfer analyses identified the immunoreactive protein as pp60c-src, and confirmed that its expression occurs at the time the first neuronal cells in the retina differentiate. We conclude from these studies that pp60c-src is the product of a developmentally regulated gene that is more important in neuronal differentiation or function than cell proliferation.     

Association of type I phosphatidylinositol kinase activity with mutationally activated forms of human pp60c-src.

Chicken embryo fibroblast cells overexpressing activated mutant forms of human pp60c-src, but not those overexpressing normal human pp60c-src, exhibited high levels of type I phosphatidylinositol (PI) kinase activity associated with pp60c-src. Levels of PI kinase activity were positively correlated with src tyrosine protein kinase activity and not with absolute levels of pp60c-src. Our results suggest that a linkage exists between certain forms of pp60c-src and the PI signal transduction pathway.     

Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus.

The c-fyn proto-oncogene is a member of a family of closely related genes of which c-src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c-fyn gene product, the c-fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c-src and pp62c-yes, pp59c-fyn is able to form a stable complex with middle-T antigen, the transforming protein of polyomavirus. The transformation-defective middle-T mutant NG59, which is unable to associate stably with pp60c-src does not associate with pp59c-fyn. In contrast to pp60c-src, complex formation with middle-T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c-fyn. These findings lead us to suggest that middle-T mediated transformation may be a consequence of the deregulation of several members of the src-family of protein tyrosine kinases.     

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.     

Investigation of factors that influence phosphorylation of pp60c-src on tyrosine 527.

Phosphorylation at tyrosine 527 of the proto-oncogene product, pp60c-src, has been proposed to decrease the tyrosine kinase activity of the enzyme. We have investigated potential factors that might influence phosphorylation at this site by making mutant variants of the pp60c-src protein. By effectively eliminating the site of N-terminal myristylation, we demonstrated that stable membrane association is not necessary for tyrosine 527 phosphorylation. Furthermore, mutational elimination of the enzymatic activity of this mutant pp60c-src protein did not alter the efficiency of phosphorylation at tyrosine 527. These data are consistent with the proposal that pp60c-src may be phosphorylated at tyrosine 527 by a cellular tyrosine kinase distinct from pp60c-src. In addition, using detergent-permeabilized cells, we established conditions that allow efficient phosphorylation of tyrosine 527 in vitro.     

Myristylation of pp60c-src is not required for complex formation with polyomavirus middle-T antigen.

Middle-T antigen (middle-T), the transforming gene product of polyomavirus, associates with several cellular tyrosine kinases, such as pp60c-src. Complex formation leads to kinase activation and is essential for cell transformation. Middle-T-associated as well as uncomplexed pp60c-src is predominantly found in the plasma membrane. We transfected mouse 3T3 fibroblasts with a mutated c-src gene (2Ac-src), allowing the expression of a protein containing alanine instead of glycine in position 2 of the primary translation product. Contrary to the wild-type c-src gene product, pp60c-src(2A) was not myristylated and accumulated in the cytoplasm instead of being transferred to cellular membranes. The mutant protein was able to associate with middle-T and was activated similarly to the wild-type c-src gene product. Both wild-type and 2A mutant protein were membrane associated upon complex formation with middle-T. This finding suggests that the putative carboxy-terminal membrane anchor sequence of middle-T is sufficient to hold middle-T-associated pp60c-src(2A) in the plasma membrane.     

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.     

Regulation of pp60c-src synthesis by inducible RNA complementary to c-src mRNA in polyomavirus-transformed rat cells.

To determine the potential role of pp60c-src in polyomavirus-transformed cells, we constructed a recombinant plasmid with the mouse metallothionein-I promoter upstream of a src gene in an anti-sense orientation. We cotransfected this plasmid into middle tumor antigen-transformed FR3T3 cells with a plasmid containing the neomycin resistance gene, and G418 resistant colonies were selected. Analysis of these cells for pp60c-src expression revealed that 50 of the 200 cellular clones screened were found to have decreased levels of c-src expression when compared with the parental middle tumor antigen-transformed cells. Three independent clones which transcribed the expected 3.6-kilobase src complementary RNA and had levels of pp60c-src kinase activity comparable to that of normal FR3T3 cells were further analyzed. In the presence of Cd2+, these clones grew significantly slower in monolayer cultures than either the parental transformed cells (FR18-1) or FR18-1 cells transfected with the neomycin resistance gene alone. The morphology of these clones in the presence of Cd2+ was distinct from that of either the parental FR18-1 cells or normal FR3T3 cells. The clones expressing the complementary src RNA were found to form fewer colonies in soft agar, form fewer foci on monolayers of normal rat cells, and form tumors more slowly following injection into syngenic rats when compared with parental FR18-1 cells. The results of these studies suggest that the level of pp60c-src kinase activity affects the growth characteristics and transformation properties of polyoma virus-transformed rat cells.     

Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation.

The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle-T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation.     

Purification and enzymatic characterization of pp60c-src from human platelets.

The purification of pp60c-src has been hampered by the low levels of protein it represents in most cells and its tendency to undergo proteolysis during purification. The discovery that the platelet expresses unusually high levels of pp60c-src has made large-scale purification from a normal source feasible. We have developed a method for the purification of intact pp60c-src to near homogeneity from human platelets and have determined the enzymatic properties of this purified protein in vitro. Rapid, high yield purification of pp60c-src from isolated platelet membranes was achieved in a two-step protocol involving sequential chromatography on an anti-pp60c-src immunoaffinity matrix and phenyl-Sepharose. This protocol yielded 0.5 mg of pp60c-src from 30 units of platelets. Using enolase as an exogenous substrate, the specific activity of the enzyme was 25 nmol P.min-1.mg-1. The Km for MnATP2- for enolase phosphorylation (2.2 microM) was higher than for the autophosphorylation of pp60c-src (0.6 microM). Maximal enzyme activity required either Mn2+ or Mg2+, and both ATP and GTP could be utilized as the phosphate donor. Evidence is shown which indicate that the autophophorylation of pp60c-src in vitro occurs through an intramolecular mechanism and that this reaction is reversible.     

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.     

AtT20 cells express modified forms of pp60c-src

We have compared the properties of pp60c-src from the mouse pituitary tumor cell line, AtT20, and from mouse fibroblasts. In vitro, pp60c-src phosphotransferase activity from AtT20 cells is 2- to 3-fold that of mouse NIH 3T3 fibroblast pp60c-src. In analyzing the reason for this elevation in specific activity, we found that pp60c-src from AtT20 cells differs structurally in at least three ways from pp60c-src in fibroblasts. First, AtT20 cells and primary rat anterior pituitary cells express low levels of the neuronal form of pp60c-src. Second, pp60c-src from AtT20 cells is phosphorylated at two additional N-terminal serine residues. Last, AtT20 pp60c-src is phosphorylated to a lower overall stoichiometry.     

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.