From the
Activation of p21
Signal transmission by many receptor tyrosine kinases involves
transient conversion of p21
Activation
of p21
More recent experiments yielded
the surprising result that, in intact flies, a mutant Drosophila Sos protein lacking the adaptor binding COOH-terminal domain was
at least as capable as full-length Sos protein in promoting R7 cell
development(19) . This effect is thought to result from
effective modulation of the p21
Equal amounts of protein from
each lysate were incubated with 30 µl of glutathione-Sepharose,
which was prebound with GST or GST-Grb2, on an end-over-end mixer at 4
°C for 2 h. GST and GST-Grb2 fusion plasmid were gifts from Dr. J.
Schlessinger (New York University, New York, NY). The Sepharose was
pelleted by centrifugation at 15,000
Drosophila Sos cDNA constructs were engineered to
evaluate the functions of its three major domains. The catalytic
domain, Cat, is defined as the region of homology to the Saccharomyces cerevisiae CDC25 protein, known to activate
yeast Ras(24) . The domain COOH-terminal to Cat containing
SH3-binding motifs is denoted as C, and the region
NH
The failure of CatHA to cause GTP loading of
p21
One explanation to
account for the data in Fig. 2is that Grb2 can also bind regions
in the dSos protein that reside in the NH
At last count, 71 proteins have been reported
to contain sequences with similarity to the PH domains of
pleckstrin(28) , but the presumed cellular partner or partners
that associate with these domains have remained elusive. Suggestions
that
The Dbl homology region of dSos represents only a small portion of
the regions in CDC24(27, 33) , Vav(27) , and Dbl (27) that appear to be required to catalyze GTP loading of
Rho-like GTP-binding proteins. The degree of sequence similarity
between the Dbl homology region in dSos and the corresponding regions
of these Rho exchangers is somewhat low, but compelling. Thus, Sos
proteins do not catalyze Rho exchange activity, but might bind such
GTP-binding proteins. This hypothesis deserves testing in future
experiments.
The key result reported here, that the Grb2 binding
region of dSos is not sufficient to allow Cat function in COS-1 or 293
cells, is not necessarily inconsistent with the recent data and
interpretation of Aronheim et al.(18) that implicates
a critical role for Grb2 in recruiting Sos proteins to receptor
complexes. However, our data reveal an additional requirement for the
NH
We thank Drs. Sean Eagan and Tony Pawson for
stimulating discussions and for providing manuscripts prior to
publication. HA peptide was synthesized in the Peptide Core Facility at
the University of Massachusetts Diabetes and Endocrinology Research
Center (funded by National Institutes of Health Grant P30DK32520). We
thank Judy Kula for excellent assistance in preparation of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
by receptor tyrosine
kinases is thought to result from recruitment of guanine nucleotide
exchange factors such as Son-of-sevenless (Sos) to plasma membrane
receptor substrates via adaptor proteins such as Grb2. This hypothesis
was tested in the present studies by evaluating the ability of
truncation and deletion mutants of Drosophila (d)Sos to
enhance [
P]GTP loading of p21
when expressed in
P-labeled COS or 293 cells.
The dSos catalytic domain (residues 758-1125), expressed without the
dSos NH
-terminal (residues 1-757) or adaptor-binding,
COOH-terminal (residues 1126-1596) regions, exhibits intrinsic
exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP
exchange activity. Here we show that this dSos catalytic domain fails
to affect GTP
p21
levels when expressed in
cultured mammalian cells unless the NH
-terminal domain is
also present. Surprisingly, the COOH-terminal, adaptor binding domain
of dSos was not sufficient to confer p21
exchange activity to the Sos catalytic domain in these cells
in the absence of the NH
-terminal domain. This function of
promoting catalytic domain activity could be localized by mutational
analysis to the pleckstrin and Dbl homology sequences located just
NH
-terminal to the catalytic domain. The results
demonstrate a functional role for these pleckstrin and Dbl domains
within the dSos protein, and suggest the presence of unidentified
cellular elements that interact with these domains and participate in
the regulation of p21
.
proteins to the
biologically active, GTP-bound state from the inactive GDP-bound
form(1) . When bound to GTP, p21
associates with Raf protein kinases, causing events that
stimulate these and multiple other protein kinases, including the
mitogen-activated protein kinases(2, 3, 4) .
These latter protein kinases catalyze phosphorylation of many cellular
proteins, resulting in their regulation. The p21
signaling cascade modulates important cellular processes
such as transcription and protein synthesis(5) .
proteins by receptor tyrosine kinases is
thought to result from recruitment of guanine nucleotide exchange
factors such as Son-of-sevenless (Sos) proteins to plasma membrane
receptor substrates via adaptor proteins such as
Grb2(6, 7, 8, 9, 10, 11, 12, 13) .
The mechanism of this recruitment seems to involve binding of the
NH
-terminal Src homology (SH)(
)
3
domain of the adaptor protein Grb2 with proline-rich motifs in the COOH
terminus of Sos proteins(14, 15) . Stimulation of
tyrosine phosphorylation of membrane receptors or receptor substrates
by growth factors provides appropriate tyrosine phosphate sites that
bind the SH2 domain of Grb2. Rapid association of Grb2 (6, 11, 16) or Sos(6, 9, 17) proteins to complexes containing such
tyrosine-phosphorylated species as EGF receptor, Shc, IRS-1, and Syp
has been demonstrated. Furthermore, Sos protein mutants engineered to
contain farnesylation or myristoylation signals exhibit plasma membrane
localization and the ability to activate p21
when expressed in NIH 3T3 cells (18). Such Sos constructs in
which the COOH-terminal, Grb2 binding domains were deleted displayed
enhanced p21
exchange activity compared to
full-length Sos(18) . These data suggest that Grb2 binding to
Sos proteins may have two functions: recruitment of Sos proteins to
plasma membrane sites and removal of inhibition of the Sos COOH
terminus on Sos catalytic activity.
signaling
pathway during R cell development. Importantly, a protein encoding only
the dSos catalytic domain was unable to mediate this response. The
ability of the mutant dSos protein containing both
NH
-terminal and catalytic domains to promote R7 cell
development required expression of an intact Sevenless
protein(19) . These data indicate that the COOH-terminal,
adaptor binding domain of Sos is not sufficient to confer to
p21
exchange activity to the Sos catalytic
domain in intact flies. The present experiments were designed to test
the role of NH
-terminal and COOH-terminal regions of dSos
in
P-labeled mammalian cells where p21
loading with [
P]GTP can be measured
directly.
Construction of pCMV5-dSosHA Deletion
Constructs
The dSos constructs engineered as described
previously (19) were modified at their COOH terminus to encode a
hemagglutinin (HA) epitope extension. The 9-amino acid peptide sequence
Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala of the influenza HA, which is
specifically recognized by the monoclonal antibody 12CA5(20) ,
was added to the 3` end of the dSos constructs coding sequences. The
dSos cDNA constructs encode the following amino acids: NCatCHA,
1-1596; NCatHA, 1-1125; NCatPHHA, 1-475 and
592-1125; PHCatHA, 433-1125; CatHA 758-1125; CatCO
HA,
1-53 and 681-1596; CO
HA, 1-53, 681-690,
and 1040-1596; NH
HA, 1-690; NCat
200HA,
1-311 and 681-1125; NCat
300HA, 1-219 and 681-1125. The
cDNA's encoding p21
, and the various dSos
mutant constructs were subcloned into the mammalian expression vector
pCMV5(21) . The p21
cDNA was a gift from
Dr. L. A. Feig.
Cell Lines and Transient Transfections
COS-1 cells
(American Type Culture Collection RL 1650) were grown in DMEM medium
containing 10% bovine calf serum, and 293 cells (American Type Culture
Collection CRL 1573) were grown in DMEM medium containing 10% calf
serum. Plates (6 cm) of COS-1 cells and 293 cells were transfected
using the CaPO method according to standard protocols. A
total of 7 µg of plasmid DNA was used per plate.
Cell Labeling and p21
Two days after
transfection, the cells were labeled with carrier-free
[Activation Assay
P]orthophosphate (1.0 mCi/ml) for 4 h at 37
°C in 1.5 ml of phosphate-free DMEM supplemented with 25 mM Hepes and 2 mM pyruvate. Analysis of labeled GTP and GDP
bound to p21
was performed as
described(13, 22) .
In Vitro Binding Assay
Spodoptera frugiperda Sf9 insect cells were cultured in Grace's complete medium
and infection with high titer baculovirus stocks of either NCatCHA or
NCatHA was carried out according to standard protocols. NCatCHA and
NCatHA cDNA constructs were subcloned into the baculovirus transfer
vector pVL1393 for expression in Sf9 cells. Frozen pellets of
baculovirus-infected Sf9 cells (NCatCHA, NCatHA, and wild-type) were
homogenized on ice using a 2-ml homogenizer (10 strokes) in 1 ml of
cold lysis buffer (phosphate-buffered saline with 10 mM NaF, 1
mM dithiothreitol (DTT), 1 mM vanadate, 1 mM
benzamidine, 1 mM phenylmethylsulfonyl chloride, and
aprotinin, pepstatin, and leupeptin at 10 µg/ml each). Homogenates
were spun in a microcentrifuge at 15,000 g for 15 min
at 4 °C. The supernatants were removed and assayed for total
protein content using the Bradford method(23) . Proteins from
each lysate were dissolved in sample buffer, subjected to SDS-PAGE, and
stained with Coomassie Brilliant Blue.
g for 2 min at 4
°C. Pellets were washed twice with (20 mM Hepes, pH 7.5,
10% glycerol, 100 mM NaCl, and 1 mM DTT), twice with
(20 mM Hepes, pH 7.5, 10% glycerol, 500 mM NaCl, and
1 mM DTT) and once with (20 mM Hepes, pH 7.5, 10%
glycerol, 100 mM NaCl, and 1 mM DTT). Proteins
remaining bound to the GST or GST-Grb2 were dissolved in SDS-PAGE
sample buffer. Samples were loaded on SDS-PAGE gels (6%) and
transferred to nitrocellulose filters. Filters were probed with anti-HA
monoclonal antibody, and antibody-bound proteins were visualized using
ECL (Amersham Corp.) according to the manufacturer's
specifications.
-terminal to Cat is denoted as N. Hemagglutinin epitope
(HA)-tagged constructs corresponding to those depicted in Fig. 1were prepared and ligated into the pCMV5 expression vector.
COS-1 (Fig. 2) or 293 cells (not illustrated) were transiently
transfected with these cDNA constructs and p21
cDNA, labeled with
P for 4 h, lysed, and
immunoprecipitated with anti-Ras monoclonal antibody. All of the
HA-tagged Sos proteins encoded by the cDNA constructs were readily
visualized by immunofluorescence microscopy when transiently expressed
in either COS-1 or 293 cells (not illustrated). Furthermore, the
heterologously expressed p21
was visualized
virtually exclusively at the cell surface by immunofluorescence
microscopy (not illustrated).
Figure 1:
Schematic representation of the
HA-tagged native and mutant Sos constructs employed in the experiment
of Fig. 2. The amino acid numbers are denoted beneath
NCatCHA.
Figure 2:
Stimulation of p21 GTP/GDP exchange in
intact cells by expression of recombinant dSos constructs NCatCHA or
NCatHA. A, COS-1 cells were transiently co-transfected with
the p21 (2 µg) and the HA-tagged dSos cDNAs (5 µg each) shown.
[P]Orthophosphate-labeled cells were then lysed,
and the cleared lysate was immunoprecipitated with anti-p21 antibody as
described (13, 22). The positions of the GTP and GDP standards are
indicated. Lane1 represents cells that were
transfected with p21 only; lane8, with NCatCHA only. B, graphic representation of p21 activation by expression of
NCatCHA or NCatHA in COS-1 cells. The GTP and GDP spots on the TLC
plate were excised, and the amount of
[
P]orthophosphate incorporated was determined
using a
counter. The ratio of GTP to GDP was calculated and
expressed as a percentage, and the values shown represent the average
of three individual experiments with verticalbars denoting standard errors. The leftmostbar on
the graph represents cells that were transfected with p21 only.
Previous work showed that the
catalytic domain of dSos (CatHA construct) is able to rescue S.
cerevisiae with the CDC25 mutation that inactivates
endogenous Ras exchange activity(19) . A similar Cat construct
of human Sos produced in Sf9 cells was also shown to be fully effective
in causing GTP/GDP exchange activity in vitro using Sf9
cell-derived K-p21
protein(25) .
Importantly, CatHA had no significant stimulatory effect on
[
P]GTP binding to p21
when expressed in intact COS-1 cells (Fig. 2, lane5). Taken together, these previous results and the data
in Fig. 2indicate that the catalytic domain of dSos possesses
intrinsic guanine nucleotide exchange activity but is unable to
catalyze this reaction when this protein domain is overexpressed in
mammalian cells.
in intact cells suggested, as expected, that
the Grb2 binding domain of dSos is necessary to carry out this
function. However, a construct (CatCHA) containing both the catalytic
and COOH-terminal domains of dSos also failed to cause p21
activation when expressed in the COS cell system (Fig. 2, lane6). Surprisingly, transfection of
a cDNA construct (NCatHA) encoding the complete NH
-terminal
and catalytic domains of dSos caused [
P]GTP
loading of p21
that was even greater than that
elicited by full-length dSos (Fig. 2, lanes 2 and 4). It should be noted that NCatHA may not actually be more
active than full-length dSosHA because Western blotting with anti-HA
antibody showed somewhat higher expression of the former (not
illustrated). Expression of either the NH
-terminal (NHA) or
COOH-terminal (CHA) domains of dSos alone had no effect on cellular
p21
[
P]GTP concentrations in
COS-1 cells. No p21
-associated
[
P]GTP could be detected when p21
cDNA was omitted from the transfection (Fig. 2, lane8). Similar data were obtained with this series
of dSos constructs using 293 cells, although the magnitude of
p21
activations by NCatCHA and NCatHA were
greater than in COS-1 cells (not illustrated).
-terminal or
catalytic domains. In order to test this possibility, HA-tagged NCat
and HA-tagged full-length dSos proteins were expressed in the
baculovirus-insect cell system, and assayed for their ability to
associate with a GST-Grb2 fusion protein. As shown in Fig. 3(lanes2 and 3), lysates of Sf9
cells expressing these dSos proteins displayed immunoreactive bands at
the expected mobilities when electrophoresed on SDS-PAGE and blotted
with anti-HA monoclonal antibody. Wild-type Sf9 cell lysates failed to
display any such immunoreactivity under similar conditions (Fig. 3, lane1). Incubation of Sf9 cell
lysates containing full-length dSos protein with GST-Grb2 fusion
protein but not GST alone resulted in the adsorption of full-length
dSos (Fig. 3, lanes6 and 7), whereas
NCatHA did not detectably associate with the GST-Grb2 fusion protein
under these same conditions (Fig. 3, lane9).
These data indicate that the ability of NCatHA to cause
p21
activation when expressed in intact COS-1 or
293 cells is not dependent upon its binding Grb2.
Figure 3:
Association
of recombinant NCatCHA but not NCatHA protein with GST-Grb2 fusion
protein. Lanes1-3 represent equal amounts of
protein from total lysate of Sf9 cells infected with wild-type, NCatHA,
and NCatCHA baculoviral stocks, respectively. The positions of NCatHA
and NCatCHA are indicated. In lanes 4-9, equal amounts
of sample were loaded, and proteins associating with GST or GST-Grb2
were detected by immunoblotting with anti-HA. The sample loaded in lane4 represents GST-Grb2 incubated with
phosphate-buffered saline alone. The sample loaded in lane5 represents GST-Grb2 incubated with lysates from
wild-type infected cells. Proteins remaining bound to the GST or
GST-Grb2 were dissolved in SDS-PAGE sample buffer. Samples were loaded
on SDS-PAGE gels (6%), and transferred to nitrocellulose filters.
Filters were probed with anti-HA monoclonal antibody, and
antibody-bound proteins were visualized using ECL (Amersham) according
to the manufacturer's specifications.
Taken together,
the results in Fig. 2and Fig. 3suggest that structural
elements within the NH-terminal domain of dSos are required
in order for the exchange activity of Cat to act on p21
in intact cells. Truncation and deletion mutants of the
HA-tagged NCat cDNA were therefore engineered as depicted schematically
in Fig. 4A, and transfected into COS-1 and 293 cells
with p21
cDNA. The design of these constructs
focused on two regions in dSos previously identified to contain
sequence similarities to domains found in pleckstrin (26) (amino
acids 475-592 in dSos) and the Rho guanine nucleotide exchange
factor Dbl (27) (amino acids 380-427 in dSos), respectively. As
found using COS-1 cells (Fig. 2), CatHA expression in 293 cells (Fig. 4) failed to significantly enhance
[
P]GTP loading of p21
compared to transfection of p21
alone. Remarkably, PHCatHA, containing the pleckstrin
homology (PH) domain NH
-terminal to Cat, greatly stimulated
cellular [
P]GTP
p21
concentrations to levels approaching those observed in
response to NCatHA (Fig. 4B, compare lanes2 and 4). Immunofluorescence microscopy of the
transfected 293 cells revealed similar levels of expression of all
constructs depicted in Fig. 4, except for somewhat higher
expression of NCatHA (not illustrated). Thus, PHCatHA may actually be
as active as NCatHA in this system, when normalized for protein
expression. In any case, these results document for the first time a
functional role of the PH domain in a p21
exchange protein. Interestingly, an NCatHA construct with
the PH domain deleted also effectively stimulated
[
P]GTP loading of p21
(Fig. 4B, lane3). Ablation
of exchange activity was observed, however, when both the PH and Dbl
homology regions of NCat were deleted (Fig. 4B, lanes5 and 6).
Figure 4:
Modulation of p21 GTP/GDP exchange in
intact cells by expression of truncation and deletion constructs of
NCatHA. A, schematic representation of the various dSos
constructs which were tested for their ability to activate p21 in
intact COS and 293 cells. B, human kidney 293 cells were
transiently transfected via the CaPO-mediated method with
p21 and the HA-tagged dSos cDNAs shown. Cells were labeled, and GDP and
GTP associated with p21
was determined (13, 22).
A simple hypothesis that
explains these data is that either PH or Dbl homology regions of dSos
are sufficient to allow the catalytic domain of dSos to interact with
cellular p21 proteins. Thus, the absence of both
of these regions in NCatHA abolishes activity, while the presence of
either domain positioned NH
-terminal to the Cat domain
confers high activity. The results presented here lead us to postulate
that interactions of the PH and Dbl homology regions with other
elements in the cellular complexes that regulate p21
proteins are necessary for localizing the catalytic domain
appropriately to operate in intact cells. The identities of the
hypothetical elements that bind PH or Dbl regions of dSos are unknown
but could be known proteins in p21
-activating
complexes or as yet unidentified cellular components. It should be
noted that our results were derived from complex intact cell systems
transiently overexpressing p21
and the Sos
constructs. Much larger increases in GTP loading of p21
are achieved under these conditions compared to growth
factor stimulation. Thus, it is not possible to draw conclusions
related to possible regulation of these hypothetical PH- or Dbl-binding
components in growth factor-mediated p21
activation.
subunits of trimeric G proteins (29) and
phosphatidylinositol derivatives (30) bind PH domains have not
yet been confirmed in intact cells. That the PH domain of Bruton
tyrosine kinase binds protein kinase C has been reported
recently(31) , but not yet extended to other PH domains. A
three-dimensional structure for the PH domain of the B-spectrin protein
has been recently reported, indicating some similarity to retinol
binding motifs(32) , but retinol does not bind this structure.
-terminal domain of dSos in p21
regulation, which may function in conjunction with or
subsequent to the association of dSos
Grb2 complexes to tyrosine
phosphates within membrane complexes (Fig. 5). Consistent with
Aronheim et al.(18) , we find evidence for an
inhibitory effect of the dSos COOH terminus on catalytic activity in
that CatCHA is unable to activate p21
(Fig. 2, lane6). Perhaps the function of
the PH or Dbl regions of dSos is to bind membrane components that
position dSos to better interact with membrane-bound
p21
, while Grb2 plays a role in relieving an
inhibitory influence of the Sos COOH terminus (Fig. 5). Strong
support of these concepts are provided by Karlovich et al.(19) using genetic complementation, showing a dominant
inhibitory function of CatC and physiological activity of NCat in
intact flies. Wang et al.(34) have also recently found
enhanced transforming activity of mutant mSos proteins with
COOH-terminal truncations. The striking similarity of results obtained
by these independent methodologies strongly reinforce the concept that
regions within the NH
-terminal region of Sos proteins are
necessary for their physiological function.
Figure 5:
Model for the
role of NH-terminal domains of dSos in facilitating GTP/GDP
exchange on p21 in intact cells. Subsequent to the binding of
Grb2
Sos complexes to tyrosine phosphate sites on receptor or
receptor substrate proteins (left), association of PH or Dbl
regions in the Sos NH
terminus to unknown components (?)
hypothetically position Sos to activate p21. Alternatively,
Sos
Grb2 complexes may be anchored to the membrane through binding
of NH
-terminal sequences prior to receptor activation and
binding of Grb2 to tyrosine phosphates (not
shown).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.