Binding of pp60v-src to membranes: evidence for multiple membrane interactions.

Membrane association of pp60v-src, the myristylated transforming protein of Rous sarcoma virus, has been shown to be a receptor-mediated process, which is inhibited by myristylated src peptides containing the N-terminal 11 amino acids of the v-src sequence (MGYsrc). By cross-linking radiolabelled MGYsrc peptide to fibroblast membranes, a 32-kilodalton membrane protein was identified as a candidate src receptor. To elucidate the potential role of p32 in binding pp60v-src, we studied the relationship between binding of MGYsrc peptide and pp60v-src polypeptide to cellular membranes. The subcellular membrane distribution of p32 was distinct from that of pp60v-src in transformed cells. Moreover, under certain defined in vitro conditions, it was possible to inhibit peptide cross-linking to p32 without significantly affecting pp60v-src membrane binding. However, when internal sequences were removed from pp60v-src, the binding characteristics of the src deletion polypeptide and MGYsrc peptide became identical. These data indicate that the presence of internal membrane binding domains influences the interaction of myristylated N-terminal src sequences with p32, and suggest that accessory binding factors might be involved in establishing stable contact between pp60v-src and the membrane phospholipid bilayer.     

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.     

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.     

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.     

Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus.

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.     

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.     

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v- src, and implicates a pivotal role for polyamines in cell transformation.     

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.     

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.     

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.     

Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor

We have identified a tyrosine kinase activity present in tumors which were raised in rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This kinase phosphorylates tyrosine on the heavy chain of IgG from tumor-bearing rabbit (TBR) sera specific for the src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we have purified this kinase over 7200-fold. The purification procedure involves detergent extraction of tumors followed by sequential column chromatography on hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, an affinity column prepared from TBR-sera, and Sephacryl S-200. The IgG kinase activity behaves as a molecule of apparent Mr = 54,000 on Sephacryl S-200 molecular sieve chromatography. Analysis of the Sephacryl fractions by SDS-PAGE indicates that a major Coomassie blue-stained band with an apparent Mr = 54,000 (p54), co-elutes with the peak of kinase activity. From 600 g of tumors, approximately 200 micrograms of p54 are obtained. We have four types of evidence which show that p54 is related to pp60src. 1) Purified p54 is capable of undergoing endogenous phosphorylation in the presence of [gamma-32P]ATP producing a 32P-labeled pp54 polypeptide which is specifically immunoprecipitated by TBR-sera and contains only phosphotyrosine. 2) Purified p54 competes with 32P-labeled pp60src for binding to TBR-IgG, indicating a degree of purification over starting material which agrees very well with the results obtained by the IgG kinase assay. 3) V8 protease digestion of pp60src and p54 suggests that they share a common 26,000 fragment. 4) Antibodies to partially purified p54 specifically precipitate pp60src from Rous sarcoma virus-transformed chicken cells.     

Rous sarcoma virus variants that encode src proteins with an altered carboxy terminus are defective for cellular transformation.

The src gene of Rous sarcoma virus (v-src) and its cellular homolog, the c-src gene, share extensive sequence homology. The most notable differences between these genes reside in the region encoding the carboxy terminus of the src proteins. We constructed mutations within the 3' end of the v-src gene to determine the significance of this region to the transforming potential of the v-src protein, pp60v-src. The mutants CHdl300 and CHis1511 contain mutations that alter the last 23 amino acids of pp60v-src, whereas the mutant CHis1545-C contains a linker insertion that alters the last 11 amino acids of pp60v-src, and the mutant CHis1545-H contains a linker insertion that results in a 9-amino-acid insertion at position 415. Plasmids bearing each of these mutations were unable to transform chicken cells when introduced into these cells by DNA transfection. In addition, the structurally altered src proteins encoded by the mutants had much-reduced levels of tyrosine protein kinase activity in vivo, as measured by autophosphorylation and phosphorylation of the 34,000-Mr cellular protein, and in vitro, as determined by measuring the level of pp60src autophosphorylation. These data indicate that the carboxy-terminal amino acid sequences play an important role in maintaining the structure of the catalytic domain of pp60v-src. In contrast, the transfection of chicken cells with plasmid DNA containing a chimeric v-c-src gene resulted in morphological cell transformation and the synthesis of an enzymatically active hybrid protein. Therefore, the carboxy-terminal sequence alterations observed in the c-src protein do not alone serve to alter the functional activity of a hybrid v-c-src protein appreciably.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

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.     

Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins.

We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (pp130) and 110,000 (pp110) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp60c-src (pp60(527)F). Most if not all of the tyrosine-phosphorylated forms of pp130 and pp110 could be immunoprecipitated from lysates with any of several src protein-specific monoclonal antibodies directed against at least three spatially distinct epitopes. Consequently, of the more than 15 prominent phosphoproteins detected on immunoblots with phosphotyrosine-specific antibodies, pp130 and pp110 were selectively removed by src protein-specific immunoprecipitation, and their presence in the immunoprecipitates appears to have been due to a direct interaction with activated src proteins. src protein variants that induce different morphological phenotypes were altered in their ability to form detergent-stable complexes with pp130 and pp110 or with pp110 alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with pp110. Expression of src variants with mutations in the A box (pp60dl92/527F) or B box (pp60dl155/527F) of the src homology region induced differences in phosphorylation of pp130 and pp110, as well as changes in their association with variant src proteins. Sequences within the B-box region appeared to be necessary for stable complex formation with pp130 and pp110 and may be involved in the interaction of activated src proteins with cellular substrates.     

N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity.

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.     

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.     

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.