Virulence determinants in vancomycin-resistant Enterococcus faecium vanB: clonal distribution, prevalence and significance of esp and hyl in Australian patients with haematological disorders.

European studies have suggested that the esp gene and other virulence factors have roles in vancomycin-resistant Enterococcus faecium (VREfm) infections. The aim of this study was to examine the relationship between the spectrum of clinical disease and putative virulence factors in vanB VREfm isolates. A multiplex polymerase chain reaction was used to amplify potential virulence genes (asa1, gel E, cylA, esp and hyl) in VREfm isolates obtained from an Australian population of haematology patients. Clonality was assessed by pulsed-field gel electrophoresis (PFGE) and automated ribotyping. Infection, requirement for intensive care unit (ICU) admission and all-cause 30-day mortality were used as clinical indicators of organism virulence. Forty-one VREfm vanB isolates (41 patients; 14 infected and 27 colonised only) were analysed. Thirty-five of these isolates were typed by PFGE, 31 of which were represented by three clusters. The esp gene was identified in 22 of 27 (81.5%) screening and 11 of 14 (78.6%) infection-associated isolates. One isolate was hyl gene positive, and no isolate contained asa1, gel E or cylA genes. VREfm infection was independently associated with host factors (underlying diagnosis of acute myeloid leukaemia, age

The membrane targeting and spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct RhoB/RhoD endosome requirements.

Src activation is a tightly regulated process which requires RhoB endosome-associated actin assembly and transit to the cell periphery. We show here that although two other ubiquitous Src family kinases (SFKs) Yes and Fyn also require intact actin filaments for peripheral membrane targeting, they display distinct spatial activation and endosomal requirements. Unlike Src, both Yes and Fyn are constitutively membrane-localized to some extent, and Fyn is present in RhoD-positive endosomes whereas Yes does not visibly colocalize with either of the endosomal markers RhoB or RhoD. By modulating amino acid acceptor sites for palmitoylation in Src, Yes and Fyn, we show that Src S3C/S6C, which is palmitoylated (unlike wild-type Src) behaves in a manner more similar to Fyn, by predominantly colocalizing with RhoD endosomes, and the targeting of both Fyn and Src S3C/S6C is inhibited by siRNA-mediated knockdown of RhoD. Moreover, Fyn C3S/C6S, which is no longer palmitoylated, behaves much more like Src by colocalizing with RhoB endosomes and by requiring RhoB for activation and membrane translocation. These data imply that distinct modes of spatial activation and membrane delivery, at least partly under the control of specific acylation attachment sequences and endosome sub-type requirements, define distinct properties of the three ubiquitously expressed SFKs.

Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification.

Human Csk Homologous Kinase (CHK), a protein of 527 amino acid residues, is involved in suppression of breast tumors. The kinase domain of CHK (amino acid residues 228 to 485) expressed with C-terminal 6HIS fusion in Pichia pastoris is heavily glycosylated. Expression of the C-terminal 6HIS fused kinase domain of CHK, with an N-terminal glutathione S-transferase fusion, in Pichia pastoris alleviated the hyperglycosylation. The expressed protein was purified by affinity chromatography to 1 mg l(-1) culture and remained active. A simple plate assay to identify colonies of P. pastoris expressing the recombinant protein is also presented.

Src family kinases and HER2 interactions in human breast cancer cell growth and survival.

Evidence from murine fibroblast models and human breast cancer cells indicates that c-Src and human EGF receptor (HER1) synergize to enhance neoplastic growth of mammary epithelial cells. To investigate whether interactions between c-Src and other HER family members may also play a role in breast tumor progression, we characterized 13 human breast carcinoma cell lines and 13 tumor samples for expression of HER family members and c-Src and examined a subset of the cell lines for Src-dependent, heregulin (HRG)-augmented, anchorage-dependent and independent growth. By immunoblotting, we found that all cell lines overexpressed one or more HER family member, and 60% overexpressed c-Src. Seventy-five per cent of the tumor tissues overexpressed HER2, while 64% overexpressed c-Src. Colony formation in soft agar was enhanced by HRG in three of five cell lines tested, a response that correlated with the presence of a c-Src/HER2 heterocomplex. This result suggests that HRG may act through both HER2 and c-Src to facilitate anchorage-independent growth. In contrast, HRG had little effect on anchorage-dependent growth in any of the cell lines tested. PP1, a Src family kinase inhibitor, reduced or ablated HRG-dependent and independent soft agar growth or anchorage dependent growth, and triggered apoptosis in all cell lines tested. The apoptotic effect of PP1 could be partially or completely reversed by HRG, depending on the cell line. These results suggest that while Src family kinases may cooperate with HRG to promote the survival and growth of human breast tumor cells, they also function independently of HER2/HRG in these processes.

Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes.

Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975-978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether Slap could be an antagonist of all Src functions. Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G(1)/S block induced by both SrcK- and a Slap mutant with a deletion of its C terminus (SlapDeltaC), but not the block induced by Slap or SlapDeltaSH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcDeltaK fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85alpha, Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapDeltaC fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

Activation of neuroendocrine L-type channels (alpha1D subunits) in retinal pigment epithelial cells and brain neurons by pp60(c-src).

The aim of this study is to characterize the subtype of tyrosine kinase-regulated L-type Ca(2+) channels in retinal pigment epithelial (RPE) cells. Ca(2+) channel alpha1D-subunits were enriched by immunoprecipitation from membrane proteins isolated from rat RPE cells. Western blot analysis of the precipitates revealed coprecipitation of pp60(c-src). In addition, in precipitates obtained with antibodies against pp60(c-src), alpha1D-subunits were identified. The same was observed in immunoprecipitations from rat brain neurons. Tyrosine phosphorylation of alpha1D-subunits was confirmed using anti-phosphotyrosine antibodies. Ba(2+) currents through L-type channels in cultured rat RPE cells were increased by intracellular application of active pp60(c-src) (30 U/ml) (heat-inactivated pp60(c-src) had no effect). Thus, L-type channels of the neuroendocrine subtype can be expressed in epithelial cells and are activated by tyrosine kinase of the src subtype. This kind of regulation is also suggested for brain-derived neurons.

Common in vitro substrate specificity and differential Src homology 2 domain accessibility displayed by two members of the Src family of protein-tyrosine kinases, c-Src and Hck.

Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.

Characterization of two different cytoplasmic protein tyrosine kinases from human breast cancer.

Two different protein tyrosine kinases were detected in the cytosolic fraction of different human tumor tissues. After partial purification, the two enzymes, which were highly active in breast tumor tissues, were characterized. One of them, soluble tyrosine kinase-1 (STK-1), represents a soluble form of the c-Src protein, which is apparently underphosphorylated on its C-terminal tyrosine residue whereas the other (STK-2) is a 48-kDa protein tyrosine kinase (PTK), which is molecularly and functionally related to the C-terminal Src kinase (Csk). These two protein tyrosine kinases clearly exhibit a different substrate specificity, and are responsible for the high tyrosine kinase activity present in the cytosolic fraction of human breast cancer. In addition, it was observed that STK-1 and STK-2 are also expressed in the breast cancer cell line, CAL-51.

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c-Src in human platelets.

Protein tyrosine phosphatase 1C (PTP1C), highly expressed in hematopoietic cells, is a soluble protein tyrosine phosphatase containing two Src homology 2 (SH2) domains at the N-terminus and two putative sites of tyrosine phosphorylation at the C-terminus. This paper reports that PTP1C and c-Src could be coimmunoprecipitated during thrombin-induced platelet activation. Moreover, association between the two signalling proteins occurred only after PTP1C had been tyrosine phosphorylated. In in vitro experiments, PTP1C bound to the SH2 domain of c-Src, suggesting that association between tyrosine phosphorylated PTP1C and c-Src was mediated by the SH2 domain of c-Src. Finally, in resting platelets, PTP1C was mainly found in the Nonidet P-40 soluble fraction whereas following thrombin-induced activation, around 17% of PTP1C was associated with the insoluble fraction.

Prolactin receptor is associated with c-src kinase in rat liver.

The mechanism of action of the pituitary hormone PRL was studied in hepatocytes of lactating rats. PRL receptor immune complexes obtained from liver lysates have an associated tyrosine kinase activity. The tyrosine kinase has been identified in isolated hepatocytes as pp60c-src. Incubation of hepatocytes with PRL induces the association of PRL receptor with pp60c-src and the resultant stimulation of its tyrosine kinase activity. Furthermore, PRL stimulates the gene expression of c-fos, c-jun, and c-src. All of these findings support the idea that the pp60c-src tyrosine kinase participates in the early steps of the PRL intracellular signaling that promotes cell growth in liver cells.

Characterization of two activated mutants of human pp60c-src that escape c-Src kinase regulation by distinct mechanisms.

Two activated transforming mutants of human pp60c-src were found to possess single point mutations within the regulatory carboxyl terminus (E527K in CY CST201) and the kinase domain (E381G in WO CST1), respectively, that do not directly interfere with either the regulatory c-Src kinase (CSK) phosphorylation site (Tyr530) or the SH2/3 domains. In vivo, both mutant proteins are hypophosphorylated on their carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitro Src kinase inactivation assay, both mutant Src proteins exhibited resistance to inactivation by CSK relative to wild-type Src. Under these in vitro conditions, E381G c-Src was found to be phosphorylated by CSK to wild-type levels, while E527K c-Src was not detectably phosphorylated. The ability of CSK to phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src was also impaired, suggesting that CSK is unable to recognize E527K c-Src as an efficient substrate. In the case of E381G c-Src, examination of whether its SH2/3 domains were accessible to the carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced ability of autophosphorylated E381G c-Src to bind to a synthetic phosphopeptide modelled from the SH2-binding region of polyoma middle-T antigen which binds to Src SH2 with high affinity. This suggests that the E381G c-Src mutation results in an altered or reduced accessibility of the SH2 domain of the autophosphorylated form of E381G c-Src and may represent a previously undescribed mode of Src activation. Further study of these and other Src mutants may offer additional new insights into the regulation of "Src family" kinases.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Anti-CD9 monoclonal antibody activates p72syk in human platelets.

NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in human platelets, whereas F(ab')2 fragments of NNKY 1-19 did not, suggesting that the stimulation of Fc gamma II receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NNKY 1-19-induced protein tyrosine phosphorylation was completely inhibited by prostaglandin I2 (PGI2). The activity of p72syk was assessed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylation was suspended. NNKY 1-19 induced a rapid and transient increase in the p72syk-associated tyrosine kinase activity that peaked at 10 s and subsided to the original level 2 min after stimulation. Coinciding with this time course, p60c-src transiently associated with p72syk. In platelets preexposed to GRGDS peptides or PGI2, NNKY 1-19 also increased the p72syk-associated tyrosine kinase activity and led to the association of p60c-src with p72syk. However, in contrast to the control without any inhibitor, the elevated tyrosine kinase activity and the associated state of the two tyrosine kinases persisted as long as 5 min after stimulation. F(ab')2 fragments of NNKY 1-19 induced changes similar to those observed with the effects of GRGDS peptides or PGI2 treatment on intact IgG NNKY 1-19 stimulation. F(ab')2 fragments of another CD9 MoAb, PMA2, had effects on p72syk essentially similar to those of NNKY 1-19. These findings suggest that the binding of anti-CD9 MoAb to CD9 on the platelet membrane per se induces an increase in the p72syk-associated tyrosine kinase activity but that Fc gamma II receptor-mediated signal(s) is required for the full activation of platelets and the appearance of tyrosine-phosphorylated proteins. The elevated intracellular cAMP level induced by PGI2 acts at a step distal to the activation of p72syk and inhibited the signal transduction pathway leading to protein tyrosine phosphorylation and aggregation. p72syk activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72syk activity induced by anti-CD9 MoAb.

Site-specific association of c-Src with epidermal growth factor receptor in A431 cells.

We have examined the interaction between c-Src and epidermal growth factor (EGF) receptor in A431 cells. c-Src was found exclusively in the Triton X-100-solubilized particulate fraction and activated up to 3-fold within 1 min after EGF treatment of the cells. Association between c-Src and EGF receptor was detected by immunoprecipitation of c-Src followed by immunoblotting with anti-EGF receptor antibody. The c-Src-EGF receptor complex was found in both EGF-treated and untreated cells, but an augmented complex formation was observed in EGF-treated cells. We have isolated the complex by DEAE-cellulose column chromatography and found that a site-specific anti-c-Src antibody, which was raised against a synthetic peptide corresponding to residues 413 to 431 of human c-Src, did not recognize the c-Src protein in the complex, while other c-Src-specific antibodies tested did. Incubation of the complex with this synthetic peptide resulted in a partial dissociation of the complex. These results suggest that the specific region of c-Src is involved in the association with EGF receptor.

pp60c-src expression in rat spermatogenesis.

A fractionation of the membrane extract of rat testes revealed the existence of pp60c-src kinase activity. The expression of pp60c-src was examined in the developing rat testes. The immunecomplex kinase assay using a monoclonal antibody specific to pp60c-src (mAb327) showed that the expression of pp60c-src kinase activity increased during the development of rat testes and declined in the adult. The increase in pp60c-src kinase activity observed during the development of rat testes was accompanied with an increase in the amount of pp60c-src protein. The peak period in the increase of pp60c-src kinase activity well coincided with the timing, when the spermatogenesis by meiosis just began. The immunohistochemical staining of pp60c-src in rat testes demonstrated that pp60c-src is most abundantly expressed in the spermatids which are the spermatogenic cells in the post-meiotic phase of the spermatogenesis. These findings strongly suggest that pp60c-src is a developmentally regulated gene product which is involved in rat spermatogenesis.

Effects of doxorubicin on pp60c-src kinase activity in polyoma virus MT antigen transformed cells.

Interaction between polyoma virus (Py) encoded middle tumor antigen (MTAg) and the cellular src gene product, pp60c-src, has been shown to be correlated with the enhancement of pp60c-src kinase activity and the transforming ability of the virus. In the present study, FR3T3 cells were transfected with plasmids encoding MTAg and used to study the effects of the antineoplastic agent, doxorubicin (DOX), on pp60c-src kinase activity. Our results showed that transfection of FR3T3 cells with MTAg results in relatively high levels of MTAg expression and enhancement of pp60c-src kinase activity several folds over that observed in the control cells. Treatment of the transformed MTF4 cells with DOX up to 5 x 10(-6) M was shown to have no effect on the in vitro phosphorylation of MTAg and pp60c-src in the drug-treated cell lysates. On the contrary, treatment of src immunocomplexes with DOX resulted in a reduction in kinase activity. The inhibition appears to be a dose-dependent competition with respect to ATP concentrations.

Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.

To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

Cell fractionation in non-ionic detergent distinguishes sub-populations of polyoma virus middle T antigen and reveals a novel form.

The transforming function of polyoma virus, middle T antigen (MT), interacts with several cellular enzymes, essential to its oncogenic activity. We have used cell fractionation to study the various MT/cellular protein complexes. We demonstrate that MT can be separated into three sub-species, dependent upon extraction in two buffers that we designate A and B: Antigen extracted from whole cells by both buffers (called MT1) is associated with most of the phosphorylated phosphatidyl-inositol kinase 85 kD subunit, pp85, and protein phosphatase 2A. Antigen (MT2), associated with the greater portion of pp60c-src, is extracted by buffer B, but not buffer A. A third population (MT3), resistant to extraction by either buffer, is not detectably associated with protein phosphatase 2A or pp85. It is, however, associated with a low level kinase activity. The interaction between pp60c-src and MT appears to influence the formation of both MT2 and MT3. MT2 fractionates with the cellular microtubule network, but does not appear to be directly associated with it. MT3, a previously undescribed population, comprises about one third of MT in wild type antigen-containing cells. It is missing in mutants incapable of interacting with pp60c-src, but exists in the absence of an interaction with pp85. We suggest that MT3 may be an intermediate in, or product of, one of the MT/pp60c-src signalling pathways, distinct from that involving pp85.

Increased expression of pp60c-src in gastric mucosa of aged rats.

The relationship between proliferative activity and the expression of pp60c-src in gastric mucosa (oxyntic gland area) of young (4-month) and aged (24-month) Fischer 344 rats was examined. Gastric mucosal proliferative activity, as assessed by 5-bromo-2'-deoxyuridine (BrdU) immunoreactive cells, was found to be 115% (p < .001) higher in aged than in young rats. This was associated with a 47% rise (p < .025) in overall tyrosine kinase (Tyr-k) activity and a 5-7-fold increase in autophosphorylation of four prominent protein bands with M(r) of 40, 55, 60, and 70 kDa in gastric mucosal membranes. A similar phenomenon was also observed for Tyr-k activity of pp60c-src in that the aged rats revealed a 69% (p < .025) higher enzyme activity and a 5-fold rise in the extent of autophosphorylation of this protein when compared with the corresponding values from young animals. Increased Tyr-k activity of pp60c-src in the gastric mucosa of aged rats could in part be due to higher levels of this protein because the relative concentration of pp60c-src, as assessed by Western blot analysis, showed a 2-5-fold increase over the young animals. In addition, the relative concentration of c-src mRNA in the gastric mucosa of aged rats was also found to be 5-6-fold higher than in young animals. We suggest that pp60c-src may play a role in regulating gastric mucosal proliferative processes in the gastric mucosa of aged rats.

Platelet-derived growth factor induces phosphatidylinositol 3-kinase release from the middle T-pp60c-src complex and association with the platelet-derived growth factor receptor.

Both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) induce mitogenesis in normal rat kidney (NRK) fibroblasts transformed by the polyoma virus middle T (pmt) oncogene. In unstimulated pmt-NRK cells phosphatidylinositol 3-kinase forms a complex with the middle T protein and pp60c-src. PDGF treatment causes a release of phosphatidylinositol 3-kinase activity from the complex and a simultaneous increase in activity associated with the PDGF receptor. In contrast after treatment with EGF the majority of phosphatidylinositol 3-kinase activity remains associated with the middle T-pp60c-src complex. Proliferation of NRK fibroblasts transformed by the v-src oncogene is already maximal, and no further stimulation is observed with either PDGF or EGF. Neither growth factor induces dissociation of the complex between phosphatidylinositol 3-kinase and pp60v-src. These observations suggest that the complex between phosphatidylinositol 3-kinase, the middle T protein and pp60c-src is dissociable, and that phosphatidylinositol 3-kinase plays different roles in mitogenic signal transduction by the PDGF and EGF receptors.