(Received for publication, August 22, 1995; and in revised form, December 19, 1995)
From the
The p120 GTPase-activating protein (GAP) is a negative regulator of Ras, which has a central role in signal transduction pathways that control cell proliferation. p120 GAP accelerates the conversion of activated Ras-GTP to its inactive form, Ras-GDP, thereby inhibiting mitogenic signaling. To examine potential contributions of p120 N-terminal sequences to regulation of its C-terminal catalytic domain, we constructed deletion mutants lacking defined regions, including the variable hydrophobic region as well as the Src homology 2 (SH2) and 3 (SH3) domains. These mutant proteins were expressed in infected Sf9 insect cells from recombinant baculoviruses and assayed in vitro for their ability to stimulate the intrinsic GTPase activity of purified Ras. While deletion of the variable hydrophobic region had no effect on p120 GAP activity, deletion of the entire SH2/SH3/SH2 region severely impaired catalytic activity toward Ras. Deletion of individual SH2 and SH3 domains within this region partially inhibited p120 GAP activity. Moreover, p120 N-terminal sequences enhanced the Ras GTPase-stimulating activity of the neurofibromin GAP-related domain. These results demonstrate that sequences in the SH2/SH3/SH2 region of p120 GAP are required for full catalytic activity toward Ras. Together with earlier findings that the p120 GAP SH2 domains mediate interactions with several GAP-associated proteins, our results suggest multiple roles for the N-terminal sequences in regulating p120 GAP catalytic activity and mitogenic signaling pathways. In addition, our results raise the possibility that SH2 domain point mutations in p120 GAP detected in some basal cell carcinomas reduce catalytic activity toward Ras and thereby contribute to oncogenesis.
p21 Ras functions as a binary switch in mitogenic signal
transduction pathways, and, as a key signaling molecule, its activation
must be tightly regulated in order to control cell proliferation (1, 2, 3, 4, 5) . Activated
Ras is bound to GTP, while the GDP-bound form is inactive. Ras can
hydrolyze bound GTP to GDP, resulting in self-inactivation; however,
this intrinsic GTPase activity is low and requires an additional factor
to stimulate the activity(6, 7) . Stimulation is
provided by Ras-specific GTPase-activating proteins (Ras GAPs), ()including p120 GAP and the product of the NF1 gene,
neurofibromin. Both of these GAPs accelerate the conversion of
activated Ras-GTP to its inactive GDP-bound form, thereby
downregulating mitogenic signaling by
Ras(7, 8, 9, 10, 11, 12) .
While the catalytic domain of p120 GAP is contained in its C-terminal portion(13) , the contribution of its N-terminal sequences to catalytic activity is not well defined. The N-terminal sequences harbor a hydrophobic region that varies among species, an SH3 domain flanked by two SH2 domains, as well as pleckstrin homology and calcium-dependent lipid binding domains(14, 15, 16, 17) . Neurofibromin contains a central catalytic domain with high sequence similarity to the p120 GAP catalytic domain, but unlike p120 GAP, it has no identifiable domains outside of this region. Even though the structural differences between p120 GAP and neurofibromin are likely to reflect different cellular functions, if either protein fails to negatively regulate Ras, altered downstream signaling events will occur. Loss of neurofibromin contributes to neoplasia and can result in von Recklinghausen's neurofibromatosis(18, 19, 20, 21) . In addition, point mutations in one of the SH2 domains of p120 GAP have been detected in basal cell carcinomas(22) , consistent with a role for these mutations in neoplasia.
We have shown previously that the SH2 domains in p120 GAP mediate its interaction with tyrosine-phosphorylated p190, which is a GAP for the Rho/Rac family of proteins(23, 24) . Although p120 GAP SH2 and SH3 domains have been shown to mediate several different protein-protein interactions(25, 26, 27) , there is also evidence suggesting that they may be involved in regulating GAP activity toward Ras. Previous studies showed that deletion of the entire N-terminal half of p120 GAP, including the SH2/SH3/SH2 region, leads to a significant loss in GAP activity, thus implicating N-terminal sequences as necessary for maximal p120 GAP catalytic activity(28) . On the other hand, different studies have suggested a possible negative regulatory role for the p120 GAP N-terminal sequences(29) . The specific domains in the p120 GAP N-terminal region that are involved in regulating catalytic activity, however, have not been defined.
To examine contributions of p120 GAP N-terminal sequences to GTPase-stimulating activity, we constructed deletion mutants lacking defined regions, including the variable N-terminal hydrophobic region and the SH2/SH3 domains. Analysis of these mutants showed that deletion of the entire SH2/SH3/SH2 region severely impaired p120 GAP catalytic activity toward Ras, while deletion of the individual SH2 and SH3 domains partially impaired GAP activity. Consistent with a positive role for the SH2/SH3/SH2 region, p120 N-terminal sequences enhanced the Ras GTPase-stimulating activity of the GAP-related domain (GRD) of neurofibromin. Conversely, a reciprocal domain swap with the neurofibromin N-terminal sequences did not restore activity of the p120 GAP catalytic domain. Our results suggest that sequences within the SH2/SH3/SH2 region of p120 GAP are necessary for maximal activity toward Ras and have important implications for the potential contribution of mutations in this region to oncogenesis.
Figure 1: p120 GAP and neurofibromin recombinants expressed from baculoviruses. Full-length bovine p120 GAP cDNA was used to make several deletion mutants. In addition to wild-type p120 GAP, constructs lacking part of the N-terminal hydrophobic region, the entire SH2/SH3/SH2 region, or the individual SH2 and SH3 domains were expressed from baculovirus recombinants in infected Sf9 cells. Chimeras of human p120 GAP and human NF1 cDNAs were also made and expressed in baculovirus-infected Sf9 insect cells. The p120 catalytic domain was fused to the N-terminal sequences of human neurofibromin (NF1-GAP). A reciprocal construct was made where the neurofibromin catalytic domain (NF1 GRD) was fused to the N-terminal sequences of p120 GAP (GAP-NF1). An additional recombinant was constructed containing the isolated GRD of neurofibromin. PH, pleckstrin homology domain; Ca, calcium-dependent lipid binding domain.
GAP catalytic activity was measured
by an in vitro filter-binding assay using purified,
bacterially-expressed p21 c-Ha-Ras loaded with
[-
P]GTP as substrate. Background GAP-like
activity in the assay was found to be negligible when the activity of
nonrecombinant baculovirus-infected Sf9 whole cell lysates was compared
with the activity of Ras alone (data not shown). Using subsaturating
Ras conditions, we compared full-length p120 GAP with two p120
recombinants,
SH and
Nterm, and the GAP activity is shown as
percent of GTP hydrolysis (Fig. 2). Deletion of the SH2/SH3/SH2
region in
SH markedly impaired p120 GAP catalytic activity,
resulting in a level of GTP hydrolysis comparable with that of
nonrecombinant baculovirus-infected cell lysates alone (Fig. 2A). In contrast, deletion of the N-terminal
hydrophobic region in
Nterm had no effect on p120 GAP activity (Fig. 2B), suggesting that this region is not essential
for full catalytic activity toward Ras.
Figure 2:
Deletion of the entire SH2/SH3/SH2 region
severely impairs p120 GAP catalytic activity toward Ras. Lysates of
cells infected with baculoviruses encoding SH or
Nterm were
normalized for expression levels to full-length p120 GAP and then
assayed for their ability to stimulate Ras intrinsic GTPase activity.
GAP activity is shown as % GTP hydrolysis with time using 80 nM [
-
P]GTP-loaded Ras (see
``Materials and Methods''). As a negative control, lysates of
Sf9 cells infected with nonrecombinant baculovirus (Baculo.)
were used to detect any background GTPase activity. Plots from both
SH (A) and
Nterm (B) represent the means of
three separate experiments with standard errors
shown.
Consistent with results obtained
under subsaturating Ras concentrations, the SH mutant was severely
impaired in its activity toward Ras compared with full-length p120 GAP (Fig. 3A). We assayed the individual p120 domain
deletion mutants and found that the
NSH2,
SH3, and
CSH2
deletion mutants were all partially impaired to similar extents in
their activity toward Ras (Fig. 3, B-D). While
the activities of the individual p120 domain deletion mutants were
higher than that of
SH, their activities were reproducibly lower
than that of full-length p120. Relative levels of GAP proteins used in
each assay are shown as insets to the graphs. These results indicate
that the presence of each domain within the SH2/SH3/SH2 region is
necessary for p120 GAP to display full catalytic activity toward Ras.
Figure 3:
The
individual NSH2, SH3, and CSH2 domains of p120 GAP contribute to
maximal GAP activity. A, clarified lysates from Sf9 cells
infected with recombinant viruses encoding full-length p120 GAP and
SH were resolved by SDS-polyacrylamide gel electrophoresis, and
Western blots were probed with monoclonal anti-GAP antibody 7D1.
Normalized proteins were subjected to GAP catalytic assays using
increasing concentrations of purified c-Ha-Ras preloaded with
[
-
P]GTP. The initial velocities of Ras-GTP
hydrolysis as measured by pmol of phosphate released/min reflect GAP
catalytic activity. Similar assays were performed on individual domain
deletion mutants: B,
NSH2; C,
SH3; and D,
CSH2. Recombinant protein levels were normalized by
Western blot analysis to full-length p120 GAP (as shown in graph insets) with monoclonal anti-GAP antibodies 6F2 (B, D) or 7D1 (C) as probes. Results represent the means
of two separate experiments, with standard errors
shown.
Figure 4: p120 GAP N-terminal sequences enhance neurofibromin activity toward Ras. A, the catalytic activity of the NF1-GAP chimera was compared with that of full-length p120 GAP after normalizing protein levels by Western blot analysis using polyclonal anti-GAP antibody 677 as probe. B, similarly, GAP-NF1 and NF1 GRD recombinants were normalized to each other by Western blot using polyclonal anti-GRD antibody as probe. Nonrecombinant baculovirus (Baculo.) infected cell lysates were used as controls for background GTPase activity. Activity is reflected by percent hydrolysis of GTP as a function of time, with 0 and 100% activity defined by the activity of GAP-NF1 at 0 and 20 min, respectively. For all constructs, assays were performed in duplicate, and plots represent the means with standard errors shown.
To determine whether any sequence fused N-terminal to the
p120 GAP catalytic domain enhances activity, a reciprocal recombinant
(NF1-GAP) was analyzed that encodes the catalytic domain of p120 GAP
downstream of the N-terminal portion of neurofibromin (Fig. 1).
Significantly, NF1-GAP had only marginally more activity than the
nonrecombinant baculovirus control for background (Fig. 4A).
Unlike what was observed with the GAP-NF1 hybrid, the N-terminal
sequences of neurofibromin did not have a positive effect upon p120 GAP
activity. This finding is consistent with the suggestion that the
enhanced activity of the p120 GAP compared with SH is due to
specific sequences within the p120 GAP N-terminal portion. Taken
together with results from the SH2 and SH3 domain deletion mutants, our
data demonstrate that the SH2/SH3/SH2 region of p120 GAP is required
for full catalytic activity toward Ras and that the N-terminal
sequences of p120 GAP stimulate neurofibromin GRD activity.
Because negative regulation of Ras activity by GAPs is one crucial mechanism the cell employs to control Ras, regulation of GAP activity is an important event for normal cell growth(8, 16, 36, 37, 38) . The N-terminal portion of p120 GAP has been implicated in a variety of GAP interactions and functions in the cell. The N-terminal hydrophobic region has recently been shown to contribute to interactions with Src-family members through its proline-rich motif(39) . The SH2 domains are responsible for multiple protein-protein interactions, including interactions between GAP and receptor tyrosine kinases(40, 41) , nonreceptor tyrosine kinases(30, 42) , as well as the GAP-associated proteins, p62 and p190(23, 43, 44, 45) . Furthermore, the intact SH2/SH3/SH2 region is capable of uncoupling a heterotrimeric G protein from muscarinic receptors(29) , while the p120 GAP SH3 domain alone blocks germinal vesicle breakdown in Xenopus oocytes (46) and inhibits carbachol-dependent NIH 3T3 transformation via muscarinic receptors(47, 48) . In addition to affecting biological functions within different cells, the entire N-terminal half of p120 GAP has been suggested to positively regulate GAP catalytic activity in one case, and possibly inhibit it in another(28, 29) . To address the question of the contribution of p120 GAP N-terminal sequences, especially its SH2/SH3/SH2 region, to p120 GAP catalytic activity toward Ras, we constructed several p120 GAP deletion mutants, expressed them in the baculovirus/Sf9 insect cell system, and assayed their ability to stimulate Ras intrinsic GTPase activity.
Consistent with earlier
results involving deletion of the entire N terminus of p120
GAP(28) , deletion of the SH2/SH3/SH2 domains significantly
impaired p120 GAP activity toward Ras. The SH mutant did not
detectably stimulate GTPase activity above that of the Ras intrinsic
GTPase activity as reflected by the nonrecombinant baculovirus control.
Other studies indicated that the isolated p120 GAP catalytic domain
displayed reduced but detectable
activity(10, 12, 13, 28) ; this
difference might be explained by different constructs or levels of
protein used in the assays. By contrast, analysis of the
Nterm
mutant revealed that deletion of this region had no affect upon p120
GAP catalytic activity. These findings suggest a specific requirement
for the SH2/SH3/SH2 region for maximal p120 GAP catalytic activity. We
examined which domains within this SH2/SH3/SH2 region were responsible
for the reduced activity of the
SH GAP construct. p120 GAP
deletion mutants lacking the individual N-terminal SH2 or SH3 or
C-terminal SH2 domains were also impaired in their ability to stimulate
Ras GTPase activity, albeit to a lesser extent. No single domain
deletion impaired p120 GAP activity as much as deleting the entire
SH2/SH3/SH2 region did, suggesting that the complete lack of activity
of the
SH mutant may be due to the cumulative effect of the
individual domain deletions.
To explore further the stimulatory role
of the p120 N-terminal sequences, we investigated whether they could
affect the GAP activity of the NF1 protein, neurofibromin. When the N
terminus of p120 GAP was fused with the GRD of neurofibromin (GAP-NF1),
this construct exhibited much higher GAP catalytic activity than the
neurofibromin GRD alone. In addition, fusion of p120 N-terminal
sequences to the neurofibromin GRD stimulated activity above that of a
near full-length neurofibromin recombinant containing the N-terminal
neurofibromin sequences as well as the GRD. ()Significantly,
the reciprocal domain swap did not restore p120 GAP activity, as shown
by analysis of the hybrid containing the N-terminal sequences of
neurofibromin and the catalytic domain of p120 GAP (NF1-GAP). This
finding, taken together with the analysis of p120 GAP deletion mutants,
points to sequences within the p120 GAP SH2/SH3/SH2 domains as being
necessary for p120 GAP to maximally stimulate GTPase activity of Ras.
While the mechanism of how the SH2/SH3/SH2 region positively influences GAP activity remains to be determined, sequences within this region may specifically interact with the catalytic domain to stimulate activity. Alternatively, it is possible that the SH2/SH3/SH2 region contributes to the overall conformational stability of the protein, and thus large deletions in this region may cause nonspecific conformational changes that alter catalytic function. Further study will be required to distinguish between these possibilities. Nevertheless, in addition to the many other roles that these domains have, our findings demonstrate that the SH2 and SH3 domains are essential for p120 GAP to display full catalytic activity toward Ras. These results are of particular interest in light of the observation that mutations in the gene encoding p120 GAP have been detected in human basal cell carcinomas(22) . Significantly, in all three cases examined, point mutations resulting in amino acid changes were detected in the C-terminal SH2 domain but not in the catalytic domain of p120 GAP. Our in vitro findings raise the possibility that these p120 GAP mutants have reduced catalytic activity, which would lead to activation of Ras and hence contribute to oncogenesis. We cannot exclude, however, that the SH2 mutations influence p120 GAP intracellular location or interaction with other proteins in vivo and in this way contribute to oncogenesis. Furthermore, it should be noted that earlier studies (3) suggested that the isolated catalytic domain of p120 GAP was at least as efficient as the full-length protein in suppressing cell transformation by Src or Ras. While the basis for this apparent discrepancy with our findings is not clear, these contrasting results suggest that the regulation of p120 GAP in intact cells may be complex. It will be of significant interest to characterize the in vitro and in vivo properties of the C-terminal SH2 domain mutants associated with basal cell carcinomas(22) . Based on our biochemical data, mutations in the N-terminal SH2 or SH3 domains might also contribute to human cancers, providing a rationale for screening tumors for mutations in the entire SH2/SH3/SH2 region of p120 GAP.