©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cooperation of Src Homology Domains in the Regulated Binding of Phosphatidylinositol 3-Kinase
A ROLE FOR THE Src HOMOLOGY 2 DOMAIN (*)

(Received for publication, October 18, 1994; and in revised form, December 21, 1994)

Burkhard Haefner (1)(§) Ruth Baxter (2)(¶) Valerie J. Fincham (1) C. Peter Downes (2) Margaret C. Frame (1)(**)

From the  (1)Beatson Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD and the (2)Department of Biochemistry, University of Dundee, Dundee DD1 4HN, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Fibroblasts transformed by the v-Src oncoprotein exhibit elevated activity of the enzyme phosphatidylinositol 3`-kinase (PI 3-kinase), which binds to, and is activated by, a wide range of receptor tyrosine kinases as well as v-Src and transforming polyoma middle T/c-Src complexes. Here we consider the role of the v-Src homology (SH) domains, SH3 and SH2, and the tyrosine kinase catalytic domain, in the stimulation of v-Src-associated PI 3-kinase activity in response to rapid activation of the oncoprotein. As shown by others, we find that the v-Src SH3 domain tightly binds the PI 3-kinase p85 regulatory subunit in normal growing chicken embryo fibroblasts. However, we also find that in transformed cells there is additional efficient binding of PI 3-kinase to the v-Src SH2 domain in a catalytically active form. Furthermore, the binding of p85 to the SH2 domain, which is almost undetectable in quiescent cells, is rapidly stimulated upon activation of temperature-sensitive v-Src and consequent cell cycle entry, demonstrating that binding is a target for regulation. We also show that v-Src-associated PI 3-kinase differs considerably from PDGF receptor-associated enzyme by a different mode of binding, a lack of substantial allosteric activation, and a dependence on the tyrosine kinase activity of v-Src. The rapidly induced binding and activation of PI 3-kinase thus provides sensitive regulation of recruitment of PI 3-kinase to its substrates and into other signaling complexes at the cell membrane, which involves all the Src homology domains.


INTRODUCTION

The c-src proto-oncogene, the cellular version of the v-src oncogene of Rous sarcoma virus, encodes the prototype of a family of cytoplasmic protein tyrosine kinases associated with the plasma membrane (reviewed by Courtneidge et al.(1993) and Fincham et al. (1994)). Src is also the prototype of proteins containing Src homology (SH) (^1)domains, i.e. SH1, the tyrosine kinase domain; SH2, which binds to phosphotyrosine residues; and SH3, which binds to proline-rich sequences in proteins. SH2 and SH3 domains have been found either alone, or in combination, in many proteins involved in signal transduction (reviewed by Pawson and Schlessinger, 1993).

There is no detailed understanding of the crucial signaling events that lead to transformation by the v-Src oncoprotein but it is clear that v-Src, transforming variants of c-Src, and activated tyrosine kinase growth factor receptors, such as the one for platelet-derived growth factor (PDGF), share common binding partners like PI 3-kinase, and known substrates such as Shc (Fukui and Hanafusa, 1989; McGlade et al., 1992a, 1992b). Much recent attention has focused on PI 3-kinase as an important binding partner and target for v-Src (reviewed by Cantley et al., 1991, Downes and Carter, 1991, Parker and Waterfield, 1992, and Stephens et al., 1993). This lipid kinase can phosphorylate phosphatidylinositol (PI), PI 4-phosphate, and PI 4,5-bisphosphate in position D-3 of the inositol ring, giving rise to products whose cellular role is still little understood. The only clue as to their function comes from in vitro experiments in which PI 3,4,5-trisphosphate was found to activate protein kinase C (Nakanishi et al., 1993).

PI 3-kinase is a heterodimer of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85) (reviewed by Fry and Waterfield, 1993). p110 is a dual specificity enzyme, able to act not only as a lipid kinase, but also as a protein serine kinase. The phosphorylation on serine 608 in the inter-SH2 domain of p85 by p110 negatively regulates PI 3-kinase (Dhand et al., 1994).

PI 3-kinase was first identified in anti-v-Src immunoprecipitates from lysates of Rous sarcoma virus-infected chicken fibroblasts (Sugimoto et al., 1984). It is also found associated with members of the protein tyrosine kinase family of growth factor receptors (Kaplan et al., 1987; Bjorge et al., 1990) and activated by proteins of the hemopoietic growth factor receptor family (Gold et al., 1994). The widespread activation of PI 3-kinase by growth factor receptors points to a common, fundamental function of this enzyme in growth factor signaling.

There is strong evidence implicating PI 3-kinase in cell transformation. The transforming ability of the polyoma middle T antigen/c-Src complex depends on its association with PI 3-kinase (Whitman et al., 1985) and PI 3-kinase binds to v-Src as well as all transforming variants of c-Src (Fukui and Hanafusa, 1989). Mutations in the v-Src SH3 domain decrease the binding of PI 3-kinase and result in a partially transformed, fusiform morphology (Wages et al., 1992), suggesting that the v-Src SH3 domain may mediate the association with PI 3-kinase. More recently, PI 3-kinase was shown to bind directly to the v-Src SH3 but not the SH2 domain expressed as glutathione S-transferase (GST) fusion proteins (Liu et al., 1993). In successive rounds of anti-phosphotyrosine immunoprecipitations from cell extracts, however, the PI 3-kinase activity able to bind v-Src is depleted (Fukui and Hanafusa, 1991). This implies that tyrosine phosphorylation of the lipid kinase is required for efficient association with v-Src, presumably mediated by the v-Src SH2 domain. Thus, the exact role of the v-Src SH domains in binding and activation of PI 3-kinase and the mechanism of regulation of binding remain unclear. Here, we investigate in detail the role of the v-Src homology domains, SH2 and SH3 alone and in combination, and the tyrosine kinase domain, in the regulation of v-Src-associated PI 3-kinase. We conclude that all of these domains contribute and that the regulation of PI 3-kinase bound to v-Src differs from that associated with activated PDGF receptor.


MATERIALS AND METHODS

Tissue Culture

Chicken embryo fibroblasts (CEF) uniformly infected with RCAN encoding the temperature-sensitive (ts) mutant v-Src proteins LA 29, LA 29A2, or LA 32 were cultured in Dulbecco's minimum essential medium supplemented with 10% newborn calf serum, 1% heat-inactivated chick serum, 10% tryptose phosphate broth, 0.375% sodium bicarbonate, 1 mM sodium pyruvate, 2 mM glutamine (all supplied by Life Technologies, Inc.). Cells were made quiescent by serum deprivation (0.2% newborn calf serum, no tryptose phosphate, no chick serum) for 4 days.

Preparation of Fusion Proteins

v-Src homology domains SH3 (amino acids 85-141), SH2 (amino acids 142-247), and combined SH3 and SH2 (amino acids 85-247) were amplified by polymerase chain reaction. BamHI and EcoRI restriction sites were generated at the ends to facilitate cloning into pGex2t (Pharmacia Biotech Inc.). The cloning products were checked by sequencing. Bacterially expressed glutathione S-transferase fusion proteins were isolated and bound to glutathione-agarose beads according to the method of Smith and Johnson(1988). Proteins were assayed using Coomassie reagent (Pierce).

Binding Assays

Semiconfluent cultures of CEF were washed twice with ice-cold phosphate-buffered saline solution and lysed in modified PLC lysis buffer (50 mM Tris, pH 7, 1% Triton, 10% glycerol, 1.5 mM MgCl(2), 1 mM EGTA, 150 mM NaCl containing 1.8 µg/ml aprotinin, 0.1 mM sodium orthovanadate, 0.5 mM NaF, 10 mM beta-glycerophosphate, 10 mM sodium pyrophosphate, and 1.25 mM phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation and protein concentrations determined by the BCA method (Pierce). 300 to 500 µg of cell protein (with or without inhibiting proline-rich peptide) were preabsorbed with 200 µg of GST for 60 min at 4 °C and the supernatant incubated with fusion protein bound to glutathione-agarose beads for 60 min at 4 °C in a volume of 1 ml. The beads were then washed four times with cold lysis buffer, proteins resolved in a 7.5% SDS-polyacrylamide gel, and Western blotting analysis performed as described below.

For PI 3-kinase activation studies, biotinylated PDGF receptor peptide (donated by Zeneca) or GST fusion proteins on glutathione-agarose beads were added to CEF lysates and incubated at 4 °C for 90 min. PDGF receptor peptide-bound PI 3-kinase was precipitated using streptavidin-agarose beads. Beads were washed and PI 3-kinase assays performed as described below.

Western Blotting Analysis

Equal amounts of clarified lysates were incubated with glutathione agarose-bound GST-fusion proteins for 2 h. The precipitates were then resolved on 10% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and probed with monoclonal anti-p85 antibody. Detection was by ECL (Amersham Corp.) following manufacturer's instructions. Quantitation of p85 binding to the fusion proteins was done by densitometry using a Molecular Dynamics computing densitometer. To examine phosphotyrosine levels in the p85 subunit of PI 3-kinase from cells expressing v-Src mutants, lysates were precipitated with anti-p85 antibody and then treated as above. Blots were then stripped following ECL instructions and reprobed with monoclonal anti-phosphotyrosine antibody PY20 (Upstate Biotechnology Inc.). To deplete cell lysates of phosphotyrosine-containing proteins, cell lysates were incubated for 6 h with excess (1:50) polyclonal rabbit anti-phosphotyrosine antibody (Affiniti) immobilized on protein A-Sepharose beads (Sigma).

Phosphatidylinositol 3-Kinase Assay

Cells were grown to around 90% confluence at the appropriate temperature, washed, rapidly lysed in PLC lysis buffer (50 mM Hepes, pH 7, 10% glycerol, 1% Triton, 1.5 mM MgCl(2), 1 mM EGTA, 150 mM NaCl containing 1.8 µg/ml aprotinin, 0.1 mM sodium orthovanadate, 0.5 mM NaF, 10 mM beta-glycerophosphate, 10 mM sodium pyrophosphate, and 1.25 mM phenylmethylsulfonyl fluoride) and protein concentrations determined by the BCA method. Equal amounts of clarified lysates were incubated either with protein A-Sepharose beads (Sigma) coated with anti v-Src monoclonal antibody EC10 (Upstate Biotechnology Inc.) or control antibody (normal mouse IgG (ICN) or monoclonal anti-p85 (Upstate Biotechnology Inc.)) for 16 h or with glutathione-agarose-bound GST fusion proteins for 2 h. Precipitates were washed once with lysis buffer, twice with phosphate-buffered saline, 1% Nonidet P-40 (Sigma), twice with 100 mM Tris/HCl, 500 mM LiCl, pH 7.6, and twice with 20 mM HEPES, 1 mM EGTA, pH 7.4, and then incubated in assay buffer (50 mM HEPES, 10 mM NaCl, 1 mM EGTA, pH 7.4) with a 3:1 sonicated mixture of phosphatidylinositol and phosphatidylserine (0.02 mg/ml) and 50 µCi of [-P] ATP, 10 mM MgCl(2) as substrates for 30 min at 30 °C. Reaction products were extracted and separated by thin layer chromatography. After autoradiography the phosphatidylinositol 3-phosphate band was scraped off the plate, counted, and presented as counts/minute. The identity of the lipid product was verified by HPLC analysis. The PI 3-kinase assay was linear under these conditions.

HPLC Analysis of Lipid Products

Lipid products of PI 3-kinase assays were deacylated and HPLC analysis performed as described previously (Carter and Downes, 1992).


RESULTS

The Binding of p85 to Fused v-Src SH3 and SH2 Domains Is Enhanced

Since mutations in SH2 (Fukui and Hanafusa, 1989), as well as SH3 (Wages et al., 1992), have been shown to influence the v-Src association with PI 3-kinase activity, we tested the ability of isolated GST-SH3 and GST-SH2 domains of v-Src, as well as a GST-SH3-SH2 fusion, to bind p85 from extracts of growing chicken embryo fibroblasts. Bacterial fusion proteins coupled to glutathione-agarose beads were incubated with extracts of growing CEF and bound proteins immunoblotted with p85 antiserum (Fig. 1A shows a short and a longer exposure of the immunoblots). As previously reported (Liu et al., 1993), the SH3 of v-Src domain bound p85 (Fig. 1A, lanes 2-4). In contrast to this previous work, we found that the SH2 domain also bound, although much more weakly (less than 10% of the amount) than SH3 (Fig. 1, panel A, lanes 5-7 and panel B). Furthermore, the SH2 domain bound both a species that co-migrated with the SH3-binding form of p85 and a faster migrating species (Fig. 1, A and C). Whether the lower species is a different gene product or an alternatively modified form of the higher molecular weight species is not clear. When the v-Src SH3 and SH2 domains were fused, enhanced binding (approximately twice the quantity bound to SH3 and SH2 combined) of p85 was observed (Fig. 1, panel A, lanes8-10 and panel B).


Figure 1: Binding of p85 to the non-catalytic domains of v-Src. A, p85 from 500 µg of protein prepared from normal growing CEF binding 2.5 nmol of GST (lane1) or 0.5, 1.0, and 2.5 nmol of SH3 (lanes2-4), SH2 (lanes 5-7), and fused SH3-SH2 (lanes 8-10) was detected by immunoblotting. Two different exposures are presented. B, densitometric quantitation of p85 binding using the longer exposure. C, immunoblot showing inhibition of p85 binding to 2.5 nmol of SH3 (lanes 1-3), SH2 (lanes 4-6), and SH3-SH2 (lanes 7-9) by 0, 100, and 250 µM proline-rich peptide from p85, respectively. Two different exposures are shown. D, densitometric quantitation of p85 binding using the longer exposure.



The enhancing effect of fusing the SH2 domain to SH3 could be either the result of direct binding of a population of p85 to SH2 or stimulation of binding to SH3 as a result of some conformational change. Thus, we examined the effect of the proline-rich peptide (amino acid residues 84-98 of bovine p85-alpha, SPPTPKPRPPRPLPV in single-letter amino acid code) on the binding of p85 to isolated and combined SH3 and SH2 domains (Fig. 1C). This peptide is an extended version of a peptide previously used by Liu et al. (1993) to demonstrate direct binding of the v-Src SH3 domain to this proline-rich region of p85. The peptide efficiently inhibited the SH3-p85 interaction (Fig. 1C, lanes2 and 3) and had no effect on the SH2-p85 interaction (Fig. 1C, lanes5 and 6). The peptide also inhibited the binding of p85 to the SH3-SH2 fusion (Fig. 1C, lanes8 and 9) albeit to a lesser extent. Inhibition of p85 binding by the proline-rich peptide, in the case of the SH3-SH2 fusion, did not reduce p85 binding to the level observed for the isolated SH2 domain (Fig. 1D). One possible explanation for this is that the combined SH3 and SH2 domains bind p85 with an increased affinity, which is greater than that for the peptide.

Enhanced PI 3-Kinase Activity Associated with SH3-SH2

The PI 3-kinase activity, which can associate with the non-catalytic domains of v-Src, was measured by incubating the GST-SH3, GST-SH2, and GST-SH3-SH2 fusion proteins with extracts prepared from growing CEF, followed by isolation of fusion proteins on glutathione-agarose. The ability of 1 mM proline-rich peptide to inhibit was also tested. This concentration was approximately 4 times that required for half-maximal inhibition of binding of PI 3-kinase activity to GST-SH3 (data not shown). We found PI 3-kinase activity associated with the SH2 domain similar to that bound to the SH3 domain (Fig. 2), despite the consistently lower binding of p85 found by immunoblotting (Fig. 1A). Possible explanations for this are that SH2-bound PI 3-kinase has a higher specific activity or that free p85 binds preferentially to SH3, while the catalytically active p85/p110 dimer binds both domains with similar efficiency. In addition, the activity bound to the SH3-SH2 fusion was considerably higher than that bound to either domain alone (Fig. 2). This is consistent with the observed increase in binding of p85 by the GST-SH3-SH2 fusion protein (Fig. 1A). The PI 3-kinase activity bound to SH3 and, to a lesser extent, SH3-SH2, but not isolated SH2, was inhibited by 1 mM proline-rich peptide from p85 (Fig. 2).


Figure 2: PI 3-kinase activity binding to 0.2 nmol of SH3, SH2 and SH3-SH2 was carried out as described under ``Materials and Methods,'' in the presence (box) and absence () of 1 mM proline-rich peptide from p85. A representative experiment from three repeats is shown.



Binding to the v-Src SH3 and SH2 Domains Results in Weaker Direct Activation of PI 3-Kinase than Binding to a PDGF Receptor Phosphopeptide

We next addressed possible activation of PI 3-kinase by direct binding to the Src homology domains, and we compared this with the activation which occurs upon binding to a tyrosine-phosphorylated peptide corresponding to a PDGF receptor sequence that contains the PI 3-kinase binding site. This peptide is GGYMDMSKDESVDY*VPMLDM (in single-letter amino acid code, where * denotes a phosphorylated residue) and corresponds to residues 738-757 of the human PDGF receptor (Kazlauskas and Cooper, 1989). We measured PI 3-kinase activity present in extracts of growing CEF, which bound the GST-SH fusion proteins and the biotinylated receptor peptide bound to streptavidin-agarose. In addition, we measured the PI 3-kinase activity present in the extract before and after binding. The ratio of ``activity bound'' to the beads to the ``activity removed'' from the extract provided a measure of the enzyme activation that occurred as a direct result of binding. Binding to the receptor phosphopeptide resulted in activation of PI 3-kinase, considerably greater than that observed upon binding to the SH3, SH2, or SH3-SH2 domains (Fig. 3A). This implies that the substantial conformational activation, which occurs upon binding PI 3-kinase to the tyrosine phosphorylated PDGF receptor, mediated by the SH2 domain of p85 (Shoelson et al., 1993), does not occur to the same extent upon binding PI 3-kinase to the Src homology domains. Consistent with the activation as a direct result of binding, the PDGF receptor phosphopeptide-associated PI 3-kinase exhibited greater specific activity than that bound by the v-Src SH3 domain (Fig. 3B). A comparison of bound enzyme activity with amount of p85 associated with the agarose beads demonstrated clearly that the receptor phosphopeptide bound considerably more PI 3-kinase activity, despite binding only about 27% of the p85 associated with the v-Src SH3 domain (Fig. 3B). In contrast to our observations with the v-Src homology domains, including SH3, it has been shown recently that peptides containing the SH3 domains of Lyn and Fyn bind to a proline-rich region of p85 and augment PI 3-kinase activity 5-7-fold (Pleiman et al., 1994). This may reflect genuine differences between the v-Src and Lyn/Fyn SH3 domains or could be due to differences in the methodology used.


Figure 3: A, activation of PI 3-kinase as a consequence of equal volumes of excess CEF protein binding to 0.5 nmol of PDGF receptor phosphopeptide and v-Src SH3, SH2, and SH3-SH2 domains. B, comparison of PI 3-kinase activity, relative to amount of associated p85, bound by the PDGF receptor (PDGFR) peptide and v-Src SH3 domain. Agarose beads (control) and GST-agarose beads (GST) were included as controls. p85 immunoblots for bound proteins are shown in lanes 1-4.



Importance of the v-Src Kinase for Associated PI 3-Kinase Activity

The weaker activation of PI 3-kinase upon binding to the Src homology domains SH3, SH2 and SH3-SH2 compared with the PDGF receptor phosphopeptide suggests that PI 3-kinase activation in response to v-Src is mediated differently from the allosteric mechanism proposed for direct binding to receptor tyrosine-phosphorylated peptides (Shoelson et al., 1993). Therefore, we examined the PI 3-kinase activity associated with various ts mutants of v-Src in CEF at restrictive (41 °C) and permissive (35 °C) temperature. The mutants used were ts LA 29, which has a temperature-sensitive kinase, ts LA 29A2, a myristylation-defective version of ts LA 29, and ts LA 32, which has functional mutations in both the SH3 and catalytic domains, which, in combination, produce a fusiform transformed morphology at permissive temperature (Stoker et al., 1984, 1986; Catling et al., 1994). CEF were uniformly infected with the replication-competent RCAN vector or RCAN-29, RCAN-29A2, or RCAN-32 as described previously (Catling et al., 1993). v-Src was isolated from lysates of cells grown at restrictive and permissive temperatures by immunoprecipitation using the monoclonal antibody EC10, and associated PI 3-kinase activity determined. The PI 3-kinase activity bound to v-Src was temperature-dependent in each case (Fig. 4A), although the degree of temperature sensitivity varied between mutant proteins. In the case of ts mutants LA 29A2 and LA 32, v-Src-associated PI 3-kinase activity at restrictive temperature was not substantially above the background ``vector only'' control. The higher level of activity associated with LA 29 at restrictive temperature has probably arisen due to its relatively higher frequency of back mutation as cells are passaged at restrictive temperature, a consequence of which is the expression of non-ts v-Src proteins in some cultures. This implies that PI 3-kinase activity associated with v-Src in transformed cells is dependent on activity of the v-Src kinase. In order to test this directly, we expressed a kinase-defective version of the mutant protein LA 32, LA 32KD, derived by mutation of Lys to Arg in the ATP binding site (described by Catling et al., 1994). The PI 3-kinase activity associated with this transformation-defective v-Src protein, although consistently above the vector alone control, was substantially reduced (Fig. 4B). These results indicate a requirement for the v-Src kinase for maximal associated PI 3-kinase activity.


Figure 4: A, PI 3-kinase activity associating with ts v-Src proteins. v-Src was immunoprecipitated using 2 µg of EC10 from equal volumes of lysates from CEF expressing RCAN vector only, RCAN-LA 29 (R29), RCAN-LA 29A2 (R29A2), and RCAN-LA 32 (R32) at restrictive and permissive temperatures. PI 3-kinase assays were performed on washed immunoprecipitates. B, PI 3-kinase activity associating with the kinase-defective (KD) LA 32 v-Src protein and the wild type (WT) positive control grown at 35 °C were similarly determined. As negative control, lysates from CEF were also immunoprecipitated with EC10. C, part A, total cell lysates were immunoprecipitated with anti-p85, separated by 7.5% SDS-PAGE and immunoblotted with anti-phosphotyrosine; part B, p85 immunoblot of total cell lysates. Shown are lysates from cells expressing the following v-Src proteins: kinase-defective (lane1, KD), RCAN vector at permissive (P) and restrictive (R) temperatures (lanes2 and 3, respectively), RCAN-LA 29 (R29) permissive and restrictive (lanes4 and 5), RCAN-LA 29A2 (R29A2) permissive and restrictive (lanes6 and 7), and RCAN-LA 32 (R32) permissive and restrictive (lanes7 and 8).



Phosphorylation of p85 on Tyrosine by ts and Kinase-defective v-Src

We also examined the tyrosine phosphorylation of p85 in cells expressing ts and kinase-defective v-Src mutant proteins by immunoprecipitating p85 from lysates of growing CEF at restrictive and permissive temperatures, blotting to nitrocellulose, and probing with both anti-p85 and anti-phosphotyrosine antibodies. Although the amount of both p85 forms immunoprecipitated was relatively invariant in the uniformly infected cultures, tyrosine phosphorylation of the p85 proteins was temperature-dependent in the case of cells expressing ts mutants (Fig. 4C). In cells expressing the kinase-inactive v-Src protein (lane1), there was no detectable tyrosine phosphorylation of p85. Thus, association of active PI 3-kinase requires the v-Src tyrosine kinase in transformed cells and correlates with tyrosine phosphorylation of p85. Both species of p85 detected from CEF cell extracts by immunoblotting, which co-migrated with those bound to the SH domains (Fig. 1, A and B), are phosphorylated on tyrosine in response to v-Src. Phosphorylation of p85 on tyrosine has also been demonstrated in 3T3 cells in response to PDGF stimulation, although the way in which this tyrosine phosphorylation regulates its activity or interaction with other proteins remains to be established (Kavanaugh et al., 1992).

The v-Src Tyrosine Kinase Rapidly Regulates the Binding of PI 3-Kinase to the SH2 Domain in Quiescent Cells

In addition to its transforming activity, v-Src can act as an intracellular mitogen. In cells made quiescent by serum deprivation, activation of ts v-Src by temperature shift results in transition from G(0) to G(1) and on to S phase and mitosis in the absence of exogenous growth factors (Bell et al., 1975; Durkin and Whitfield, 1984; Welham et al., 1990; Catling et al., 1993). The temperature-dependent association of PI 3-kinase in cells transformed with ts v-Src implicates the lipid kinase as a potential downstream intermediate in the transduction of v-Src tyrosine kinase activity into its biological consequences. We therefore examined the kinetics of association of active PI 3-kinase with ts LA 29 v-Src in CEF that had been made quiescent at restrictive temperature and stimulated for various times by shift to permissive temperature. Fig. 5A demonstrates that there was a very rapid stimulation of v-Src-associated PI 3-kinase activity upon temperature shift. The kinetics of stimulation parallels the activation of the LA 29 v-Src tyrosine kinase, which is detectable at 10 min and continues to rise at 30 min and 1 h after shift to permissive temperature (shown by Catling et al., 1993). Thus, association of PI 3-kinase activity with v-Src is a rapid consequence of activating the v-Src kinase, suggesting that this may be an early event in the pathways leading to v-Src-induced mitogenesis and transformation. In addition, p85 tyrosine phosphorylation was increased upon temperature shift over the same time course (Fig. 5B), suggesting that this was responsible for the increase in v-Src-bound PI 3-kinase activity.


Figure 5: A, stimulation of v-Src-associated PI 3-kinase activity in response to activation of ts v-Src kinase by temperature shift. The PI 3-kinase activity associated with immunoprecipitated v-Src was determined from quiescent CEF expressing ts LA 29 at restrictive temperature and stimulated by shift to permissive temperature for 10, 20, 30, and 60 min. B, immunoblot of total p85 (upperpanel) and tyrosine-phosphorylated p85 (lowerpanel) in CEF upon activation of v-Src for 0, 10, 20, 30, and 60 min.



In order to determine whether this increase was attributable to the SH3 or SH2 domain of v-Src, we examined the PI 3-kinase activity associated with GST-SH3 or GST-SH2 in response to activation of the v-Src kinase by temperature shift. The increase in v-Src-associated PI 3-kinase activity observed is almost certainly due to stimulated binding to the SH2 domain, since binding to GST-SH2 was similarly enhanced while binding to GST-SH3 was invariant (Fig. 6A). To test our hypothesis that tyrosine phosphorylation of p85 provides a mechanism for association with v-Src via the SH2 domain, lysates of CEF expressing ts LA 29 v-Src at permissive temperature were depleted of phosphotyrosine-containing proteins by immunoprecipitation with anti-phosphotyrosine antibody before incubation with GST-SH2 or GST-SH3. We found that p85 binding to SH2 but not SH3 requires tyrosine phosphorylation of p85 (Fig. 6B). In this experiment, the SH3 domain bound a small amount of the faster migrating form of p85 (Fig. 6B, top, short exposure). This was seen in some of our binding experiments and may reflect variations in the state of the cells or the experimental conditions.


Figure 6: A, PI 3-kinase activity associated with GST-SH3 and GST-SH2 over a 60-min time course of v-Src stimulation. B, p85 binding to GST-SH2 or GST-SH3 without (-) or with (+) depletion of phosphotyrosine-containing proteins by anti-phosphotyrosine immunoprecipitation of lysates from CEF expressing ts LA 29 at permissive temperature. Two exposures are shown.



To establish whether activation of the v-Src kinase directly regulates binding to the SH3 and SH2 domains, we prepared protein extracts from CEF expressing ts LA 29 v-Src, which had been made quiescent at restrictive temperature and stimulated by shift to permissive temperature for various times. The ability of p85 to bind to GST-SH3, GST-SH2, and GST-SH3-SH2 was determined by incubation of the extracts with the bacterial fusion proteins, followed by elution and detection by immunoblotting (Fig. 7). The ability of p85 to bind the SH3 domain was detectable in quiescent cultures (Fig. 7, lowerpanel (long exposure), lane1) and largely unaffected by the activation of the v-Src kinase (closedarrow; Fig. 7, lanes4, 7, and 10), consistent with no regulation of the proline-rich sequence-mediated binding upon cell stimulation. However, p85 binding to the SH2 domain, which was detectable from lysates of growing CEF, was almost undetectable in quiescent cultures (Fig. 7, lowerpanel (long exposure), lane2), was rapidly stimulated by 30 min after shift to permissive temperature (Fig. 7, lane5) and remained elevated at 60 min and 120 min (Fig. 7, lanes8 and 11). In particular, the faster migrating form of p85 (arrow) was the more abundant species associating with the SH2 domain early after induction (Fig. 7, lowerpanel (long exposure), lanes5 and 8). Although the slower migrating form of p85 (closedarrow) from unstimulated cells bound the SH3-SH2 fusion, p85 binding to SH3-SH2 was responsive to activation of the v-Src kinase by stimulation of binding of the faster migrating form of p85 (arrow; Fig. 7, upperpanel (short exposure), lanes6, 9, and 12). We have shown that this species preferentially, but not exclusively, binds the v-Src SH2 domain (Fig. 1, 6B, and 7), implying that the increase in its binding to the SH3-SH2 fusion in response to activation of the v-Src kinase is mediated by the SH2 domain. Thus, the contribution of the SH2 domain to the binding of the regulatory subunit of PI 3-kinase to the non-catalytic domains of v-Src varies according to the physiological growth state of the cells; in quiescent (Fig. 7) and exponentially growing normal cells (Fig. 1), binding is largely mediated by the SH3 domain, while in transformed cells soon after stimulation with an activating tyrosine kinase, the SH2 domain contributes significantly to p85 binding.


Figure 7: p85 from quiescent CEF. Immunoblot shows p85 from quiescent CEF expressing ts LA 29 at restrictive temperature (41 °C) (lanes 1-3) and stimulated by shift to permissive temperature (35 °C) for 30 (lanes 4-6), 60 (lanes7-9) and 120 min (lanes 10-12), associating with the v-Src SH3, SH2, and SH3-SH2 (SH3/2) domains. Two exposures are shown.




DISCUSSION

Studies to date have implicated either the SH3 (Wages et al., 1992; Liu et al., 1993) or the SH2 domain (Fukui and Hanafusa, 1989) of v-Src in the interaction with PI 3-kinase. This led us to examine in detail the requirements for the binding and activation of PI 3-kinase by v-Src and to compare them to those proposed for the PDGF receptor.

In the experiments presented, p85 from growing normal CEF bound to the v-Src SH3 domain and, although less strongly, to the SH2 domain. The SH2 domain bound a faster migrating form of p85 in addition to a species that co-migrated with the SH3-bound form. Both species were phosphorylated on tyrosine in cells transformed by v-Src. We also observed enhanced binding of p85 to fused SH3 and SH2 domains, as present in the intact v-Src protein. In line with these findings, an apparent synergism has been proposed for p56, where the presence of an adjacent SH2 domain facilitated binding of PI 3-kinase from T cells to the p56 SH3 domain (Prasad et al., 1993). In cells fully transformed by v-Src, binding of p85 to the SH2 domain was considerably increased. Consistent with this, altered specificity has been observed for PI 3-kinase binding to p56, mediated by the SH3 domain in normal CEF and the SH2 domain in Rous sarcoma virus-transformed CEF (Vogel and Fujita, 1993). The SH2 domain-associated PI 3-kinase had a higher specific activity, possibly as a consequence of tyrosine phosphorylation of the p85 regulatory subunit. Upon stimulation of CEF by activation of the v-Src tyrosine kinase, p85 binding to the SH2 domain was rapidly increased. In particular, the faster migrating form of p85 from transformed cells, which preferentially but not exclusively binds the SH2 domain, displayed enhanced binding to both SH2 and the SH3-SH2 fusion. This enhanced binding was reflected in an increase in the PI 3-kinase activity, which could associate with the SH2 domain in vitro and v-Src protein in vivo, implying that p85 binding to the non-catalytic domains of v-Src, in particular SH2, contributes to the formation of active v-Src-PI 3-kinase complexes at the cell periphery. The rapid increase in v-Src-associated PI 3-kinase activity demonstrates that the increased ability of p85 to bind SH2 is an early event that correlates with phosphorylation of p85 on tyrosine. This is supported by the observation that depletion of cell lysates of phosphotyrosine-containing proteins results in a considerable reduction in SH2- but not SH3-bound p85. Furthermore, these observations suggest that both SH3 and SH2 domains contribute to the binding of PI 3-kinase and that binding to the SH2 domain is responsive to intracellular signals.

The proposed mode of interaction between PI 3-kinase and v-Src is quite distinct from that suggested for the binding of p85 to the PDGF receptor, which is mediated by interaction of the p85 SH2 domains with phosphotyrosine residues in the receptor tail. In addition to the different modes of binding, the apparent regulation of PI 3-kinase activity as a consequence of binding was also different. In the case of binding to a tyrosine phosphopeptide encompassing a PDGF receptor PI 3-kinase binding site, the enzyme was substantially activated, an effect that is obviously independent of tyrosine phosphorylation. Activation of PI 3-kinase upon binding to the Src homology domains was much weaker. Thus, in contrast to PI 3-kinase binding to the PDGF receptor (Shoelson et al., 1993), allosteric activation of PI 3-kinase does not occur to any great extent upon binding to v-Src. We therefore addressed the role of the v-Src catalytic domain in stimulation of v-Src-associated PI 3-kinase activity.

Using a kinase-defective and three ts mutants of v-Src, we demonstrated that v-Src tyrosine kinase activity is required for maximal v-Src-associated PI 3-kinase activity in transformed cells and correlates with phosphorylation of p85 on tyrosine. Since cellular PI 3-kinase bound directly to isolated v-Src SH domains, it seems likely that association of the small amount of PI 3-kinase activity consistently observed with the kinase-defective v-Src occurs through binding of active enzyme to its SH domains. These findings are consistent with the requirement for phosphorylation of PI 3-kinase by another Src family member, p56, in Jurkat T cells (von Willebrand et al., 1994). Thus, in addition to the differences in mode of binding and allosteric activation, v-Src-bound and PDGF receptor-associated PI 3kinase also differ in their tyrosine kinase requirement.

Association with PI 3-kinase is likely to be of considerable importance for the biological activity of v-Src. Here we have refined the understanding of the regulation of v-Src-associated PI 3-kinase. We have demonstrated that all Src homology domains cooperate to positively regulate the binding of PI 3-kinase in response to activation of the oncoprotein. Before ts v-Src kinase activity is switched on, PI 3-kinase is bound predominantly by the SH3 domain. Upon activation of the v-Src tyrosine kinase, additional efficient binding to the SH2 domain occurs in a catalytically activated form. The rapid nature of the induced binding of activated PI 3-kinase thus provides a very sensitive means of recruiting the lipid kinase to the vicinity of its substrates and signaling complexes at the cell membrane. Any role for the SH3 and SH2 domains of p85 in the binding of PI 3-kinase to v-Src remains unknown. The activation of PI 3-kinase in response to the v-Src oncoprotein may also involve the catalytic subunit p110, a possibility that we have not addressed here.


FOOTNOTES

*
This work was supported in part by the Cancer Research Campaign (United Kingdom). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a studentship from the Cancer Research Campaign (United Kingdom).

Supported by a studentship from the Agriculture and Food Research Council (United Kingdom).

**
To whom correspondence should be addressed. Tel.: 44-41-942-9361; Fax: 44-41-942-6521.

(^1)
The abbreviations used are: SH, Src homology; PI, phosphatidylinositol; GST, glutathione S-transferase; CEF, chicken embryo fibroblast(s); ts, temperature-sensitive; HPLC, high performance liquid chromatography; PDGF, platelet-derived growth factor.


ACKNOWLEDGEMENTS

We thank Andy Catling and Nigel Carter for their input to this work in the early stages, John Wyke and Philip Cohen for critical reading of the manuscript, and Sam Crouch for help with photography.


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