(Received for publication, October 18, 1994; and in revised form, December 21, 1994)
From the
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.
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) ()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.
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.
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-, 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.
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 () and
absence (
) of 1 mM proline-rich peptide from p85. A
representative experiment from three repeats is
shown.
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.
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).
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.
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.