(Received for publication, May 30, 1995; and in revised form, August 3, 1995)
From the
The 14.3.3 protein is a ubiquitous and abundant
arachidonate-selective acyltransferase and putative phospholipase
A
, which self-assembles into dimers and binds to c-Raf-1
and other polypeptides in vitro and in intact cells. The
14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a
fairly reproducible molar ratio that is probably
1. Purified
baculoviral recombinant Raf, despite its preassociated 14.3.3
polypeptide, binds additional recombinant 14.3.3
polypeptide in vitro, in a saturable and specific reaction, forming a
complex that is resistant to 1 M LiCl. A two-hybrid analysis
indicates that 14.3.3
binds primarily to Raf noncatalytic
sequences distinct from those that bind Ras-GTP, and in vitro 14.3.3
binds to Raf without inhibiting the Ras-Raf
association or Raf-catalyzed MEK phosphorylation. Deletion analysis of
14.3.3
(1-245) indicates that the 14.3.3 domain responsible
for binding to Raf extends over the carboxyl-terminal 100 amino acids,
whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As
with Ras, the 14.3.3
polypeptide does not activate purified Raf
directly in vitro. Moreover, expression of recombinant 14.3.3
in COS cells beyond the substantial level of endogenous 14.3.3
protein does not alter endogenous Raf kinase, as judged by the activity
of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3
with recombinant Myc-tagged Raf in COS cells does increase
substantially the Myc-Raf kinase activity achieved during transient
expression, which is attributable primarily to an increased level of
Myc-Raf polypeptide, without alteration of Myc-Raf specific activity or
the activation that occurs in response to epidermal growth factor or
12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence
that 14.3.3 actively participates in Raf activation in situ is
provided by the finding that although full-length 14.3.3
binds
active Raf in situ, truncated versions of 14.3.3, some of
which bind Raf polypeptide in situ nearly as well as
full-length 14.3.3
, are recovered in association only with
inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to
one or more sites on c-Raf. Overexpression of 14.3.3
enhances the
expression of recombinant Raf, perhaps by stabilizing the Raf
polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3
polypeptides are unable to undergo activation in situ,
indicating that 14.3.3 participates in the process of Raf activation by
mechanisms that remain to be elucidated.
An important insight into the initial step in Raf activation was the discovery that Raf binds directly to the GTP-bound form of Ras (1, 2, 3) . This Raf-Ras-GTP interaction does not result directly in Raf activation, inasmuch as addition of Ras GTP to inactive, baculoviral recombinant Raf in vitro does not alter Raf kinase activity. Presumably, Ras GTP functions in situ to translocate Raf to the surface membrane so as to enable its activation by other processes. Support for this model is provided by the demonstration that fusion of plasma membrane targeting (CAAX) sequences onto the Raf carboxyl terminus is transforming and bypasses the need for Ras in Raf activation; a large increase in the activity of membrane-associated Raf is observed in growth factor-deprived cells, and EGF stimulates Raf CAAX activity a further 10-fold in a Ras-independent reaction(4, 5) .
The inability of Ras to directly
activate Raf, together with the finding that mitogen activation of Raf
becomes Ras independent if Raf is targeted directly to the plasma
membrane, implies that physiologic activation of (Ras bound) Raf
requires Raf interaction with other plasma membrane components, e.g. lipids, polypeptides, or both. Ghosh et al.(6) reported that the Raf amino-terminal noncatalytic
sequences bound to liposomes in a phosphatidylserine-dependent reaction
that is independent of Ca and diacylglycerol. In this
report, we describe the binding of c Raf-1 in vitro and in
situ to the 14.3.3
polypeptide, an arachidonate-selective
acyl transferase and putative phospholipase A2(7) . We define
the Raf domain employed for the binding of 14.3.3
in situ and the 14.3.3
domains necessary for self-association and
Raf binding; we find that while carboxyl-terminal fragments of 14.3.3
bind Raf in situ nearly as well as full-length 14.3.3, only
the latter is found in association with catalytically active Raf
polypeptides in situ.
cDNAs encoding murine 14.3.3 were isolated from a
murine T cell DNA library by two-hybrid expression cloning according to
Durfee et al.(8) , using the c-Raf sequences
1-25/305-648 as bait (see ``Results''). cDNAs
encoding rat Erk-1, human MEK-1, and human C-Ha-Ras were expressed in Escherichia coli as GST (
)fusion proteins using the
p-GEX kg vector (9) and purified by glutathione-agarose
affinity chromatography. The free Erk-1 and 14.3.3
polypeptides
were obtained after thrombin cleavage. Recombinant Raf polypeptide
containing a hexahistidine tag at the carboxyl terminus was expressed
in Sf9 cells using a recombinant baculovirus and purified by nickel
chelate affinity chromatography. Active baculoviral Raf kinase was
obtained by co-infection with baculoviruses encoding v-Ras and
v-Src(10) .
The transacylation activity of the recombinant
14.3.3 was measured according to Zupan et
al.(7) . The Raf kinase assay was performed as previously
described(11, 12) .
The binding in vitro of various polypeptides to GST or GST fusion proteins immobilized on glutathione-Sepharose was carried out at 30 °C for 30 min in buffer A containing 25 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol; polypeptide concentrations are described in the figure legends. The beads were washed in excess binding buffer three times; the retained polypeptides were eluted directly into SDS-containing buffer, separated by SDS-PAGE, and analyzed by protein staining, immunoblot, or autoradiography as described.
The association of polypeptides in
situ was assessed during transient expression in COS M7 cells and
transfected by the DEAE-dextran method. The cDNA sequences encoding Raf
were inserted into two mammalian expression vectors; Myc-Raf contains a
33-amino acid epitope from human c-Myc, known to be reactive with the
monoclonal antibody 9B7.3(13) , appended to the Raf amino
terminus, and inserted into pMT2. Raf was also expressed as a GST
fusion protein using the vector pEBG, which encodes glutathione S-transferase driven by the EF1 promoter/enhancer. The
cDNA encoding 14.3.3
was introduced unmodified into the vector
CMV5, into the pEB vector (lacking the glutathione S-transferase sequences) with a 9-amino acid epitope from the
influenza hemagglutinin (HA epitope, (14) ) added to its
carboxyl terminus, and into pEBG for expression in situ as a
GST fusion. Deletion mutation of the 14.3.3
was made by
polymerase chain reaction from the 5`- and 3`-ends of the cDNA. The
polymerase chain reaction products were subcloned into the pEBG vector,
and the structures were verified by DNA sequence analysis.
All
transfections utilized a total of 20 µg of DNA; 48 h after
transfection, extracts were prepared by homogenization in a buffer
containing 25 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 25 mM -glycerophosphate, 1 mM sodium vanadate, 1% Triton
X-100, and proteinase inhibitors. Immunoprecipitations were carried out
for 1 h at 4 °C using the monoclonal antibody 12CA5 for the HA
epitope or the anti-Myc monoclonal antibody 9B7; immune complexes were
harvested with protein G-Sepharose. GST-Raf and GST-14.3.3 fusions were
recovered using glutathione-Sepharose beads. Immunoblots of Raf were
carried out using 9B7.3 for Myc-Raf or a polyclonal antipeptide
antibody raised to the carboxyl-terminal 12 amino acids of human
c-Raf-1. Immunoblot of 14.3.3 was carried out using a polyclonal
antibody raised to cleaved, purified recombinant murine 14.3.3
.
L--1-palmitoyl-2-arachidonyl
(arachidonyl-1-
C) phosphatidylethanolamine, and
[
-
P]ATP were obtained from DuPont NEN.
Glutathione-Sepharose 4B was from Pharmacia Biotech Inc.
Seeking proteins other than Ras and related small GTPases
that interact with Raf, we utilized the Raf sequences corresponding to
1-25/305-648 in a two-hybrid screening(8) . This
truncated Raf polypeptide, also known as BXB-Raf(15) , is a
transforming protein that encodes a dispensable region of the Raf amino
terminus (residues 1-25) fused to the Raf carboxyl-terminal 344
amino acids, which includes the entire catalytic domain (residues
335-627) flanked by short segments of amino-terminal (residues
305-334) and carboxyl-terminal (residues 628-648)
noncatalytic sequences. 20 His and LacZ
cDNAs were recovered in a screen of 2.5
10
transformants. Five of these cDNAs gave much more rapid
complementation of LacZ
activity than the remaining
cDNAs; the former all encoded MEK1 or MEK2. Among the remaining 15
cDNAs were 5 that encoded polypeptide sequences 100% identical to the
rat
isoform of the 14.3.3 polypeptide, and that differed from a
human platelet PLA
polypeptide by a single conservative
substitution(7) . Inasmuch as 14.3.3 proteins have been
reported to copurify or associate with a relatively large number of
proteins(16) , and 14.3.3
has been identified as a
protein cofactor for a number of enzymes in vitro, including
the ADP-ribosylation of Ras and other small GTPases by exoenzyme
S(17) , we examined several proteins other than Raf for their
ability to associate with 14.3.3 in the two-hybrid system. No
interaction of 14.3.3
with p70 S6 kinase and amino-terminal
regulatory domain of protein kinase C
(residues 1-245) or
c-Ha-Ras (1-185) was detected (data not shown). The relative
selectivity of the 14.3.3-Raf association led us to undertake a further
characterization of this interaction.
The two-hybrid method was
employed to identify the region on Raf that interacts with 14.3.3
in comparison to MEK1 and Ras, proteins known to interact with Raf in a
physiologic context. The Ras binding site has previously been localized
to Raf residues 50-150(3, 18) , whereas neither
MEK nor 14.3.3
interacts with Raf 1-257. MEK, a known Raf
substrate, interacts strongly with the BXB-Raf and holo-Raf
(1-648) but not at all with Raf 1-332 (Table 1). By
contrast, 14.3.3
interacts weakly with BXB-Raf and holo-Raf
(1-648) but associates strongly with the Raf 1-332 (Table 1). Thus, 14.3.3
binds in situ most avidly
to a segment of Raf between amino acids 257 and 332, a noncatalytic
region distinct from those that bind to Ras or MEK.
Figure 1:
Recombinant
14.3.3 catalyzes autoacylation from
[
C]arachidonyl sn-2-phosphatidylethanolamine. GST, GST-14.3.3
, and
cleaved 14.3.3
(5 µg each) were incubated with L-
-1-palmitoyl-2 ([
C]arachidonyl
phosphatidylethanolamine, 52.8 µM, 125,400 cpm/pmol) at 37
°C for 3 min, mixed rapidly with SDS sample buffer, and heated to
90 °C for 3 min. The samples were subjected to SDS-PAGE and
subsequent fluorography. Lanes1-3, Coomassie
Blue stain; lanes4-6, autoradiogram of
[
C]arachidonate. Lanes1 and 4, glutathione S-transferase; lanes2 and 5, GST-14.3.3
; lanes3 and 6, thrombin cleaved and purified 14.3.3
.
Raf 1
containing a hexahistidine tag at its carboxyl terminus was expressed
in Sf9 cells by baculoviral infection and purified by nickel chelate
chromatography, either as a catalytically inactive polypeptide (Fig. 2, lane6) or in a catalytically active
form, as a result of coinfection with baculoviral v-Ras plus v-Src (Fig. 2, lanes1 and 3-5)(10) . An antiserum against prokaryotic
recombinant murine 14.3.3 , reactive primarily with epitopes in
the amino-terminal half of 14.3.3
(Fig. 5C, upperpanel), readily immunoblots the 28- and 30-kDa
14.3.3 polypeptides endogenous to Sf9 (Fig. 2, lane2) and COS cell (Fig. 7A, upperpanel) extracts; immunoblot of several Raf-1 isolates (Fig. 2, lanes2-6) shows that both the
active (lanes3-5) and inactive (lane6) Raf polypeptides purified from Sf9 cells are recovered
in association with endogenous Sf9 14.3.3 polypeptides. Both the
recombinant Raf and Sf9 14.3.3 polypeptides are readily visualized on
Coomassie Blue-stained gels of the preparations of purified Raf (Fig. 2, lane1); although the relative
Coomassie Blue binding per unit mass for c-Raf-1 and 14.3.3 is unknown,
the comparable staining intensities observed for Raf and the
co-purified 14.3.3, together with the general tendency of acidic
polypeptides such as 14.3.3 to stain weakly with Coomassie Blue,
suggests that the molar ratio of 14.3.3 to Raf in these isolates is at
least 1. Moreover, a relatively constant ratio of the two polypeptides
is recovered in different isolates of Raf, whether active (Fig. 2, lanes3-5) or inactive (Fig. 2, lane6).
Figure 2:
Copurification of 14.3.3 and c-Raf-1 from
insect Sf9 cells. Sf9 cells were infected with baculovirus encoding
human (His) 6-tagged c-Raf-1 alone or plus baculoviral encoded v-Ras
and/or v-Src. The recombinant Raf was purified by nickel chelate
affinity purification and subjected to SDS-PAGE prior to
immunoblotting. Lane1, Coomassie Blue stain of
SDS-PAGE gel corresponding to the isolate shown in lane5. Lanes2-6, the upper part was
immunoblotted with an antiserum against carboxyl-terminal 12 amino
acids of human c-Raf-1, and the lower part was immunoblotted with an
antiserum against mouse 14.3.3 . Lane2, Sf9
cell extract without infection; lane3, purified Raf
coinfected with v-Src; lane4, Raf coinfected with
v-Ras; lane5, Raf coinfected with v-Ras plus v-Src; lane6, inactive baculoviral Raf; mwm,
molecular weight markers.
Figure 5:
Deletion analysis of functional domains of
14.3.3 . Truncations of 14.3.3
were constructed by the
polymerase chain reaction. The polymerase chain reaction products were
subcloned to pEBG vector and expressed as GST fusions in COS cells. A, schematic diagram of GST-14.3.3
variants. B,
dimerization domain of 14.3.3
. cDNAs encoding the GST-14.3.3
variants shown in A were cotransfected with a vector encoding
a full-length 14.3.3
polypeptide tagged at its amino terminus
with a Myc epitope. Extracts were prepared 48 h later; the GST fusion
proteins were purified by GSH affinity chromatography and resolved on
SDS-PAGE gel. A Coomassie Blue-stained gel is shown in the upperpanel, and an immunoblot using anti-Myc antibody 9E10.2
is shown in the lowerpanel. Lane1, GST; lane2, GST-14.3.3
(1-245); lane3, GST-14.3.3 (1-80); lane4, GST-14.3.3 (1-140); lane5, GST-14.3.3 (1-180); lane6,
GST-14.3.3 (79-245); lane7, GST-14.3.3
(139-245); lane8, GST-14.3.3 (179-245). mwm, molecular weight markers (from the top) 200,
116, 97.4, 66.2, 45, 31, and 21.5 kDa. C, Raf binding domain
of 14.3.3
. GST (lane1) or GST-14.3.3 fusions (lanes2-8 as in B) were coexpressed
with Myc-Raf in COS cells and purified by GSH affinity chromatography.
The toppanel is immunoblot of the GSH-Sepharose
isolates using a polyclonal antiserum raised against recombinant 14.3.3
; note that the major epitopes are in the amino-terminal half of
the 14.3.3
polypeptide. The middlepanel is an
immunoblot of the GSH affinity isolates using the anti-Myc antibody 9E
10.2. The bottompanel displays the Raf kinase
activity of the Myc-Raf polypeptides associated with the
GSH-Sepharose-bound, GST-14.3.3 polypeptides. After purification of
GST-14.3.3-Raf complex, the Raf kinase activity was measured by the
addition of magnesium, [
-
P]ATP (0.1
mM, 2000 cpm/pmol), GST-MEK-1 (20 µg/ml), and Erk-1
polypeptide. The basal activity of the substrate GST-MEK 1, incubated
with Erk-1, magnesium, [
-
P]ATP, and
GSH-Sepharose beads, is shown in lane0.
Figure 7:
Effect of 14.3.3 on the expression
and activity of Myc-Raf in situ. Vectors encoding GST (lanes1, 5, 9), GST-14.3.3
(1-245) (lanes2, 6, 10),
GST-14.3.3 (139-245) (lanes3, 7, 11), or Raf (1-257) (lanes4, 8, 12) were cotransfected with Myc-Raf (lanes1-12) into COS cells; 48 h later, the cells were
placed in medium containing 0.5% serum, and after a further 18-h
incubation period, they were treated with carrier (lanes1-4), EGF (10 ng/ml, lanes5-8), or TPA (500 nm) (lanes9-12) for 15 min, rinsed, and extracted. Aliquots
of the cell extracts, normalized for protein content, were subjected to
anti-Myc immunoprecipitation (A, 3rdpanel from top, and bottompanel) and
GSH-Sepharose affinity chromatography (B, allthreepanels). A, the toppanel shows an anti-14.3.3 immunoblot of the cell
extracts. The panel secondfromthetop shows an immunoblot of the cell extracts using the anti-Myc
antibody 9 E10; the Myc-Raf band is indicated. The panelthirdfromthetop, and the bottompanels are analyses of the anti-Myc
immunoprecipitates prepared from the extracts shown in the toptwopanels; the panelthirdfromthetop is a Myc immunoblot of the
Myc immunoprecipitate; the Myc-Raf polypeptide is indicated. The bottompanel displays an assay of the Raf kinase
activity found in the Myc-Raf immunoprecipitates, estimated by the
phosphorylation of recombinant GST-MEK 1, coupled to the
phosphorylation of recombinant Erk-1 as described in Fig. 5C, bottompanel. B,
the proteins retained by GSH-Sepharose affinity chromatography of
extracts prepared from serum-deprived (lanes1-3), EGF-treated (lanes4, 5), and TPA-treated (lanes6, 7)
COS cells, transfected with a vector encoding Myc-Raf (lanes1-7) together with GST (lane1),
GST-14.3.3 (1-245) (lanes2, 4, 6), or GST-14.3.3 (139-245) (lanes3, 5, 7)), are analyzed for 14.3.3 content (by
immunoblot with anti-14.3.3, upperpanel), Myc-Raf
content (by anti-Myc immunoblot, middlepanel), and
Myc-Raf kinase activity (by coupled phosphorylation assay with
[
-
P]ATP, GST MEK, and Erk-1 substrates, lowerpanel). Lane8 in the lowerpanel contains GST-MEK,
[
-
P]ATP substrates with empty GSH-Sepharose
beads.
Despite the presence of
considerable Sf9-derived 14.3.3 already bound to the baculoviral
recombinant Raf-1 polypeptide, such Raf preparations bind in vitro to a GST-14.3.3 fusion protein but not to GST alone (Fig. 3, upperpanel, lanes1 and 2); preincubation of Raf with cleaved, purified E. coli recombinant 14.3.3
polypeptide prevents the
subsequent binding of Raf by immobilized GST-14.3.3
, whereas
preincubation of Raf with bovine serum albumin has no effect (Fig. 3, upperpanel, lanes1, 3, 4). The ability of 14.3.3 to bind
active baculoviral Raf was verified by the demonstration that
GST-14.3.3 can specifically immobilize essentially all of the MEK
phosphorylating activity in a preparation of active Raf (Fig. 3, lowerpanel, lanes1-4), and
the complex of active Raf and GST-14.3.3 is resistant to washing with 1 M LiCl. Extensive Raf autophosphorylation in vitro did not interfere with Raf binding to GST-14.3.3
(Fig. 3, upperpanel, lanes6 and 7); inactive Raf polypeptide (identifiable by its
slightly faster mobility in SDS-PAGE) also binds specifically to
GST-14.3.3
(Fig. 3, upperpanel, lane5).
Figure 3:
Binding of Raf to GST-14.3.3 in
vitro. Upperpanel, baculoviral Raf activated by
coinfection with v-Ras and v-Src (
0.2 µM, lanes1-4, 6, and 7) or unactivated
(
0.3 µM, lane5) and purified by
nickel chelate chromatography was incubated with immobilized GST-14.3.3
(20 µg/ml settled beads, lanes1 and 3-6) or GST (20 µg/ml beads, lanes2 and 7) directly (lanes1, 2,
and 5-7) or after preincubation with a 100-fold molar
excess of purified 14.3.3
(lane3) or bovine
serum albumin (lane4). In lanes6 and 7, the activated Raf was subjected to
autophosphorylation in vitro in the presence of magnesium (10
mM), [
-
P]ATP (100 µM,
30 °C for 30 min) prior to addition of GST-14.3.3 (lane6) or GST (lane7). In lanes1-5, Raf was detected by anti-Raf immunoblot; in lanes6 and 7,
P-Raf was
detected by autoradiography. Lower panel, baculoviral Raf,
activated in situ by coinfection with v-Ras and v-Src, was
purified and mixed with GST-14.3.3 (lanes1 and 3) or GST (lanes2 and 4)
immobilized on GSH-Sepharose. Aliquots of the supernatants (lanes1 and 2) and washed GSH-Sepharose beads (lanes3 and 4) were assayed for Raf kinase
activity by phosphorylation of GST-MEK (20 µg/ml)
[
-
P]ATP (0.1 mM, 2000
cpm/pmol).
These results demonstrate that
baculoviral recombinant Raf, although purified as a complex with Sf9
14.3.3, binds additional 14.3.3 in vitro, and this binding is
saturable. The ability of GST-14.3.3 to specifically adsorb essentially
all the Raf polypeptides, both active (Fig. 3) and inactive,
from such preparations indicates that GST-14.3.3 must bind Raf
polypeptides that already contain bound Sf9 14.3.3. Although
displacement of or dimerization with the preassociated Sf9 14.3.3 is
possible, the most plausible model envisions more than one binding site
on Raf for 14.3.3. Thus, the ability of 14.3.3 to interact with
Raf in the two-hybrid yeast expression system certainly reflects the
direct binding of the two polypeptides; however, the number of 14.3.3
binding sites on Raf and their precise localization remain to be more
fully defined.
Figure 4:
Association of recombinant Raf and 14.3.3
in intact COS cells. Two cDNA encoding tagged Raf polypeptides
were constructed, one in the vector pEB, encoding full-length Raf fused
at its amino terminus to the carboxyl terminus of glutathione S-transferase, and a second in the vector pMT2 encoding a Myc
epitope fused to the Raf amino terminus. In lanes1-4, pEB encoding GST-Raf (lanes1, 3, 4) or GST (lane2) was cotransfected into COS cells with pMT2 encoding an
HA-tagged 14.3.3
(lanes1-3) or the pMT2
HA vector (lane4). Similarly in lanes5-8, pMT2 encoding Myc-Raf (lanes5, 7, 8) or pMT2 Myc vector (lane6) was cotransfected with vector encoding HA-tagged
14.3.3
(lanes5-7) or empty pMT2 HA
vector (lane8). After 48 h, cells were extracted,
and recombinant polypeptides were purified using GSH-Sepharose (lanes1 and 2), anti-Myc monoclonal
antibody 9B7.3 (lanes5 and 6) for direct
isolation of recombinant Raf, or anti-HA epitope monoclonal antibody
12CA5 for isolation of recombinant 14.3.3 (lanes3, 4, 7, and 8). Raf polypeptide in each
isolate was detected by immunoblot with an anti-COOH-terminal Raf
peptide antibody. Note that recovery of recombinant Raf using
GSH-Sepharose (lane1) or anti-Myc
immunoprecipitation (lane5) is comparable to that
achieved by immunoprecipitation of HA 14.3.3
(lanes3, 7).
Two
tagged versions of recombinant Raf-1 were constructed by introduction
at the Raf amino terminus of a Myc epitope (13) or by fusion to
GST. After cotransfection of HA-tagged 14.3.3 with either version
of Raf into COS cells, anti-HA 14.3.3
immunoprecipitates (Fig. 4, lanes3, 4, 7, 8) contain substantial immunoreactive Raf-1, ranging from 30
to 100% of the amount of Raf polypeptide recovered from an identical
aliquot of the COS cell extract by anti-Myc immunoprecipitation (Fig. 4, lanes5 and 6) or by binding
to GSH-Sepharose ( Fig. 4lanes1 and 2). The anti-Myc immunoprecipitates of Raf-1 contained in
addition to the recombinant 30-kDa HA-tagged 14.3.3
polypeptide
some endogenous 28-kDa 14.3.3 polypeptide, as visualized by immunoblot
with anti-14.3.3 antiserum (not shown). Thus, a substantial portion of
recombinant Raf is recovered in association with the coexpressed 14.3.3
, indicating clearly that the 14.3.3
associates in situ with most or all of the recombinant Raf-1 in COS cells.
The
domains of 14.3.3 responsible for dimerization and Raf
association were determined by coexpression in COS cells of Myc-Raf or
Myc-14.3.3 with GST-14.3.3 (1-245) and a series of 14.3.3
fragments constructed as GST fusions (Fig. 5A). The
transiently expressed GST-14.3.3 fusions were purified from COS cell
extracts using GSH-Sepharose, and the isolates were evaluated for the
presence of the cotransfected full-length Myc-tagged 14.3.3 polypeptide (Fig. 5B) or Myc-Raf (Fig. 5C). The
Coomassie Blue stain of the purified, COS recombinant GST-14.3.3
fusions and their associated polypeptides is shown in Fig. 5B, upperpanel. The variation
in Coomassie Blue staining of the GST-14.3.3 fusions reflects the
variability in their expression in situ rather than in their
recovery, inasmuch as recovery of the irrelevant endogenous COS
polypeptides that bind to GSH-Sepharose (designated as A and B, Fig. 5B, upperpanel) is
identical in all lanes. The binding of cotransfected Myc-tagged 14.3.3
polypeptides to the GST-14.3.3 fusions is evident as the 30-kDa
Coomassie band seen just above bandA in lane2 (absent in lane1) and more faintly
in lanes3-5. The identity of this band as
Myc-14.3.3 is verified by anti-Myc immunoblot (Fig. 5B, lowerpanel). These results indicate that although a
free NH
terminus is not critical for 14.3.3
self-association, the amino-terminal 80 amino acids of 14.3.3 are
sufficient to confer some 14.3.3 self-association, and nearly full
self-association is seen with GST-14.3.3 (1-140). Reciprocally,
deletion of the amino-terminal amino acids from 14.3.3 markedly reduces
its ability to self-associate; little (Fig. 5B, lowerpanel, lane7) or no (Fig. 7B) self-association is detectable with deletion
of the amino-terminal 138 amino acids, and the carboxyl-terminal 14.3.3
fragment 179-245, although very well expressed, is unable to
associate at all with full-length Myc-14.3.3 (Fig. 5B, lowerpanel, lane8).
As to the
effects of truncation on the ability of GST-14.3.3 to bind
cotransfected Myc-Raf, the 14.3.3 amino-terminal 139 residues are
largely dispensable; optimal Myc-Raf recovery is observed with
GST-14.3.3 (139-245) (Fig. 5C, middlepanel, lanes2, 7), and
considerable Myc-Raf is recovered with GST-14.3.3 (179-245) (Fig. 5C, middlepanel, lane8), whereas no binding of Myc-Raf occurs to GST-14.3.3
(1-140) (Fig. 5C, middlepanel, lane4). Each of these GST-14.3.3 isolates was also
assayed for the presence of active Raf kinase, estimated by its ability
to phosphorylate and activate GST-MEK (Fig. 5C, bottompanel). Notably, only the GST-14.3.3
(1-245), i.e. full-length 14.3.3, is associated with
catalytically active Raf polypeptide. Thus, although GST-14.3.3
(139-245) retains as much Myc-Raf polypeptide as GST-14.3.3
(1-245), the Raf associated in situ with the truncated
14.3.3 is devoid of catalytic activity. The failure of active Raf to
bind to truncated 14.3.3 polypeptides in situ probably cannot
be attributed to a lack of 14.3.3 dimerization, inasmuch as the Myc-Raf
bound to GST-14.3.3 (1-180) (Fig. 5C, middlepanel, lane5), which dimerizes quite
well with Myc-14.3.3 (Fig. 5B, bottompanel, lane5), also lacks detectable
kinase activity (Fig. 5C, bottompanel, lane5). Taken together, these
results establish that although the carboxyl-terminal 65 amino acids of
14.3.3 are sufficient to mediate association with Raf, only the
full-length 14.3.3 sequences are found in association with
catalytically active Raf polypeptides in situ.
We
next examined the ability of 14.3.3 to modulate the activation of Raf
kinase in vitro and in situ. An initial step in Raf
activation in situ involves its binding to GTP-Ras. The
effects of 14.3.3 polypeptide on the Ras-Raf interaction was
assessed in vitro. The full-length Raf protein binds
specifically to immobilized GST-Ras-GTP. Addition of 14.3.3
at
>50-fold molar excess to Raf does not inhibit the binding of Raf to
GST-Ras-GTP (Fig. 6, upperpanel, lanes1 and 2). In addition, after washing the
immobilized GST-Ras-Raf complex, 14.3.3
is seen to have been
retained by the GST-Ras, but only if Raf is present (Fig. 6, lowerpanel, compare lanes1 and 2). These data demonstrate that 14.3.3
does not bind to
Ras directly nor displace Raf from Ras, but rather it is capable of
binding Raf in vitro so as to allow the formation of a ternary
complex with Ras and Raf.
Figure 6:
Raf binds 14.3.3 and Ras
simultaneously. Upperpanel, GST-Ras, charged with
GTP (71 nM, lanes1, 2) or GST (lanes3, 4), were incubated with
baculoviral recombinant Raf alone (45 nM, lanes1, 3) or Raf (45 nM) plus 14.3.3
(3.6 µM) (lanes2, 4). After
washing, the polypeptides bound to GSH-Sepharose were resolved by
SDS-PAGE, and the retained Raf polypeptides were detected by immunoblot
with an anti-Raf COOH-terminal peptide antibody. Lowerpanel, the recombinant 14.3.3
polypeptide was
incubated with GST-Ras-GTP (lanes1, 2) or
GST (lane3) in the presence of Raf (lane1) or without Raf (lanes2, 3). After washing the bead, bound polypeptides were resolved
by SDS-PAGE, and the retained 14.3.3 polypeptides were detected by
immunoblot with an antibody to GST-14.3.3. Lane4 contains an aliquot of 14.3.3
as a positive control.
Residual reactivity of this antiserum toward GST polypeptide sequences
is evident in the blot.
Addition of 14.3.3 to inactive Raf
purified from Sf9 cells does not activate Raf-catalyzed MEK
phosphorylation (not shown). This negative result was obtained despite
preincubation of Raf and 14.3.3
polypeptide in the presence of
various combinations of GTP-Ras (both bacterial and baculoviral, fully
processed Ras), phospholipid micelles prepared from bovine brain
lipids, Mg
(10 mM), Ca
(0.1 mM), and ATP (100 µM), for 30 min
prior to and after the addition of recombinant GST-MEK1. Inasmuch as a
reliable in vitro assay for the activation of Raf kinase is
not yet available and 14.3.3 and Raf associate in situ during
transient expression, the influence of 14.3.3
on Raf activation
was examined in situ by cotransfection. To examine the effects
of 14.3.3 overexpression on the regulation of endogenous Raf (and other
mitogen-responsive MEK activators), 14.3.3 was cotransfected in COS
cells with an HA-tagged Erk-1 reporter. Overexpression of 14.3.3
severalfold above the already substantial level of endogenous 14.3.3
polypeptides did not alter recombinant Erk-1 activity in serum-deprived
cells or in response to TPA or EGF (data not shown). We next examined
the effects of 14.3.3
on the activity of recombinant Myc-Raf. COS
cells transfected with Myc-Raf and various truncated forms of 14.3.3
were either serum deprived or stimulated with mitogens prior to
harvest, and the Myc-tagged Raf-1 recombinant was immunoprecipitated
and assayed for MEK kinase activity. Coexpression of Myc-Raf with
GST-14.3.3 (1-245) increased the MEK kinase activity recovered in
a Myc immunoprecipitate by about 2-3-fold, both in serum-deprived
cells (Fig. 7A, bottompanel, compare lane1 to lane 2) and in response to EGF (Fig. 7A, bottompanel, compare lane5 to lane 6); EGF itself gave
2-3-fold activation of Myc-Raf (Fig. 7A, bottompanel, compare lane1 to lane 5). The ability of GST-14.3.3 to increase Myc-Raf
activity is not due to Raf activation, as occurs with EGF or TPA, but
appears to be attributable to an increased Myc-Raf polypeptide
abundance, as seen by immunoblot of the whole cell extract (Fig. 7A, 2ndpanel from top, compare lanes1 to 2, 5 to 6, 9 to 10) and in the Myc
immunoprecipitates (Fig. 7A, 3rdpanel from top). Such an increase in Myc-Raf expression and
recovery of activity was observed repeatedly on cotransfection with
GST-14.3.3 (1-245) and was present but less pronounced with the
truncated 14.3.3 GST fusion proteins that were capable of binding
Myc-Raf in proportion to their somewhat lesser expression than
GST-14.3.3 (1-245) (Fig. 7A, toppanel and 2ndpanel from top,
compare lanes2 to 3, 6 to 7, 10 to 11).
Based on the finding that
GST-14.3.3 (139-245) bound Myc-Raf strongly but did not associate in situ with active Raf (Fig. 5C, lowerpanel), we examined whether GST-14.3.3 (139-245)
could interfere with Myc-Raf activation. Fig. 7B demonstrates that although nearly equal amounts of Myc-Raf are
recovered with GST-14.3.3 (1-245) and GST-14.3.3 (139-245) (Fig. 7B, middlepanel), the latter
is completely devoid of kinase activity (Fig. 7B, lowerpanel, lanes3, 5,
and 7). Nevertheless, whereas coexpression with GST-14.3.3
(1-245) increased Myc-Raf abundance (Fig. 7A, 2nd and 3rdpanels from top) and
activity (Fig. 7A, bottompanel) in
parallel, the expression of GST-14.3.3 (139-245) had a lesser
effect on Myc-Raf expression (Fig. 7A, 2nd and 3rdpanels from top), and neither inhibited
nor activated overall Myc-Raf activity (Fig. 7A, bottompanel). It is likely that the high levels of
14.3.3 endogenous to COS (and other cells) prevent the recombinant
GST-14.3.3 (139-245) fragment from interfering with the
activation of most Raf polypeptides, whereas those that become
associated with GST-14.3.3 (139-245) are clearly excluded from
the activation process. The inactive Raf polypeptides bound to
GST-14.3.3 (139-245) were not activated by the addition of
recombinant 14.3.3 (1-245) in vitro (not shown).
The 14.3.3 class of 29-33-kDa polypeptides has been
found to copurify with a broad array of proteins and has been
repeatedly rediscovered as activators (e.g. of tyrosine
hydroxylase), inhibitors (e.g. of protein kinase C), or
cofactors (exoS) for a number of enzymes in
vitro(16) . Several recent reports (19, 20) have described an interaction of Raf with
14.3.3 polypeptides in vitro and in intact yeast similar to
that described here. All studies of the Raf 14.3.3 interaction
including the present report have employed one or both as recombinant
polypeptides, and it is possible that the interaction observed in
vitro, or even in situ examining the overexpressed
recombinant polypeptides, may not accurately reflect certain aspects of
the interaction between the endogenous 14.3.3 and Raf polypeptides in situ. An apparent activation of recombinant human c-Raf-1
kinase in intact yeast occurs concomitant with overexpression of
recombinant mammalian 14.3.3. Furthermore, disruption of the gene
encoding the Saccharomyces cerevisae 14.3.3 homolog, Bmh1,
abrogates the ability of recombinant c-Raf-1 to rescue a
Ste11-deficient strain containing a Raf-responsive mutant Ste7. Fantl et al.(21) found that microinjection or
overexpression of 14.3.3 in Xenopus oocytes led to an
increase in the activity of endogenous or recombinant Raf-1. Li et
al.(22) reported that transient expression of 14.3.3 in
NIH 3T3 cells had little effect on the activity of cotransfected
reporters known to be responsive to active Raf-1; however, the ability
of transiently expressed Raf-1 or BXB Raf-1 to activate these reporters
was substantially augmented by cotransfection with 14.3.3. We find that
although overexpression of 14.3.3 in cultured mammalian cells (COS or
293) has no effect on endogenous c-Raf-1 abundance or activity,
measured directly or by the activity of a cotransfected Erk-1 reporter
(not shown), coexpression of 14.3.3 with recombinant Myc-Raf results in
greater Raf kinase activity (Fig. 7), much as found by Li et
al.(22) Significantly however, we find that this increase
in Raf kinase activity is largely or entirely attributable to a
14.3.3-induced increase in the expression and abundance of recombinant
Raf rather than an increase in Raf-1 specific activity. It seems
probable that the ability of recombinant mammalian or endogenous yeast
14.3.3 to enhance the activity of mammalian Raf-1 when the latter is
expressed in the heterologous milieu of S. cerevisae or Xenopus oocytes may be attributable, in part or in whole, to
an ability of 14.3.3 to enhance Raf-1 polypeptide abundance, perhaps e.g. by stabilizing the recombinant Raf-1 polypeptide.
Several observations nevertheless suggest that 14.3.3 may
participate more directly in Raf-1 activation. Irie et al.(20) observed that addition of a maltose binding
protein-14.3.3 fusion protein directly to mammalian c-Raf-1,
immunoprecipitated from yeast extracts, gave a 3-4-fold increase
in MEK kinase activity. Takai and colleagues (23) have
purified a MEK activator (REKS) from Xenopus oocyte cytosol
that is activated in vitro approximately 2-fold by direct
addition of GTP Ras and about 1.3-fold by 14.3.3 protein; addition of
14.3.3 in the presence of maximal GTP Ras gives a further 2-fold
activation in REKS activity. Li et al.(22) observed
that although recombinant 14.3.3 and Ras added directly to inactive
baculoviral Raf do not alter Raf kinase activity, the further addition
of a crude cellular extract results in a Ras-14.3.3-dependent,
2-3-fold activation of Raf-1 kinase. The biochemical mechanisms
that underlie in vitro ``activation'' of Raf by
14.3.3 are not known, and the extent to which they reflect the ability
of 14.3.3 to ``stabilize'' the Raf polypeptide in
vitro, comparable to the effects that underlie the enhanced
expression seen on cotransfection, is also not known. The present
results, however, provide one persuasive piece of evidence that 14.3.3
participates in Raf activation beyond its ability to bind to and
``stabilize'' the Raf polypeptide. Carboxyl-terminal
fragments of 14.3.3, which bind Raf nearly as well in situ as
full-length 14.3.3 and which also provide some enhanced Raf expression in situ, are nevertheless recovered from cells in association
only with catalytically inactive Raf polypeptides, whereas full-length
14.3.3 is recovered with catalytically active Raf kinase. This result
suggests that the Raf-binding 14.3.3 fragments have lost a function
critical to the activation of Raf. This function does not appear to be
their ability to dimerize, inasmuch as the Raf polypeptides associated
with GST-14.3.3 (1-180), which dimerizes normally (Fig. 5B, lane5), are not
catalytically active (Fig. 5C, lane5). We suggest that full-length 14.3.3 contributes to Raf
activation either by recruiting an as yet unidentified polypeptide, by
providing an intrinsic catalytic function, or both. The present data
show that in addition to its ability to bind Raf concomitantly with
Ras, the recombinant 14.3.3 polypeptide binds phospholipid and
cleaves the sn-2 acyl bond. Whether the acyl transferase
function of 14.3.3
is contributory to its role in the regulation
of Raf function is not known. Thus, 14.3.3 polypeptides participate in
the regulation of Raf activity; however, their specific biochemical
function in Raf activation remains to be elucidated.
Subsequent to
the completion of these studies, the structures of 14.3.3 (24) and
(25) crystals were reported. Both 14.3.3
isoforms exhibited dimeric structures; each monomer was composed of
nine helical segments arranged in antiparallel arrays. The dimer
interface is created by highly conserved, primarily hydrophobic
residues from the four amino-terminal helices, suggesting that 14.3.3
heterodimers will form readily. The carboxyl-terminal five helices of
each monomer (which provide the Raf binding domain as demonstrated in
the present report) are folded so as to provide within the dimer a
cavity whose internal face is composed of (primarily hydrophilic)
residues that are highly conserved in all 14.3.3 isoforms and whose
external surface is provided by nonconserved residues. Future studies
will determine the contributions of specific residues on each of these
surfaces to the interactions of 14.3.3 with its polypeptide partners
and phospholipids.