(Received for publication, September 21, 1994; and in revised form, November 18, 1994)
From the
Phosphatidylinositol 3`-kinase (PI 3`-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3`-kinase in vitro. However, activation by the mono-phosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3`-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3`-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3`-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3`-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3`-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3`-kinase by tyrosyl-phosphorylated proteins requires occupancy of both SH2 domains in p85.
Phosphatidylinositol 3`-kinase (PI 3`-kinase) ()is a
lipid kinase that has been implicated in the regulation of cell growth
by growth factor receptors and oncogene products(1) . PI
3`-kinase phosphorylates phosphatidylinositol at the D-3 position of
the inositol ring(2) , and stimulation of cells with mitogens
such as platelet-derived growth factor or transformation of cells with
polyoma middle T antigen leads to increases in the levels of the lipid
products PI 3,4-P
and PI
3,4,5-P
(3, 4) . The function of these
lipids has not yet been determined, but their low abundance and rapid
appearance in mitogen-stimulated cells suggests that they may serve as
intracellular second messengers.
PI 3`-kinase is a heterodimer
composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic
subunit (p110)(1) . During activation of PI 3`-kinase by
tyrosine kinase receptors such as the platelet-derived growth factor or
colony stimulating factor-1 receptors, the SH2 domains of p85 bind
directly to specific phosphorylated YXXM motifs present in the
cytoplasmic domains of the receptors (reviewed in (5) ). In the
case of the insulin receptor, PI 3`-kinase binds to phosphorylated
YXXM motifs in the substrate IRS-1 to a greater extent than to
the receptor itself(6, 7) . The binding of receptors
or substrates containing phosphorylated YXXM motifs activates
the lipid kinase in vitro and in intact cells(6) .
Activation of PI 3`-kinase in vitro can be mimicked by
synthetic phosphopeptides that contain the YXXM motif, and
this peptide-mediated activation correlates with conformational changes
in the p85 subunit(6, 8, 9) . Thus,
regulation of PI 3`-kinase catalytic subunit p110 appears to require
conformation changes in the regulatory subunit p85, which are driven by
SH2 domain binding to phosphorylated YXXM motifs. Recently,
several additional mechanisms of activation have been described,
including the binding of Src family SH3 domains to p85 (10) and
the binding of p21 to p110(11) . In
contrast, PI 3`-kinase activity is reduced 80% by phosphorylation of
Ser
in p85 and can be restored by treatment with
phosphatase 2A(12, 13) .
In our previous study(6) , we found that activation by mono-phosphorylated YXXM peptides is significantly less potent than activation by the multiply phosphorylated IRS-1; based on these data, we proposed that full activation of PI 3`-kinase requires occupancy of both SH2 domains. This hypothesis has been supported by studies showing that bis-phosphopeptides exhibit enhanced activation of PI 3`-kinase relative to mono-phosphopeptides(8, 14, 15) . In the present study, we have directly tested our hypothesis using recombinant PI 3`-kinase containing disabling mutations in the N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domain reduces phosphopeptide binding to p85 and reduces maximal PI 3`-kinase activation by 50%. Mutation of both SH2 domains abolishes both binding and activation. These data demonstrate that full activation of PI 3`-kinase requires occupancy of both SH2 domains in the p85 regulatory subunit.
Figure 1: PI 3`-kinase activation by wild-type and denatured IRS-1. Native P-IRS-1 or denatured RCM-P-IRS-1 was incubated with immunopurified PI 3`-kinase for 30 min at 4 °C. The immunoprecipitates were washed and assayed for PI 3`-kinase activity as described. All determinations were done in triplicate, and the data are the mean ± S.E. from three experiments.
Figure 2:
PI 3`-kinase binding and activation by
mono- and bis-phosphorylated YXXM peptides. A, a
glutathione S-transferase fusion protein containing both SH2
domains from p85 was incubated with P-labeled P-Y628 in
the presence of the indicated concentrations of unlabeled
phosphorylated mono-phosphorylated (P-Y628) or bis-phosphorylated
(P-Y608/P-Y628) peptide for 30 min at 4 °C. The fusion protein was
immobilized on glutathione-Sepharose, the beads were pelleted, and
bound radioactivity was measured by Cerenkov counting. The data are
representative of two separate experiments. B, PI 3`-kinase,
immunopurified from quiescent Chinese hamster ovary cells with anti-p85
antibody and protein A-Sepharose, was incubated with
mono-phosphorylated (P-Y628) or bis-phosphorylated (P-Y608/P-Y628)
YXXM peptide at the indicated concentrations for 30 min at 4
°C. PI 3`-kinase was assayed as described in the continued presence
of the peptide. All determinations were done in triplicate, and the
data are the mean ± S.E. from three
experiments.
Figure 3: Model of enhanced PI 3`-kinase activation by bis-phosphopeptides. A, activation of PI 3`-kinase by tyrosyl phosphoproteins requires the occupancy of only the upper SH2 domain. However, the interaction of the bis-phosphopeptide with the two SH2 domains increases its net binding affinity and leads to an increased occupancy of the single activating site. Occupancy of this second site does not itself add to the magnitude of activation. B, activation of PI 3`-kinase requires occupancy of both SH2 domains. Partial activation occurs when either SH2 domain is occupied, and full activation occurs only when both sites are occupied.
To differentiate these two models, we produced
recombinant PI 3`-kinase by co-infection of Sf-9 cells with baculovirus
constructs coding for p110 plus wild-type p85, or p85 containing
disabling mutations in the N-terminal, C-terminal, or both SH2 domains.
In the mutant p85 constructs, the arginine residue in the invariant
FLVRES motif was mutated to alanine (Arg and Arg
in the N- and C-terminal SH2 domains, respectively). This
substitution has been shown to inhibit phosphopeptide binding to the
Abl SH2 domains without causing global structural changes(24) .
If the model in Fig. 3A is correct, then maximal
activation of recombinant PI 3`-kinase containing one or two functional
SH2 domains should be equivalent. If the model in Fig. 3B is correct, then the magnitude of PI 3`-kinase activation at
saturating levels of P-IRS-1 or phosphopeptide should be reduced in the
constructs containing a single functional SH2 domain.
Wild-type and
mutant p85 was co-expressed in Sf-9 cells with wild-type p110. All of
the mutants were expressed at similar levels and were shown by
co-immunoprecipitation experiments to form stable dimers with p110. The
ability of recombinant wild-type and mutant p85 to bind to tyrosyl
phosphopeptides was determined using an I-labeled analog
of P-Y628 that contains the photo-activable amino acid
benzoylphenylalanine in the Y+1 position (Fig. 4).
Cross-linking of p85 SH2 domains by
I-labeled BPA-P-Y628
was completely inhibited by 100 µM unlabeled P-Y628 and
was therefore specific (data not shown). Incubation of
I-labeled BPA-P-Y628 with SF-9 cell lysates containing
wild-type p85, followed by irradiation at 350 nm for 60 min at 4
°C, lead to the labeling of a single 85-kDa band (Fig. 4, lanee). No labeling was observed in lysates from
control Sf-9 cells or cells expressing p110 alone (Fig. 4, lanesa and f). Cross-linking of p85 was
significantly reduced in lysates from Sf-9 cells producing N- or
C-terminal SH2 p85 mutants (Fig. 4, lanesb and c, respectively) and was barely detectable in lysates
containing p85 mutated at both SH2 domains (Fig. 4, laned). When the data were normalized to the amount of p85 in
each lysate by immunoblotting with anti-p85 antibody, mutation of
either the N- or C-terminal SH2 domains reduced phosphopeptide
cross-linking by 50%. However, these data do not take into account
possible differences in the binding affinity of the N- and C-terminal
SH2 domains in intact p85 nor the possible differential yields of
covalent labeling of each SH2 domain upon photolysis, and a comparison
of the relative decrease in binding to the different single mutants is
therefore only approximate.
Figure 4:
Labeling of recombinant p85 with I-labeled BPA-P-Y628. Lysates from control or
baculovirus-infected SF-9 cells were incubated with
I-labeled BPA-P-Y628 for 30 min at 4 °C, followed by
irradiation at 350 nm for 60 min on ice. The proteins were separated by
SDS-polyacrylamide gel electrophoresis (12.5% resolving) and visualized
by autoradiography. Labeling of p85 was quantitated using a Molecular
Dynamics phosphorimager. Toppanel: lanea, control cells; laneb, p110 plus
mutant p85 (R358A); lanec, p110 plus mutant p85
(R649A); laned, p110 plus mutant p85 (R358A/R649A); lanee, p110 plus wild-type p85; lanef, p110 alone. Bottompanel, labeling
of p85 in Sf-9 lysates was normalized for the expression of p85, as
determined by immunoblotting with anti-p85 antibody. WT, wild
type.
We then examined PI 3`-kinase activation in lysates from cells infected with baculovirus constructs producing wild-type p110 and wild-type or mutant p85 (Fig. 5). Incubation of lysates containing wild-type p85/p110 dimers with saturating concentrations of tyrosyl-phosphorylated IRS-1 (100 nM) lead to a 3.5-fold activation of PI 3`-kinase. In contrast, no activation was detected in control Sf-9 lysates or in lysates containing only p110. Mutation of either the N- or C-terminal SH2 domain of p85 reduced activation by 50%, whereas mutation of both domains completely eliminated activation by P-IRS-1.
Figure 5: Activation of recombinant PI 3`-kinase by IRS-1 and phosphopeptide. Sf-9 cells were infected with baculovirus encoding p110 alone or in combination with wild-type or mutant p85. Lysates were prepared from control cells (CTL), cells infected with p110 alone (p110), or cells co-infected with p110 and the N-terminal SH2 p85 mutant (R358A), the C-SH2 mutant p85 (R659A), the double-SH2 p85 mutant (R358A/R659A), or wild-type p85 (WT). Lysates were incubated with tyrosyl-phosphorylated IRS-1 (100 nM) for 30 min at 4 °C and then assayed for PI 3`-kinase activity. All determinations were done in duplicate, and the data are the mean ± S.E. from four experiments.
SH2 domains have been implicated in the regulation of an increasing number of proteins involved in signal transduction by growth factors and oncogene products(25) . For the PI 3`-kinase, the binding of its SH2 domains to phosphorylated IRS-1 or peptides containing phosphorylated YXXM motifs can directly regulate its activity(6) . We and others (6, 8, 14, 15) have shown that peptides or proteins containing multiple phosphotyrosine residues bind and activate PI 3`-kinase with enhanced potency relative to mono-phosphopeptides. However, these studies cannot differentiate between a mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. In the present study, we show that point mutations in either the N- or C-terminal SH2 domains of p85 inhibit phosphopeptide binding to p85 and reduce the activation of PI 3`-kinase by IRS-1 by 50%. These data are the first direct demonstration that full PI 3`-kinase activation by tyrosyl phosphoproteins requires occupancy of both SH2 domains.
As we have
previously noted, a number of the receptors that activate PI 3`-kinase
have pairs of tyrosine phosphorylation sites that may be involved in
the bivalent binding of p85 SH2 domains(6) . Thus, the kinase
insert region of the platelet-derived growth factor -receptor
contains two tyrosine phosphorylation sites, both of which are
important for PI 3`-kinase
binding(26, 27, 28, 29) . Recent
studies(8, 30) have also suggested that two
phosphotyrosine residues are required for activation of PI 3`-kinase by
the hepatocyte growth factor receptor and polyoma middle T antigen.
Mutation of Tyr
but not Tyr
or Tyr
within the colony stimulating factor-1 receptor kinase insert
domain blocks PI 3`-kinase binding (30) ; however, activation
of PI 3`-kinase was not examined and may require an additional
phosphotyrosine residue. In contrast, in vitro activation of
PI 3`-kinase by a peptide containing the motif Y
THM from
the insulin receptor C terminus is unaffected by the phosphorylation of
the neighboring Tyr
(31) . The ability of a
second phosphotyrosine residue to augment the activation by a
phosphorylated YXXM motif presumably depends on the primary
sequence surrounding the second phosphotyrosine residue or perhaps the
distance between the residues. In this regard, the optimal spacing
between phosphotyrosine residues for PI 3`-kinase activation has not
been determined. The two phosphotyrosine residues in the IRS-1-derived
peptide examined here are 20 amino acids apart, whereas the
phosphotyrosine residues in activating peptides derived from polyoma
middle T and the hepatocyte growth factor receptor are 7 residues
apart; those in the insulin receptor bis-phosphopeptide, which does not
show enhanced activation relative to mono-phosphopeptides, are only 6
residues apart.
Little is known about the native tertiary structure of p85, but these observations suggest that the phosphopeptide binding sites of the p85 SH2 domains must lie quite close to each other. The demonstration that p110 binds to a region between the two SH2 domains of p85(32, 33, 34) suggests an intriguing model in which occupancy of both SH2 domains causes a conformational change in the intervening region and activates p110. In contrast, a recent report showed that a construct containing either SH2 domain plus the intervening region could bind to the p110 catalytic subunit and mediate phosphopeptide-stimulated activation (35) ; it is not clear how the structure of this truncated construct relates to that of the intact p85 molecule. The resolution of these questions must await a better structural characterization of the intact regulatory and catalytic subunits of PI 3`-kinase.