Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.     

The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.

A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.     

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.     

Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling.

SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two amino-terminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression of dominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erk1) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated pp125FAK.     

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.     

Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.     

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.     

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.     

The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity.

Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.     

Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic precursors and their progeny. The Ph chromosome arises from a translocation that deregulates the c-ABL protein tyrosine kinase, giving it transforming potential and increased kinase activity. We observed a unique 39-kD tyrosine phosphoprotein (pp39), previously reported in blastic CML cell lines, in neutrophils from 50 cases of chronic phase CML. This protein was prominently and constitutively tyrosine-phosphorylated in CML neutrophils and was not phosphorylated in normal neutrophils. Stimulation of normal neutrophils with cytokines and agonists did not induce tyrosine phosphorylation of proteins migrating in the region of pp39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. The pp39 was not phosphorylated in hematopoietic cells from healthy donors or from patients with Ph chromosome-negative myeloproliferative disorders. Using micro amino acid sequencing of purified preparations of pp39, we identified pp39 as CRKL protein, which is consistent with recent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells. Our results suggest that pp39 CRKL in CML neutrophils may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an early stage of myeloid differentiation when the ABL kinase is active. CRK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signalling pathways. Although the CRKL protein is present in normal neutrophils, it is not tyrosine-phosphorylated, and the inability to induce such phosphorylation in normal neutrophils suggests a special role of this phosphoprotein in the pathogenesis of CML. Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention.     

Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin.

Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization.     

Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets.

Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.     

Src homology region 2 domains direct protein-protein interactions in signal transduction.

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.     

Identification, cloning, and characterization of a novel human T-cell- specific tyrosine kinase located at the hematopoietin complex on chromosome 5q

Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonucleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encoding a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECI mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECII mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31-32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31-32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces

Infected cell protein 0 (ICP0) is a 775-residue multifunctional herpes simplex virus protein associated with numerous functions related to transactivation of gene expression and repression of host defenses to infection. We report that an uncharted domain of ICP0 located between residues 245 and 510 contains multiple SH3 domain binding motifs similar to those required for binding to CIN85, the M(r) 85,000 protein that interacts with Cbl. CIN85 and Cbl are involved in endocytosis and negative regulation of numerous receptor tyrosine kinases. We report that ICP0 binds CIN85 in a reciprocal manner and that the complexes pulled down by ICP0 also contain Cbl. We tested the role of ICP0 in the down-regulation of receptor tyrosine kinases by using epidermal growth factor receptor (EGFR) as a prototypic receptor. In transfection assays, ICP0, in the absence of other viral genes, down-regulated EGF-dependent expression of a reporter gene (luciferase). ICP0 also down-regulated both total and cell surface levels of EGFR in EGF-independent manner. In wild-type virus-infected cells, the surface levels of EGFR were also decreased in the absence of EGF stimulation. Stimulation by EGF enhanced the decrease in surface EGFR. We conclude that ICP0 encodes SH3 domain binding sites that function to down-regulate signaling pathways associated with receptor tyrosine kinases. The results suggest that ICP0 precludes signaling to the infected cells through the receptor tyrosine kinases.     

Nonreceptor tyrosine protein kinase pp60c-src in spatial learning: synapse-specific changes in its gene expression, tyrosine phosphorylation, and protein-protein interactions

c-src is a nonreceptor tyrosine protein kinase that is highly concentrated in synaptic regions, including synaptic vesicles and growth cones. Here, we report that the mRNA signal of pp60c-src is widely distributed in the rat brain with particularly high concentrations in the hippocampus. After spatial maze learning, up-regulation of c-src mRNA was observed in the CA3 region of the hippocampus, which was accompanied by increases in pp60c-src protein in hippocampal synaptosomal preparations. Training also triggered an increase in c-src protein tyrosine kinase activity that was correlated with its tyrosine dephosphorylation in the synaptic membrane fraction. After training, pp60c-src from hippocampus showed enhanced interactions with synaptic proteins such as synapsin I, synaptophysin, and the type 2 N-methyl-d-aspartate receptor, as well as the cytoskeletal protein actin. The association of pp60c-src with insulin receptor in the synaptic membrane fraction, however, was temporally decreased after training. Furthermore, in vitro results showed that Ca(2+) and protein kinase C might be involved in the regulation of protein-protein interactions of pp60c-src. These results suggest, therefore, that pp60c-src participates in the regulation of hippocampal synaptic activity during learning and memory.