Regulation by the autophosphorylation site in overexpressed pp60c-src.

We show that overexpressed pp60c-src is phosphorylated at Tyr-416 and has increased specific kinase activity when isolated from cells incubated with vanadate, a tyrosine phosphatase inhibitor. This supports the hypothesis that transient Tyr-416 phosphorylation modulates the activity of overexpressed pp60c-src in vivo. Mutagenesis indicates that Tyr-416 modulates pp60v-src activity as well.     

p59fyn and pp60 c-src modulate axonal guidance in the developing mouse olfactory pathway

The Src-family tyrosine kinases p59^fyn and pp60^c-src are localized on axons of the mouse olfactory nerve during the initial stages of axonal growth, but their functional roles remain to be defined. To study the role of these kinases, we analyzed the trajectory of the olfactory nerve in E11.5 homozygous null mutant mice lacking single src or fyn genes and double mutants lacking both genes. Primary olfactory axons of single and double mutants exited the olfactory epithelium and projected toward the telencephalon, but displayed differences in fasciculation. The fyn-minus olfactory nerve had significantly more fascicles than the src-minus nerve. Most strikingly, the primary olfactory nerve of src/fyn double mutants showed the greatest degree of defasciculation. These defects, identified by NCAM labeling, were not due to apparent changes in the size of the olfactory epithelium. With the exception of the src-minus mice, which had fewer fascicles than the wild type, no obvious differences were observed in coalescence of vomeronasal axons from mutant mice. The mesenchyme of the double and single mutants exhibited only subtle changes in laminin and fibronectin staining, indicating that the adhesive environment of the mesenchyme may contribute in part to defects in fasciculation. The results suggest that signaling pathways mediated by p59^fyn and pp60^c-src contribute to the appropriate fasciculation of axons in the nascent olfactory system, and comprise partially compensatory mechanisms for axonal adhesion and guidance. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 53–63, 1998     

Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src

The retroviral oncogene v-src arose by transduction of the cellular gene c-src. The similarity between these genes raised the possibility that c-src might be able to elicit neoplastic growth. We explored this by constructing a chimeric plasmid that allows the expression of chicken c-src. A rat cell line containing ten times the normal intracellular level of pp60c -src was isolated after transfecting rat-2 cells with the chimeric DNA. These cells produce the protein encoded by c-src ( pp60c -src) in quantities at least three times greater than required to achieve transformation by the product of v-src ( pp60v -src). The cells remain phenotypically normal, contain actin cables, and do not grow in soft agar. However, transfection of the cell line containing elevated cells of pp60c -src or Rat-2 cells with a molecular clone of v-src produces cells that exhibit properties of biologically transformed cells: round morphology, disrupted actin cables, and ability to grow in soft agar.     

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.     

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.     

Activity of pp60c-src and association of pp60c-src, pp54/58lyn, pp60fyn, and pp72syk with the cytoskeleton in platelets activated by collagen

Collagen stimulation of platelets induced an increase in the specific activity of pp60c-src immunoprecipitated from the Triton-soluble fraction. The earliest time after collagen stimulation that an increase in pp60c-src activity was observed was 30 s. However, the maximum activity of pp60c-src in the Triton-soluble fraction was observed 60 s after collagen stimulation. At this time an approximately twofold increase of pp60c-src activity towards phosphorylation of KVEKIGEGTYGVVKK specific peptide and enolase and a 4.5-fold increase towards phosphorylation of pp60c-src itself was measured. Furthermore, the majority of pp60c-src as well as pp54/58lyn, pp60fyn, and pp72syk were found in the Triton-soluble fraction in resting platelets. Collagen induced, to different extents and velocities, translocation of all of these proteins from the Triton-soluble fraction to the Triton-insoluble, cytoskeleton-rich, platelets fraction. These results provide direct evidence that collagen stimulation of platelets increases the tyrosine kinase activity of pp60c-src and suggest that the platelet cytoskeleton plays an important role in collagen-induced signal transduction by localizing signaling molecules.     

pp60c-src in human melanocytes and melanoma cells exhibits elevated specific activity and reduced tyrosine 530 phosphorylation compared to human fibroblast pp60c-src.

Elevated levels of pp60c-src tyrosine kinase activity have been implicated in both tumorigenesis and cell differentiation. We have found a 2- to 4-fold elevation in pp60c-src specific activity in certain human melanoma cell lines compared to human foreskin fibroblasts. This activation of pp60c-src did not appear to be related to melanoma tumor progression, because when normal human epidermal melanocytes were examined, it was found that they contained pp60c-src having a 7-fold elevation in specific activity compared to pp60c-src from human fibroblasts. It was determined that pp60c-src from melanocytes was not the neuronal form, pp60c-src+. Melanocyte pp60c-src exhibited a reduced level of phosphorylation on its carboxyl-terminal regulatory site, tyrosine 530, which might be responsible for its elevated specific activity. These results suggest that, in melanocytes, regulation of tyrosine 530 phosphorylation-dephosphorylation favors activation of pp60c-src. This activation may be involved in the growth, differentiation, or function of human melanocytes.     

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.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

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.     

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.     

In vivo effect of sodium orthovanadate on pp60c-src kinase.

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.     

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.     

The complex of polyoma virus middle-T antigen and pp60c-src.

We have recently proposed that the transforming protein of polyoma virus, middle-T antigen, forms a complex with pp60c-src. Here we provide additional evidence for the existence of the complex using both monoclonal antibodies specific for middle-T and antibodies raised against synthetic peptides corresponding to sequences from both middle-T and pp60c-src. The complex was retained during partial purification of middle-T and was stable to incubation under various conditions. A survey of a number of mutants of middle-T antigen showed that there was a complete correlation between the ability of middle-T to accept phosphate in the in vitro kinase reaction and the presence of a middle-T: pp60c-src complex. This result is in accord with our hypothesis that middle-T itself is not a protein kinase but rather that pp60c-src phosphorylates middle-T. All mutant forms of middle-T antigen capable of transformation had associated pp60c-src. The middle-T of two non-transforming mutants (hr-t mutants) did not have associated pp60c-src, whereas other non-transforming middle-T species did associate with pp60c-src. We propose that the complex plays an essential role in transformation by polyoma virus, but that the existence of the complex per se may not be sufficient.     

Activation of the pp60c-src kinase during differentiation of monomyelocytic cells in vitro.

The proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60c-src tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60c-src kinase.     

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.     

The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA.

A cellular src (c-src) cDNA clone was isolated from a chicken embryonic brain cDNA library and characterized by DNA sequence analysis. Comparison with the published sequence of a chicken genomic c-src clone indicated that the brain cDNA clone contained an 18-base-pair insertion located between exons 3 and 4 of the c-src gene. The six amino acids encoded by the insertion caused an alteration in the electrophoretic mobility of the c-src gene product similar to that of the structurally distinct form of the src protein detected in neuronal cultures.