Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity.

We have examined human and rabbit blood platelets for expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp60c-src kinase activity was determined by an immune-complex kinase assay that uses enolase as the substrate, and pp60c-src protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-src-specific kinase activity and pp60c-src protein compared to the levels found in other tissues. pp60c-src was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple protein species other than pp60c-src were also phosphorylated on tyrosine in the membrane phosphorylation reactions, and phosphotyrosine represented approximately equal to 80% of the total phosphoamino acid residues phosphorylated in the membranes. These results indicate that tyrosine kinases represent the major protein phosphorylating enzymes detected in isolated platelet membranes. Although the association of tyrosine kinase activity with many viral oncogene products and cellular growth hormone receptors has suggested a role for these enzymes in the regulation of cell proliferation, these results indicate that the expression of high levels of tyrosine kinase activity is not exclusively associated with proliferating cells.     

c-src gene product in developing rat brain is enriched in nerve growth cone membranes.

Differentiating rat neurons express high levels of the protooncogene product pp60c-src, a 60-kDa tyrosine kinase of unknown function encoded by c-src. pp60c-src was found to be concentrated at least 9-fold in membranes from a subcellular fraction of nerve growth cones, the motile tips of outgrowing neuronal processes. Indirect immunofluorescence staining of cultured chick retinal explants showed pp60c-src in neuronal growth cones and processes, with the antigen particularly concentrated in growth cones of long neurites. pp60c-src in growth cone membranes was an active tyrosine-specific protein kinase with elevated tyrosine-specific protein kinase activity and reduced electrophoretic mobility characteristic of the form of pp60c-src in central nervous system neurons. pp60c-src was present at lower levels in subcellular fractions from mature rat brain but synaptosomal membranes were not enriched. Preferential localization of an active form of pp60c-src in nerve growth cone membranes and persistence of pp60c-src in mature neurons suggest that this tyrosine kinase is important in growth cone-mediated neurite extension and synaptic plasticity.     

Phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide of pp60c-src.

The major site of tyrosine phosphorylation of the transforming protein of Rous sarcoma virus, pp60v-src (tyrosine-416), is different from the major site of tyrosine phosphorylation of its nontransforming normal cellular counterpart, pp60c-src. We have shown that antibodies against a synthetic peptide modeled on the carboxyl-terminal 13 residues of pp60c-src specifically immunoprecipitate the major phosphotyrosine tryptic peptide of pp60c-src from both chicken and rat fibroblasts. These experiments localize the major site of tyrosine phosphorylation to one or more of the three tyrosine residues in the carboxyl-terminal tryptic peptide at positions 511, 519, and 527 of the amino acid sequence of chicken pp60c-src. Tyrosines-519 and -527 are in the carboxyl-terminal 19-amino acid segment of pp60c-src that is deleted and replaced by an unrelated sequence in pp60v-src. It is possible that phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide may be involved in the normal regulation of pp60c-src. The absence of this phosphorylation site in pp60v-src may, in part, contribute to its oncogenic properties.     

Reduced tyrosine kinase specific activity is associated with hypophosphorylation of pp60c-src in cells infected with avian erythroblastosis virus.

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that causes erythroblastosis and sarcomas in chickens and transforms immature erythroid cells and fibroblasts in culture. AEV encodes two oncogenes, v-erbA and v-erbB, whose products are closely related to the thyroxine receptor and the epidermal growth factor receptor, respectively. Since tyrosine protein kinases have been implicated in the process of normal growth signal transduction, we wished to study the possible consequences of the expression of these mutated, growth-regulating receptor genes on the activity of the cellular tyrosine kinase pp60c-src. A continuous cell line from AEV-infected quail embryo fibroblasts was derived that exhibited a typical transformed phenotype and expressed the viral oncogene products, p75gag-erbA and gp66-68erbB. Using an immune-complex kinase assay, we found that the specific activity of pp60c-src in AEV-transformed quail cells was decreased by a factor of 6-30 relative to that found in uninfected quail cells. A concomitant 50-80% reduction of 32Pi incorporation into the pp60c-src protein from radiolabeled, transformed cells was also observed, indicating a relationship between hypophosphorylation and diminished enzyme activity. Partial proteolytic phosphopeptide analysis revealed a decrease in phosphorylation of both serine- and tyrosine-containing peptides, suggesting an activation of specific phosphatases or inhibition of specific kinases in the AEV-transformed quail cells. Similar results were found in pp60c-src precipitated from AEV-transformed chicken and rat cells.     

Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets.

We previously demonstrated that platelets express high levels of the tyrosine protein kinase pp60c-src. By a quantitative immunoblot assay, it is shown in this report that pp60c-src represents 0.2-0.4% of total platelet protein. The expression of high levels of pp60c-src in platelets correlated with high levels of total cell phosphotyrosine. Unstimulated platelets were shown to possess numerous phosphotyrosine-containing proteins by immunoblot analysis using antibodies that specifically recognize phosphotyrosine residues. To examine whether the pattern of phosphotyrosine-containing proteins changes upon platelet activation, lysates from thrombin- and phorbol ester-treated platelets were subjected to immunoblot analysis. Novel phosphotyrosine-containing proteins were detected within seconds following platelet stimulation. These results suggest that tyrosine phosphorylation, perhaps mediated by pp60c-src, may be involved in events associated with platelet activation.     

pp60c-src expression in the developing rat brain.

We have studied pp60c-src expression in the striatum, hippocampus, and cerebellum of the developing rat brain. In the striatum, pp60c-src protein kinase activity peaks during embryonic development and then declines in the adult. The peak activity occurs in the striatum on embryonic day 20 (E20) when it is 18- to 20-fold higher than the activity in fibroblasts and 4- to 5-fold higher than the activity in the striatum at E15 or in the adult striatum. In the hippocampal region, pp60c-src activity reaches a maximum shortly after birth but remains high throughout life. On postnatal day 2 (P2) the activity in the hippocampus is 9- to 13-fold higher than the activity in fibroblasts and twice as high as the activity in the hippocampus at E18. In the cerebellum, the kinase activity remains constant from E20 onward and is 6- to 10-fold higher than that observed in fibroblasts. The increase in pp60c-src kinase activity observed during the development of the striatum and hippocampus is due to an increase in the amount of pp60c-src protein and to an increase in the specific activity of the kinase. The increase in specific activity in these regions coincides with the peak periods of neurogenesis and neuronal growth. In the striatum, we have found that the increase in pp60c-src activity also parallels the increase observed in culture as embryonic striatal neurons differentiate. Taken together, our results are consonant with the idea that pp60c-src is the product of a developmentally regulated gene that is important for the differentiation and/or the continuing function of neurons.     

Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src).

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp60v-src and pp60-c-src are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v-src and pp60c-src. We have therefore characterized the sites of tyrosine phosphorylation in the two proteins. Tyrosine phosphorylation of pp60v-src in infected cells occurs mainly (if not entirely) at residue 419 in the deduced amino acid sequence of the protein. Surrounding this residue is the sequence Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg. This peptide is distinguished by the fact that three out of the four amino acids that precede the phosphorylated tyrosine are acidic in nature. These results define what may prove to be a widely used site for tyrosine phosphorylation in the regulation of cellular function. The same site was phosphorylated when partially purified pp60v-src was used in a phosphotransfer reaction in vitro. The results with pp60c-src were more complex. The site of tyrosine phosphorylation in vitro appeared to be the same as that found in pp60v-src. By contrast, phosphorylation of pp60c-src in vivo apparently occurred at a different, and currently unidentified, tyrosine residue. It is therefore possible that pp60v-src and pp60c-src respond differently to regulatory influences in the intact cell.     

Activation of pp60c-src protein kinase activity in human colon carcinoma.

The tyrosine-specific protein kinase activity of pp60c-src molecules obtained from human colon carcinoma tissues and tumor-derived cell lines was found to be elevated over that from normal colon tissues or cultures of normal colon mucosal cells. The elevated pp60c-src protein kinase activity in tumor tissues and in cultured colon carcinoma cells does not appear to result solely from an increase in the abundance of the c-src-encoded protein, suggesting that the specific activity of the pp60c-src tyrosine phosphotransferase is enhanced. These results raise the possibility that activation of the pp60c-src protein kinase may contribute to the genesis of human colon tumors.     

pp60c-src kinase expression in brain of adult rats in relation to age.

Although there is evidence of alterations in brain protein phosphorylation patterns with age, it is not known if the protein kinases that phosphorylate only at tyrosine residues are involved in these changes. For this reason, we examined the age-related expression of pp60c-src, a tyrosine protein kinase enriched in neural tissues, in whole brain of adult Fischer-344 rats. The pp60c-src kinase activity was immunoprecipitated using a monoclonal antibody and the incorporation of [32P] from radiolabeled ATP into an exogenous substrate (casein) measured. The results showed that there was a substantial amount of pp60c-src kinase activity in brain of the adult animals ranging in age from 4 to 23 months and that it was not significantly different among these groups. Also, immunoprecipitates obtained under conditions of monoclonal antibody excess and utilized for immunoblot analysis indicated that the relative levels of the pp60c-src protein were unchanged in the same animals. These results suggest that, at the whole brain level, the pp60c-src kinase has a stable turnover and that a high amount of activity is biologically important in brain of adult rats through early senescence.     

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.     

Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen.

We have examined the in vitro phosphorylation of cellular src protein (pp60c-src) molecules associated with the polyoma virus middle-sized tumor antigen in polyoma virus-transformed cells. These pp60c-src molecules possessed an enhanced tyrosyl kinase activity, migrated aberrantly on NaDodSO4/polyacrylamide gels, and contained a novel site of tyrosine phosphorylation within the amino-terminal region of the molecule. The pp60c-src molecules not associated with the middle-sized tumor antigen were phosphorylated exclusively on a tyrosine residue within the carboxyl-terminal domain of pp60c-src. A similar modified form of the middle-sized tumor antigen-associated pp60c-src protein was detected in lysates from polyoma virus-transformed cells labeled in vivo with [32P]orthophosphate in the presence of sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatases.     

Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.

Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.     

The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor.

We have observed a modification of the cellular protein kinase pp60c-src, elicited in murine 3T3 fibroblasts by platelet-derived growth factor (PDGF). The modification occurred rapidly after addition of PDGF to the culture medium and was first detected as a reduction in the electrophoretic mobility of a portion of the pp60c-src molecules. A similarly modified form of the viral homologue pp60v-src occurs in vivo in the absence of stimulation by PDGF. The occurrence of modified forms of both pp60c-src and pp60v-src was associated with a novel phosphorylation at tyrosine in the amino-terminal domains of the proteins. The time-course and dose-response for this modification of pp60c-src paralleled PDGF-induced increases in phosphorylation of pp36, a major cellular substrate for several tyrosine-specific protein kinases. In parallel experiments, treatment of cells with PDGF increased the kinase activity of pp60c-src in an immunocomplex assay. These results suggest pp60c-src may play a role in the mitogenic response to PDGF.     

Identification of the tyrosine protein kinase from LSTRA cells by use of site-specific antibodies.

The lymphoma cell line LSTRA contains an elevated level of tyrosine protein kinase activity. It has been suggested that this elevated level of activity is due to the presence of a phosphoprotein with a molecular weight of 56,000 (pp56, formerly referred to as a 58,000-dalton protein). This paper describes the preparation of antibodies against pp56 through the use of a synthetic peptide that contains the sequence around the site of tyrosine phosphorylation in pp56, which is identical to the phosphorylation site in pp60src. These antipeptide antibodies specifically immunoprecipitated 32P-labeled pp56 from detergent extracts of LSTRA cells. In immunoblotting experiments, pp56 was the major antigen detected in the particulate fraction from LSTRA cells by the antipeptide antibodies. The antibodies were also used to show that the level of pp56 is greatly elevated in LSTRA cells. Incubation of the detergent extract of the particulate fraction from LSTRA cells with the antipeptide antibodies resulted in inhibition of most of the LSTRA cell tyrosine protein kinase activity. These results indicate that pp56 is the tyrosine protein kinase whose activity is elevated in LSTRA cells. This enzyme may be a member of the large family of protein kinases involved in the regulation of cell growth.     

Involvement of pp60c-src with two major signaling pathways in human breast cancer.

The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.     

Nucleophosmin-anaplastic lymphoma kinase of anaplastic large-cell lymphoma recruits, activates, and uses pp60c-src to mediate its mitogenicity

Anaplastic large-cell lymphomas (ALCLs) are lymphomas of T or null phenotype often associated with a chromosomal translocation, t(2;5)(p23;q35). This translocation leads to the expression of a hybrid protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). NPM-ALK possesses a constitutive tyrosine kinase activity responsible for its oncogenic property through activation of downstream effectors such as phospholipase C gamma (PLC-gamma) and the type IA phosphoinositide 3-kinase. Here, we show that the Src-kinases, particularly pp60(c-src), associate with and are activated by NPM-ALK expression in various cells, and in cell lines established from patients with ALCL. The kinase activity and the tyrosine 418 of NPM-ALK are required for its association with Src-kinases. Y418F mutation of NPM-ALK impaired its association with Src-kinases and strongly reduced the proliferation rate of Ba/F3 cells. In agreement, Src-kinase inhibitors or pp60(c-src) siRNA significantly decreased the proliferation rate of NPM-ALK-positive ALCL cell lines. Moreover, using active or inactive forms of pp60(c-src) and NPM-ALK, we provide evidence that NPM-ALK is a potential substrate of pp60(c-src). Overall, our data place Src-kinases as new important downstream effectors of NPM-ALK and as attractive potential therapeutic targets for new ALCL treatment.     

Heat-shock protein hsp90 governs the activity of pp60v-src kinase.

During or immediately after synthesis in vertebrate cells, the oncogenic protein-tyrosine kinase pp60v-src associates with the approximately 90-kDa heat-shock protein (hsp90). In this complex, pp60v-src is not functional as a kinase. When pp60v-src is subsequently found inserted into the plasma membrane, it is active as a kinase and is no longer associated with hsp90. We have taken advantage of genetic manipulations possible in Saccharomyces cerevisiae to investigate the function and specificity of the association between hsp90 and pp60v-src. Expression of pp60v-src is known to be toxic to S. cerevisiae cells. We find that this toxicity is due to a very specific effect on growth, arrest at a particular point in the cell cycle. In cells expressing v-src, a mutation that lowers the level of hsp90 expression (i) relieves cell cycle arrest and rescues growth, (ii) reduces the level of tyrosine phosphorylation mediated by pp60v-src, (iii) changes the pattern of tyrosine phosphorylation, and (iv) reduces the concentration of pp60v-src. We conclude that hsp90 does not simply suppress pp60v-src kinase activity during transit to the plasma membrane, as previously suggested, but also stabilizes the protein and affects both its activity and specificity. This function of hsp90 is highly selective for pp60v-src: the same hsp90 mutation has no effect on the activity or specificity of the exogenous pp160v-abl tyrosine kinase; similarly, it does not affect the specificity and has only a very small effect on the activity of the exogenous pp60c-src kinase.     

Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.

A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.     

NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1

Phosphorylation regulates the function of ligand-gated ion channels such as the N-methyl d-aspartate (NMDA) receptor. Here we report a mechanism for modulation of the phosphorylation state and function of the NMDA receptor via an inhibitory scaffolding protein, RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. Peptides that disrupt the interactions between RACK1, NR2B, and Fyn induce phosphorylation and potentiate NMDA receptor-mediated currents. Therefore, RACK1 is a regulator of NMDA receptor function and may play a role in synaptic plasticity, addiction, learning, and memory.