From the Departments of Biochemistry and
§ Zoology and Genetics, Iowa State University, Ames, Iowa
50011 and the ¶ Department of Pharmacology, University of North
Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Ha-Ras undergoes post-translational modifications
(including attachment of farnesyl and palmitate) that culminate in
localization of the protein to the plasma membrane. Because palmitate
is not attached without prior farnesyl addition, the distinct
contributions of the two lipid modifications to membrane attachment or
biological activity have been difficult to examine. To test if
palmitate is able to support these crucial functions on its own, novel
C-terminal mutants of Ha-Ras were constructed, retaining the natural
sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the
Ext61L and ExtWT proteins were modified in a dynamic fashion by
palmitate, without being farnesylated; bound to membranes modestly
(40% as well as native Ha-Ras); and retained appropriate GTP binding
properties. Ext61L caused potent transformation of NIH 3T3 cells and,
unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with
the six lysines but lacking palmitates was inactive. Thus, farnesyl is
not needed as a signal for palmitate attachment or removal, and a
combination of transient palmitate modification and basic residues can
support Ha-Ras membrane binding and two quite different biological
functions. The roles of palmitate can therefore be independent of and
distinct from those of farnesyl. Reciprocally, if membrane association
can be sustained largely through palmitates, farnesyl is freed to
interact with other proteins.
In its GTP-bound conformation, Ha-Ras activates several signal
transduction cascades that control gene expression and actin cytoskeleton organization. The most well studied of these pathways, the
Raf/mitogen-activated protein kinase cascade, involves a series of
cytoplasmic serine/threonine kinases; another utilizes several members
of the Rho family of GTP-binding proteins that regulate cell
morphology. In NIH 3T3 mouse fibroblast cells, activated forms of
Ha-Ras induce mitogenesis and transformation; in the rat
pheochromocytoma cell line PC12, Ha-Ras activation triggers neuronal
differentiation. More important, membrane localization of Ha-Ras is
critical for either of these distinct biological activities. One
important consequence of membrane binding is to enable Ha-Ras to act as
a GTP-dependent, membrane-localized docking site for
effector proteins, such as Raf kinase.
The correct targeting of Ha-Ras to the inner surface of the plasma
membrane requires a series of post-translational modifications at the
protein's C terminus. These reactions include attachment of the
isoprenoid farnesyl to a cysteine residue (Cys-186) located four
residues from the C terminus, followed by removal of the C-terminal
tripeptide and methylation of the newly exposed carboxyl group of the
farnesylated cysteine (1). The final step is palmitoylation of two
nearby C-terminal cysteines (Cys-181 and Cys-184) (2, 3).
The duties of these lipids has been inferred principally from mutant
Ras proteins that lack the palmitates, but retain the isoprenoid, or
that lack all three lipids entirely. The decreased membrane binding
observed with these modification mutants has led to the model that
membrane binding is a primary role for the lipids attached to Ha-Ras.
However, the function(s) of farnesyl and palmitate, individually, have
not been completely elucidated.
The consequences of farnesyl attachment are particularly important
because prenylation is the first step in processing, putting farnesylation in temporal control of subsequent modifications. Without
an isoprenoid attached to Cys-186, the Ha-Ras protein remains soluble,
and the adjacent Cys-181 and Cys-184 do not become palmitoylated. An
isoprenoid has been shown to be a prerequisite modification for
recognition by both the membrane-bound protease and methylase enzymes,
which further modify the C terminus (4, 5). It has been suggested that
a putative palmitoyl acyltransferase may also require the presence of a
farnesyl group (6), but the authenticity of this enzymatic activity has
not been confirmed. Farnesyl thus functions in maturation of Ras
proteins by acting as part of a signal sequence for and allowing
interaction of Ras with at least two membrane-bound processing enzymes.
The most well recognized role for farnesyl is its participation in Ras
membrane binding. Prenylation is required for initiating the transition
of the cytosolic precursor to the membrane-bound form. Whether farnesyl
also plays a role in sustaining or directing submembrane localization
has not been clarified. Farnesyl by itself does not confer high
affinity membrane binding to Ras peptides in vitro (7-9),
nor does it suffice for targeting of the protein to plasma membranes in
intact cells (10-12). The farnesyl group appears to depend heavily on
a second mechanism to assist its efforts in membrane binding. Two types
of "secondary" membrane-binding signals have been identified: a
hydrophobic type involving palmitoylation of nearby cysteine residues,
as is found in Ha-Ras, N-Ras, and Ki-Ras4A; and an ionic type, based on
the series of basic residues just N-terminal of the CAAX
sequence, as found in Ki-Ras4B (2, 13).
A number of studies have now suggested that farnesyl may have an
additional role besides initiating membrane attachment. The farnesyl
group is proposed to bind to specific membrane proteins (14), a process
that might enhance Ras/membrane interaction or that might be needed for
activation of Ras effector proteins. Good evidence has been presented
that a structural or conformational epitope found in the prenylated
form of Ha-Ras is important (in addition to the role of Ras in Raf
membrane targeting) for activation of Raf-1 through the kinase's
cysteine-rich zinc finger domain (15-17). In vitro analysis
has suggested that farnesylation of Ha-Ras may also be needed for human
SOS1 to promote guanine nucleotide exchange (18). Earlier studies using
an activated yeast Ras2 protein revealed that mutation of the
farnesylation site decreased the interaction between Ras2 and adenylyl
cyclase (19, 20). Thus, these studies imply that the prenyl structure
of Ras may be involved in and perhaps required for specific
associations of Ha-Ras and its regulatory or target proteins. How
potential roles in both lipid bilayer binding and protein interactions
can be performed simultaneously or sequentially remains an important, unanswered question.
The duties for which the palmitates of Ha-Ras are used are much less
clear. Palmitate certainly has sufficient hydrophobicity to support
membrane binding of an Ha-Ras protein, but the requirement for prior
farnesylation has prevented study of the role(s) of the Ha-Ras
palmitates independent of isoprenoid. It is possible that
palmitoylation of Cys-181 and Cys-184 of Ha-Ras is only a nonspecific
(although required) secondary membrane attachment signal that simply
enables more substantial or sustained levels of membrane binding of a
farnesylated Ha-Ras protein. More direct, biochemical studies of
palmitoylation have been thwarted because the enzymes that might attach
palmitates to Ha-Ras, or similar proteins, have proved to be extremely
difficult to isolate (6, 21, 22). In vivo requirements for
palmitate attachment have been examined in a large series of C-terminal
mutant Ha-Ras proteins, all of which became palmitoylated (11). These
studies identified amino acids that appeared to be novel signals for
intracellular trafficking, but no consensus sequence for palmitate
attachment was found. However, these mutant proteins still retained an
intact CAAX motif and were farnesylated, so it remained
possible that farnesyl might be part of an otherwise enigmatic signal
for palmitoylation. Because several of these farnesylated and
palmitoylated mutant Ha-Ras proteins were mis-localized to internal
membranes within the cell, one clear result was that acquisition of
both C-terminal lipids is not sufficient to assure correct plasma
membrane targeting.
However, based on observations that C181S/C184S mutants of Ha-Ras
(which retain farnesyl but lack palmitate) localize poorly to the
plasma membrane, several studies have suggested that it is palmitate
rather than farnesyl that is largely responsible for Ha-Ras membrane
binding. In the Xenopus oocyte maturation assay and also in
transformation of NIH 3T3 cells, Ha-Ras proteins modified only by
farnesyl have poor biological activity, indicating that acquisition of
palmitates is important functionally (9-11, 23, 24). Using a yeast
plasmid-loss assay, Mitchell et al. (25) studied a series of
yeast Ras2 proteins that showed that the combined effect of C-terminal
basic amino acids and palmitoylation of cysteine residues was
sufficient to support Ras-dependent growth, independent of
prenylation. More important, the biological function of these yeast
Ras2 proteins correlated with their ability to be palmitoylated. These
results show that palmitate is an important contributor to Ha-Ras
membrane binding and function and imply that, despite its chronological
precedence, farnesyl depends upon palmitate to support these
activities. However, it remains unclear whether palmitate mutually
requires the farnesyl or plays an independent but complementary role in
specific membrane association and biological activity of Ha-Ras.
To enable study of requirements for palmitate addition and to determine
if palmitates could direct specific plasma membrane targeting, sustain
Ha-Ras membrane binding, and support biological function, a novel
mammalian Ha-Ras protein was constructed, designed to have C-terminal
palmitates, but no isoprenoid. The results indicate that, although
farnesyl needs the assistance of palmitate for membrane binding and
full biological function, palmitates can support substantial
farnesyl-independent activity. The results begin to define distinct
roles for Ha-Ras farnesyl and palmitate and suggest that palmitate is
more than just an energetic form of membrane tether that serves the
needs of the farnesyl and may have unique and dynamic biological roles
of its own.
Mutants and Plasmids--
Ext61L, ExtWT,
Ext61L(C181S/C184S/C186S), ExtWT(C181S/C184S/C186S), and Ha-Ras61L were
expressed from pcDNA3 (Invitrogen, Carlsbad, CA). To construct
Ext61L, an S190K substitution followed by the addition of five lysine
residues was introduced into Ha-Ras with a Q61L activating mutation by
oligonucleotide-directed polymerase chain reaction mutagenesis. The
construction used an oligonucleotide 5'-GGGGGGATCCACCATGACAGAATACAAGCTT-3', which was fully complementary to
the 5'-sequences of human Ha-Ras, and the mutagenic oligonucleotide 5'-GGGGGGATCCTCACTTCTTCTTCTTCTTCTTGAGCACACACTTGCAGCT-3',
which reproduced the nucleotides complementary to the
3'-sequences of Ha-Ras and introduced the six lysine codons
(underlined), a termination codon, and a BamHI restriction
enzyme recognition site. The resulting pcDNA3-Ext61L cDNA was
inserted as a BamHI fragment into the BamHI site
of pcDNA3 and encoded the complete Ha-Ras protein with a C-terminal
polylysine tail (Fig. 1A). pcDNA3-ExtWT is identical to
pcDNA3-Ext61L, except that it encodes the non-activated cellular form of Ha-Ras with the normal glutamine residue at position 61. Ext61L(C181S/C184S/C186S) and ExtWT(C181S/C184S/C186S) were constructed in a similar fashion using the mutagenic oligonucleotide
5'-GTACTCTAGATCACTTCTTCTTCTTCTTTTTTAACACACTCTTACTGCTCATAGAGCCAGGACC-3' to introduce the six lysine codons (underlined) and to change codons
181, 184, and 186 from Cys to Ser (double underlined). The cDNA was
inserted as a BamHI-NotI fragment into pcDNA3.
Cell Culture, Transfection, and Transformation Assays--
PC12
cells were maintained at 5% CO2 in RPMI 1640 medium
supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum (both from Hyclone Laboratories, Logan, UT) . Twenty-four hours
before DNA transfection, PC12 cells were plated onto laminin (10 µg/ml; Life Technologies, Inc.)-coated 60-mm tissue culture dishes at
a density of 106 cells/dish and grown overnight.
Transfection was performed using LipofectAMINE reagent (Life
Technologies, Inc.) as described by the manufacturer. NIH 3T3 cells
were grown at 10% CO2 in Dulbecco's modified Eagle's
medium supplemented with 10% calf serum (Hyclone Laboratories). Cells
were transfected with plasmid DNAs encoding Ext61L or ExtWT Ras
proteins using the calcium phosphate precipitation technique (11, 26).
Transformed foci were quantified after 7-14 days. Transfected cells
were also selected in growth medium containing G418 (Geneticin, Life
Technologies, Inc.) at 500 µg/ml to establish cell lines that stably
expressed the mutant proteins.
Preparation of Subcellular Fractions--
Subcellular fractions
were prepared by lysis of cells in hypotonic buffer (0.1 M
Tris, pH 7.4, 0.5 M MgCl2, 1 mM
Pefabloc, 1 µM leupeptin, 2 µM pepstatin,
and 0.1% aprotinin (Calbiochem)) and addition of NaCl to adjust the
ionic strength to 0.15 M, followed by ultracentrifugation
for 30 min at 100,000 × g as described previously
(26). Hydrophobic (e.g. lipid-modified) proteins were
isolated by separation into detergent (hydrophobic) and aqueous (hydrophilic) fractions by Triton X-114 lysis and phase separation as
described (2). For testing release of proteins from the P100 membrane
fraction, the supernatant (S100 = S1) was set aside, and the
pellet was resuspended in 1 ml of hypotonic buffer supplemented with
0.5 M NaCl and incubated on ice for 30 min. The suspension was centrifuged at 100,000 × g; the supernatant (S2)
was collected; and the pellet was incubated with the high salt buffer a
second time and centrifuged, yielding a third supernatant (S3). The
pellet from the second salt wash (P3) was resuspended in 1 ml of
radioimmune precipitation assay buffer and centrifuged, and the
radioimmune precipitation assay extract (S4) removed. The proteins in
the separate S1-S4 supernatants were precipitated with 10 ml of
acetone for 1 h at 4 °C, collected by centrifugation at 3000 rpm for 30 min, and dissolved in 100 µl of electrophoresis sample
buffer. Equal volumes of each fraction were separated by
SDS-PAGE.1
Radiolabeling, Immunoprecipitation, and Immunoblotting--
For
protein labeling, cells were incubated with 200 µCi/plate
Tran35S-label (ICN, Costa Mesa, CA) in Dulbecco's modified
Eagle's medium lacking cysteine/methionine for 18 h. To examine
isoprenoid attachment, cells were pretreated with 50 µM
compactin for 30 min and then labeled for 18 h with 100 µCi/ml
[3H]mevalonolactone (American Radiolabeled Chemicals, St.
Louis, MO) in the continued presence of compactin (27). Palmitate
incorporation and subsequent depalmitoylation were measured by labeling
cells for 4 h with 1 mCi/ml [3H]palmitate (NEN Life
Science Products) in medium containing nonessential amino acids, 25 µg/ml cycloheximide, and 10% calf serum and then incubating for
varying times in nonradioactive medium containing 200 µM
palmitic acid. After radiolabeling, samples were prepared for
subcellular fractionation as described above, or cells were lysed
directly in high SDS/radioimmune precipitation assay buffer (50 mM Tris, pH 7.0, 0.5% SDS, 1% Nonidet P-40, 1% sodium
deoxycholate, 150 mM NaCl, and 50 mM aprotinin
(Calbiochem)) for immunoprecipitation. Extracts of cell fractions were
incubated on ice for 1 h with Ha-Ras-specific mouse monoclonal
antibody 146-3E4 (Quality Biotech Inc., Camden, NJ).
Immune complexes were recovered with Staphylococcus aureus
cells (Pansorbin, Calbiochem) coated with goat anti-mouse heavy and
light chain IgG; washed; resuspended in special sample buffer (2% SDS,
10 mM NaPO4, pH 7.0, 10% glycerol, 0.05%
dithiothreitol, and 0.02% bromphenol blue); resolved by SDS-PAGE;
transferred to polyvinylidene difluoride membrane; and, as needed,
sprayed with EN3HANCE (NEN Life Science Products) for
fluorography or developed for immunoblot analysis. For immunoblotting,
after separation by SDS-PAGE, proteins were transferred
electrophoretically to polyvinylidene difluoride membranes, and
nonspecific protein binding was blocked by incubating the membrane in
1.25% nonfat dry milk in Tris-buffered saline. Membranes were probed
with Ha-Ras-specific monoclonal antibody 146-3E4. Biotinylated
secondary antibodies (anti-mouse; Vector Laboratories, Inc.,
Burlingame, CA) were used with development by alkaline phosphatase
(Vector Laboratories, Inc.) using the manufacturer's protocol.
Immunofluorescence--
PC12 cells were plated at low density on
serum-coated coverslips in six-well dishes and transfected with Ext61L
DNA as described above. Following fixation with freshly prepared 2%
paraformaldehyde, cells were treated with a blocking solution
containing 0.4% bovine serum albumin and 3% horse serum to block
nonspecific antibody interactions and 0.05% Triton X-100 and 0.05%
Tween 20 to permeabilize the cells (28). Cells were then treated with a
1:20 dilution of Ha-Ras-specific rat monoclonal antibody Y13-172,
followed by a 1:50 dilution of fluorescein isothiocyanate-conjugated
rabbit anti-rat secondary antibody (Cappel/Organon-Teknika, Durham,
NC), both diluted in blocking solution; mounted in
diazabicyclo[2.2.2]octane/glycerol solution to prevent fading; and
viewed by confocal microscopy.
GTP/GDP Determination--
Confluent cultures of PC12 cells were
grown overnight in 1% dialyzed calf serum. Cells were then
radiolabeled with 0.5-1 mCi/ml 32Pi (NEN Life
Science Products) for 4 h in phosphate-free medium containing 1%
calf serum. Cells were rinsed with phosphate-buffered saline and lysed
in 0.6 ml of cold GTP lysis buffer containing 50 mM
Tris-HCl, pH 7.4, 20 mM MgCl2, 150 mM NaCl, 0.5% (v/v) Nonidet P-40, 20 µg/ml aprotinin, 1 mM EGTA, and 1 mM
Na3VO4. Insoluble material was removed by
centrifugation at 750 × g for 3 min. Supernatants were
cleared with 50% (v/v) bovine serum albumin-coated charcoal in lysis
buffer, and then Ha-Ras proteins were captured by immunoprecipitation using GTP lysis buffer. Ha-Ras and its bound nucleotides were eluted by
heating to 60 °C for 5 min in a minimal volume of buffer containing
20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2% (w/v)
SDS, 2 mM GDP, and 2 mM GTP. The eluants were
cleared by centrifugation at 10,000 × g for 5 min, and
then samples were spotted on polyethyleneimine cellulose plates
(J. T. Baker Inc.) and separated with 0.75 M KH2PO4, pH 3.4. Plates were dried and exposed
to preflashed film using screen enhancers. The film images of GDP and
GTP were scanned and quantified using the program ImageQuant (Molecular
Dynamics, Inc.), and the percent of an Ha-Ras protein that contained
bound GTP was calculated using the following formula: % with GTP = GTP/(GTP + 1.5 GDP).
Ext61L cDNA Produces a Stable Protein That Is Not
Prenylated--
Multiple studies that had examined a great many
mutations within and immediately N-terminal of the CAAX box
of non-prenylated Ha-Ras or Ki-Ras4B proteins had all failed to detect
membrane binding (a biochemical surrogate for potential
palmitoylation). The only successful examples of palmitoylated forms
that lacked isoprenoid were the yeast Ras2 mutants in which the
CAAX box was extended with a series of basic amino acid
residues (25). This work had suggested that lengthening and introducing
positively charged residues enabled a yeast Ras2 protein to contact
membranes and to become palmitoylated. The mammalian ExtRas proteins
were therefore designed with a lysine in the X position of
the CAAX box, which was anticipated would produce a
nonfunctional prenylation motif, and five additional lysine residues to
provide an ionic platform for interaction with acidic membrane phospholipids.
The initial experiments determined the stability and size of the Ext61L
protein and whether the carboxyl-terminal CVLKKKKKK sequence (Fig.
1A) prevented the Ext61L
protein from being prenylated. This was important because it was
possible the Ext61L protein would be vulnerable to proteolytic removal
of these basic residues, which would remove the structure that was
meant to initiate and assist membrane binding. Also, removal of five
lysines would potentially recreate a CAAX box motif (CVLK)
that might permit prenylation. This was not considered likely as the
presence of a lysine at the X position had previously been
found to prevent prenylation of a Ki-Ras4B protein (29). If such
trimming and prenylation were to occur, it could be detected because it
would produce a smaller protein, similar in size to mature native
Ha-Ras protein. An additional way to detect, indirectly, if prenylation
was occurring was to treat cells with compactin. Compactin inhibits
isoprenoid synthesis and hence decreases Ras prenylation, preventing
the change in mobility and leading to the accumulation of the
unmodified precursor form of Ha-Ras in the cytosol (1).
To test these possibilities, COS cells expressing Ext61L were labeled
metabolically with [35S]methionine in the presence or
absence of compactin. Cytosolic and membrane-containing fractions were
prepared, and proteins were separated by SDS-PAGE and detected by
fluorography. The Ext61L protein was easily detected and had the
appropriate, slightly slower mobility than the endogenous Ha-Ras
protein of COS cells, indicating that the C-terminal residues of Ext61L
were retained (Fig. 1B). More important, a significant
portion (40%) of the Ext61L protein was detected in the
membrane-containing fraction and had the same apparent size as the
cytosolic protein. This similarity in size of proteins in the S100 and
P100 fractions indicated indirectly that the extension protein was not
farnesylated. In addition, compactin did not decrease the amount of
Ext61L protein that was present in the P100 fraction. Without compactin
treatment, >90% of the small amount of endogenous Ha-Ras protein in
untransfected or transfected COS cells (Fig. 1B) was found
in the membrane fraction. In the compactin-treated cells, the amount of
membrane-bound endogenous Ha-Ras decreased; the increased amount of the
precursor form in the cytosol was obscured by the large amount of
soluble Ext61L protein.
To evaluate more directly whether the Ext61L protein remained
unmodified by isoprenoid, COS cells expressing this protein or
Ha-Ras61L were labeled with [3H]mevalonic acid, and the
Ras proteins were isolated by immunoprecipitation. No incorporation of
radioactivity could be detected in the Ext61L lane (Fig. 1C,
lane 2). However, labeling of cells expressing Ha-Ras61L confirmed that an Ha-Ras protein with an intact
CAAX motif could be prenylated (Fig. 1C,
lane 1). In addition, immunoblot detection of the same
membrane confirmed that the Ext61L protein was expressed (data not
shown). These results indicated that the Ext61L protein was not prenylated.
Ext61L Binds Membranes Rapidly and Is Targeted to the Plasma
Membrane--
Although a significant amount of Ext61L protein was
found in the membrane-containing fraction in the cells, the extent of binding of the Ext61L protein was far less than the >95% binding attained by endogenous Ha-Ras with the natural version of the C
terminus. Previously constructed C-terminal mutants of v-Ha-Ras had
also shown decreased or delayed membrane binding compared with v-Ha-Ras
with the normal C-terminal residues and modifications (11). To examine
if the decreased membrane association of Ext61L reflected inefficient
trafficking or attachment to membranes, the speed with which newly
synthesized Ext61L traversed the cytosol and achieved membrane
association was assessed. Replicate dishes of cells were metabolically
labeled with [35S]methionine/cysteine for 10 min, and
then one plate was incubated in nonradioactive medium for an additional
30 min. Subcellular fractions were prepared, and Ha-Ras
immunoprecipitates were formed. Within the 10-min pulse period, 27% of
the newly synthesized Ext61L protein had already translocated to the
membrane (Fig. 2A). After the
30-min chase, Ext61L in the membrane-containing fraction had increased
to 44% of the total protein and therefore to the same level seen at
steady state. This speed of membrane binding was similar to or even
faster than that seen with a v-Ha-Ras protein that undergoes both
farnesylation and palmitoylation (11). The addition of the basic
residues to Ext61L therefore did not impede normal trafficking or delay
membrane attachment.
Some of the previously mentioned v-Ha-Ras C-terminal mutants, which by
biochemical fractionation techniques were membrane-associated, were
subsequently found to be mis-localized to internal rather than plasma
membranes (11). This intracellular trapping was correlated with poor
biological activity. As a more definitive way to assess attachment to
specific membranes, immunofluorescence was used to visualize the
subcellular localization of Ext61L in intact single cells. A series of
confocal laser microscopic images of PC12 cells expressing Ext61L
exhibited a clear defined signal of Ext61L at the plasma membrane (Fig.
2B). No staining of internal membranes was observed. These
data, taken together with the biochemical data described above,
indicated that somewhat less than half of the Ext61L protein succeeded
in associating with membranes and, more specifically, that these
membrane-bound molecules were located at the plasma membrane.
Ext61L Associates with Membranes Largely through Hydrophobic
Interactions--
The lysines at the terminus of ExtRas were
envisioned to participate in ionic interactions with acidic
phospholipids of the plasma membrane and thus to initiate membrane
binding that would enable palmitoylation. To determine if ionic forces
might also contribute to maintenance of the association between the
Ext61L protein and cellular membranes, addition of a concentrated salt solution to isolated membranes was used to disrupt ionic interactions (2). Membranes from transfected COS cells were first separated from the
cytosol, suspended in 0.5 M NaCl for 60 min, and then again
isolated by sedimentation. Less than 10% of the membrane-associated Ext61L protein was released by the salt extraction procedure (S2) (Fig.
3A). The basic residues of
Ext61L thus appeared to contribute in only a small way to long-term
stability of membrane association.
Because ionic forces could not account for the substantial amount of
protein that partitioned into the P100 fraction at steady state, the
ability of detergent to extract the membrane-bound Ext61L protein was
used to further explore if the portion of Ext61L that resisted salt
washout was bound to membrane through hydrophobic forces. The
salt-washed membranes were again washed with 0.5 M NaCl
(S3) and then resuspended in SDS-containing buffer. The
detergent-released material (S4) was separated from the residual
detergent-insoluble material by centrifugation. For Ext61L, ~75% of
the membrane-associated protein was released after SDS extraction (S4)
(Fig. 3A). The palmitoylated and farnesylated endogenous
Ha-Ras protein in untransfected COS cells resisted the salt washout and
was released by detergent in the S4 fraction.
To further determine if the Ext61L protein displayed any hydrophobic
character suggestive of a lipid modification, transfected cells were
also processed by Triton X-114 phase separation. This procedure
separates cellular proteins, based on overall hydrophobic character,
into either an aqueous (hydrophilic) or detergent (hydrophobic) fraction. For the Ha-Ras protein, whose amino acid sequence is hydrophilic, Triton X-114 phase separation has been used to distinguish forms that have or that lack lipids. Cytosolic (S100) and
membrane-containing (P100) fractions were prepared from PC12 cells
expressing Ha-Ras61L or Ext61L proteins. The membranes were further
partitioned by resuspending the P100 fraction in 1% Triton X-114 and
warming the sample to trigger phase separation. For the Ha-Ras61L
protein, with the native version of the C terminus, the precursor form (not yet lipid-modified) was present in the cytosol, whereas the mature
Ha-Ras61L protein (farnesylated and palmitoylated) appeared in the
detergent phase (Fig. 3B). For Ext61L, ~60% of the
protein again was cytosolic, and roughly half of the ~40% of the
protein that sedimented in the P100 fraction (in multiple experiments, varying from 15 to 25% of the total protein) further partitioned into
the detergent phase, suggesting that this minor portion of the
expressed protein possessed hydrophobic character (Fig.
3B).
The Ext61L Protein Is Palmitoylated--
The two cysteines
(Cys-181 and Cys-184) that are palmitoylated in the Ha-Ras61L protein
are retained in Ext61L, as is Cys-186, which is normally modified by
farnesyl. Thus, the Ext61L protein has three potential sites for
palmitate attachment. To determine whether or not Ext61L was
palmitoylated, NIH 3T3 cells expressing Ext61L and Ha-Ras61L,
respectively, were labeled for 3 h with [3H]palmitate, followed by a chase in nonradioactive
medium. At the indicated times, immunoprecipitates were formed, and
palmitate labeling of the Ext61L and Ha-Ras61L proteins was detected by fluorography. The Ext61L protein not only incorporated palmitate, but
did so at a rate similar to the Ha-Ras61L protein; both required ~3 h
for maximum [3H]palmitate labeling (data not shown).
During the chase, the depalmitoylation rates for the two proteins were
also similar (Fig. 4). When
immunoblotting was used to account for variations in Ras protein
recovery in the samples on the membrane (data not shown), both Ext61L
and Ha-Ras61L lost half of their incorporated palmitate between 2 and
4 h. Similar results were also obtained using PC12 cells
transiently expressing either Ext61L or Ha-Ras61L (data not shown). The
palmitoylated form of the Ext61L protein was detected only in the
membrane-containing cellular fraction, with no evidence of a partially
(de)palmitoylated protein being released into the
cytosol.2 Thus, although the
Ext61L protein was not prenylated, it could be palmitoylated, and the
palmitate displayed an apparently normal turnover rate. This indicated
that the presence of the polybasic domain did not prevent the Ext61L
protein from being accessible to either acylating or deacylating
enzymes.
Ext61L Transforms NIH 3T3 Cells--
Soluble Ha-Ras61L protein has
been reported to act as a dominant-negative inhibitor of Ha-Ras
transformation of NIH 3T3 cells (30, 31), presumably by binding and
trapping effector proteins, such as the Raf-1 protein kinase, in the
cytosol. Because a large amount of Ext61L was cytosolic, it was
possible that the biological effectiveness of Ext61L would be dampened
by this pool of cytosolic protein. The ability of the Ext61L protein to
stimulate mitogenesis and transformation of NIH 3T3 cells was examined
to ascertain if the combination of palmitate and basic residues was
capable of supporting a biological function of Ha-Ras. Ext61L DNA was potently transforming and caused robust focus formation (~1100 foci/µg of DNA) (Table I). This
activity was equivalent to the transforming activity of DNA encoding
the Ha-Ras61L form with the native lipid modifications. The cellular
version of the ExtRas protein, ExtWT, did not cause focus formation in
NIH 3T3 cells, even when the cells were transfected with up to 2 µg
of DNA. This lack of activity was similar to the low transforming
activity of the normally lipidated form of Ha-Ras, p21WT. The
morphology of transfected NIH 3T3 cells expressing Ext61L was easily
distinguished from that of cells expressing the normally lipidated 61L
protein: foci produced by 61L were typically swirling, spreading
colonies of transformed cells, whereas the Ext61L-induced foci
contained clumps of cells that tended to round up and pull away from
the plastic substratum and contained unusually long, spindle-shaped, elongated cells with a refractile appearance (data not shown).
To determine if Ext61L expression also triggered anchorage-independent
growth, NIH 3T3 cells stably expressing Ha-Ras61L or Ext61L were plated
in soft agar and assayed for the ability to form colonies. Like
Ha-Ras61L-transformed cells, Ext61L-transformed cells readily formed
colonies in soft agar (data not shown). All together, these results
demonstrated that the non-prenylated but palmitoylated Ext61L protein
produced the aberrant growth properties characteristic of an oncogenic
Ha-Ras protein in NIH 3T3 cells.
Ext61L Causes Neurite Extension in PC12 Cells--
Transformation
of NIH 3T3 cells is a hallmark of oncogenic Ha-Ras activity that
culminates in focus formation. To assess a different aspect of Ras
biological function, the ability of the Ext61L protein to cause
differentiation of PC12 cells was examined. In PC12 cells, expression
of activated Ras proteins is characterized by the cessation of mitosis,
unique rearrangements of the actin cytoskeleton, and extension of
neuron-like processes in a program of events similar to those triggered
by exposure to nerve growth factor (32, 33). Only activated forms
of Ras have been found to produce this response, whereas normal
cellular Ras has no effect (34-36). More important, Ras must be
membrane-bound to cause neurite extension, as non-lipidated forms, even
if activated, are incapable of triggering differentiation (37, 38).
PC12 cells transfected with DNA encoding a cellular, normally lipidated
form of Ha-Ras (p21WT) continued to proliferate and showed the limited
adherence and round shape characteristic of the parental PC12 cells
(Fig. 5, a and b).
PC12 cells transfected with DNA encoding the constitutively active,
prenylated and palmitoylated Ha-Ras61L protein developed neurites that
attained a length of greater than two cell bodies after ~48 h (see
Fig. 5 legend). There were two or occasionally three of these axon-like
extensions/cell, which continued to elongate over the next 2-3 days.
However, at 48 h, only 21% of these extensions were longer than
100 µm (see Fig. 5 legend). The processes were smooth and, during the
first days, generally extended linearly, with a single growth cone on each and little branching (Fig. 5e). The amount of DNA
necessary to generate this rate and extent of response by Ha-Ras61L was titrated and found to require 1 µg of DNA. The morphology of PC12 cells expressing Ha-Ras61L was similar to that of the cells treated with 50 ng/ml nerve growth factor, although the nerve growth factor response required 2-3 days for neurite extension to be established and
progressed more slowly, over 8-10 days (data not shown).
In contrast, PC12 cells transfected with 1 µg of DNA encoding the
Ext61L protein developed multitudes of neurites (Fig. 2B). As little as 50 ng of DNA encoding the Ext61L protein caused
development of visible neurites as early as 24 h after
transfection (Fig. 5f). These processes were longer than
those produced by 61L and extended rapidly, exceeding within 2 days
even the final length of those in the 61L cultures (Fig.
5e). Cells expressing the Ext61L protein often produced four
or five extensions/cell, and 84% of these extensions were longer than
100 µm 48 h after transfection (see Fig. 5 legend). Furthermore,
as the differentiation of these cells progressed, the extensions became
highly branched, wandered in several directions, and displayed numerous
fine filopodia along their length. In addition, Ext61L brought about a
flattening of the cell body, with large lamellipodia, areas of
adherence, and veils of attachment. A prominent feature of cells
expressing the Ext61L protein was the accumulation of numerous large,
intracellular vesicles. The biological activity of Ext61L was therefore
unexpectedly robust, and the morphological changes it caused were an
exaggerated version of those caused by the normally lipidated Ha-Ras61L protein.
To determine if the conspicuous activity of Ext61L could be ascribed to
particularly efficient protein expression, the amount of Ext61L or
Ha-Ras61L in the membrane/detergent fraction prepared from
differentiated PC12 cells transfected as described for Fig. 5 was
examined more closely. In the Ext61L-expressing cells, the amount of
protein in the detergent fraction was less than the amount
present in the detergent fraction of the Ha-Ras61L-expressing cells
(Fig. 3B). However, despite expressing smaller amounts of membrane-associated protein, the culture transfected with Ext61L DNA
exhibited more extravagant neurite extensions than the parallel culture
of cells expressing Ha-Ras61L protein (Fig. 5, e and
f). Additional experiments using an even smaller amount of
Ext61L DNA (10 ng) showed that exaggerated neurite morphology was still produced when the total amount of Ext61L protein (cytosolic + aqueous + detergent) was much less (approximately one-tenth) than the
total amount of protein that was present in the parallel culture transfected with Ha-Ras61L DNA (data not shown). Thus, even with smaller amounts of membrane-bound protein, Ext61L continued to exceed
Ha-Ras61L activity and to display unusual characteristics in the
neurites it produced. Therefore, in two assays that test quite
different outcomes of Ha-Ras activity, transformation and differentiation, there was certainly no evidence that the residues appended to the C terminus or the large amount of cytosolic Ext61L protein diminished biological function.
The Cellular Form of the Extension Protein ExtWT Causes
Differentiation of PC12 Cells--
The unusual activity of the Ext61L
protein in PC12 cells raised the possibility that the cellular
(wild-type) form of ExtRas might generate a response in PC12 cells.
Transfection of PC12 cells with as little as 250 ng of ExtWT DNA caused
production of neurites, with an accelerated time course that was
intermediate between that of Ha-Ras61L and Ext61L and with a morphology
characteristic of and as unusual as the Ext61L protein (Fig.
5g). This amount of ExtWT DNA caused production of protein
amounts that were less than those produced in parallel cultures
expressing Ha-Ras61L protein (data not shown). The quite appreciable
activity of the ExtWT protein contrasted with the complete inactivity
of normal Ha-Ras with the native C-terminal residues and lipid
modifications. Transfection of cultures with 40 times larger amounts of
DNA (10 µg) for p21WT produced abundant protein expression (data not
shown), but caused no neurite extension (Fig. 5b) (36).
Thus, these changes in the Ha-Ras C terminus appeared to have also
increased the biological activity of a cellular form of Ha-Ras to cause PC12 cell differentiation.
Palmitoylation Is Required for ExtRas-mediated Differentiation of
PC12 Cells and Membrane Localization--
Previous observations have
established that prenylation and either palmitoylation or a stretch of
basic amino acids are required for membrane localization and biological
activity of Ras proteins (2, 10). To determine if the exaggerated
activity of Ext61L could be ascribed to the polylysine motif, ExtRas
proteins that could not be palmitoylated (Ext61L(C181S/C184S/C186S) and
ExtWT(C181S/C184S/C186S)) were constructed. The C181S/C184S/C186S
mutants were unable to induce differentiation of PC12 cells (Fig. 5,
c and d). Thus, the C-terminal polylysine motif,
without palmitate, did not support Ha-Ras biological activity.
The subcellular localization of Ext61L and ExtWT(C181S/C184S/C186S) was
also examined by subcellular fractionation with Triton X-114 phase
separation. As shown in Fig. 3, the ExtRas(C181S/C184S/C186S) proteins,
which lack palmitates but retain the lysines, were localized completely
to the cytoplasm. Thus, the polylysines alone were not sufficient to
maintain association of ExtRas(C181S/C184S/C186S) with the plasma
membrane, and stable membrane attachment of ExtRas appeared to
require palmitoylation.
Ext61L and ExtWT Bind GTP/GDP Appropriately--
One possible
explanation for the pronounced activity of the ExtWT and Ext61L
proteins was that the changes at the C terminus altered access of
nucleotide exchange proteins or GTPase-activating proteins, which
regulate the ability of Ha-Ras to bind GTP and allowed the accumulation
of the active form of these proteins, especially ExtWT. To test if the
levels of GTP bound to ExtRas proteins were elevated, transfected PC12
cells were labeled with 32Pi, and radioactive
nucleotides bound to Ha-Ras immunoprecipitates were separated by
thin-layer chromatography. In differentiated PC12 cells, 83% of the
expressed 61L protein and 82% of the Ext61L protein were in the
GTP-bound form (Fig. 6). In a second
experiment, nucleotides bound to cytosolic and membrane-associated
forms of the proteins were analyzed. The portion of the membrane-bound form of Ext61L that had GTP (82%) was the same as the portion that
bound GTP in the cytosolic form (data not shown), suggesting that
GTPase-activating proteins were equally (in)effective on the Ext61L
protein in either location. Thus, both the soluble and membrane-bound
forms of Ext61L were, to an extent similar to Ha-Ras61L, predominantly
in the active conformation.
For the ExtWT protein, 8% of the protein contained GTP (Fig. 6). This
is equivalent to the portion found using this isolation protocol on an
overexpressed, cellular Ha-RasWT in NIH 3T3 cell lines, where 8% was
also in the GTP-bound form (data not shown). Again, both cytosolic and
membrane-associated ExtWT proteins appeared to have similar GTP/GDP
ratios (data not shown). Thus, the ExtWT protein was primarily in the
GDP state, as appropriate for a cellular form of Ha-Ras, and showed no
evidence that the altered C terminus had increased interactions with
exchange factors or decreased interactions with GTPase-activating
protein(s) to the point that could explain its potent differentiating activity.
Targeting and stable localization of Ha-Ras to the plasma membrane
coincide with acquisition of biological activity. However, the
possibility of distinct or independent contributions of the two lipid
modifications to targeting, binding, or function has been difficult to
examine. We have developed a unique form of Ha-Ras that can be
palmitoylated without the requirement for prior farnesylation. The
C-terminal changes in this protein do not appear to have altered the
basic GTP binding properties of this protein, and the protein causes
unexpectedly potent biological function of two quite different types.
These studies support the possibility that palmitate may have
structural and biological roles of its own, independent of isoprenoid.
Requirements for Palmitate Attachment and Removal--
To produce
a palmitoylated but non-prenylated protein, an artificial polylysine
domain was attached to the C terminus of Ha-Ras. The polylysine tail
was designed to be (and was apparently successful as) an ionic platform
through which the otherwise soluble, non-prenylated Ha-Ras protein
could initiate membrane contact and undergo subsequent palmitoylation,
which then promoted tighter and more sustained interaction. This tail
motif partially mimics the larger polybasic region found just internal
to the carboxyl terminus of the Ki-Ras4B protein, which has no
cysteines that can be palmitoylated. However, in Ki-Ras4B, the simple
presence of a C-terminal polybasic domain, without farnesylation, does
not support membrane binding (2, 29, 39, 40).
The ExtRas proteins share several properties with a non-farnesylated
mutant yeast Ras2 protein with the C-terminal sequence CCIIKLIKRK (mutated CAAX box underlined). This
mutant Ras2 protein is also palmitoylated (at the cysteine previously
used for farnesyl attachment as well as the natural adjacent site),
binds membranes, and is biologically functional (25). Why having a
series of positively charged residues beyond the natural C-terminal end of Ha-Ras or Ras2 might permit palmitoylation remains to be studied, but this placement does appear to be a successful method to circumvent the need for prenylation and to permit palmitate attachment and may be
applicable to other proteins as well.
The data presented here demonstrate that Ha-Ras can be palmitoylated at
the natural C-terminal sites without being farnesylated. Our own
studies have indicated that palmitoylation of Ha-Ras, although proposed
to be catalyzed enzymatically, has no discernible primary amino acid
structure that functions as a potential signal sequence (11). The
successful palmitoylation of the non-prenylated Ext61L protein clearly
indicates that a farnesyl is not a required part of a recognition motif
for any potential Ha-Ras acyltransferases.
A dynamic balance of addition and removal is a central property of
Ha-Ras palmitate modification and the characteristic that most clearly
differentiates it from prenylation. The Ext61L protein has three
cysteines that are potential sites for palmitoylation. Whether all of
these sites are used or to what extent each is modified is unknown, as
current methods to directly examine palmitate stoichiometry are
unsatisfactory. The abundant amount of cytosolic Ext61L protein
indicates that palmitoylation of this protein is less complete than
that of the native form. The polylysine extension on Ext61L is
compatible with dynamic, transient acylation-deacylation and does not
prevent access of thioesterases to the C terminus. Future studies can
now be designed to remove lysine residues and to replace each cysteine
individually to more clearly define favored sites for palmitate
attachment as well as the requirements for palmitate removal.
Properties Needed for Membrane Interaction--
For Ha-Ras,
farnesyl is a rather poor membrane tether: only about 10% of the
protein is membrane-bound when a farnesyl is the only lipid present (2,
11). With only palmitate and lysines present, Ext61L interacts with
membranes to a greater extent than Ha-Ras(C186S), a property presumably
due to the combination of hydrophobic and ionic signals in Ext61L.
However, the sizable pool of cytoplasmic Ext61L protein, which has the
lysines but no palmitates, suggests that the lysine residues on their
own do not contribute greatly to long-term membrane interactions. The
solubility of the mutant Ext61L(C181S/C184S/C186S) supports this
inference, as does the yeast Ras2 extension protein, where mutation of
the cysteines to serines, leaving the polybasic residues intact,
abolishes membrane interaction (25). These results argue that firm
membrane interaction of an Ha-Ras protein does not require a
permanently attached lipid and that it is largely the palmitates, despite their continuous cycles of attachment and removal, that support
the stable interaction of the Ext61L protein with membranes. This
capacity of palmitates has not previously been demonstrated. Thus, if
palmitates can assume responsibility for bilayer interactions, farnesyl
does not need to be permanently engaged in this function and becomes
available for additional interactions with proteins within the membrane
or with effectors. These results emphasize that palmitate and farnesyl
may have distinct, perhaps sequential, but complementary roles in
membrane binding and function of Ha-Ras.
However, the combination of palmitates and lysines clearly is not as
efficient as the >95% achieved by the natural farnesyl plus palmitate
modification. One possible explanation for this modest amount of
membrane-bound Ext61L protein is that, in the absence of a tethering
farnesyl, the dynamic turnover of the palmitates now sets a new, lower
equilibrium point for membrane attachment/release. The amount of
membrane-bound ExtRas is likely to depend heavily on the rates of
palmitoylation and deacylation. The development of the ExtRas proteins
should now allow events that may regulate Ha-Ras palmitoylation and
membrane interactions to be addressed more clearly.
Other C-terminal alterations in Ha-Ras have produced proteins that are
located on intracellular, possibly Golgi, membranes (11). Such data
suggest that unexplored determinants of plasma membrane targeting are
located in the C terminus of Ha-Ras and may include the sites of
palmitoylation. That region remains unaltered in the Ext61L protein and
may continue to impart specific plasma membrane interaction signals
because immunofluorescence showed strong plasma membrane localization
and no staining of internal membranes. Additional studies can now
determine if the GTP-bound Ext61L and GDP-bound ExtWT proteins are
associated with membrane subdomains (e.g. "rafts" or
caveolae) and how their localization compares with that of Ha-Ras
protein with the natural lipid modifications. The ExtRas proteins will
permit study of the specific roles of palmitate and farnesyl in this compartmentation.
Influence of the Ha-Ras C Terminus on Biological
Function--
Early studies had suggested that the C-terminal lipids
on Ras proteins were only a convenient method for membrane attachment, were not required for more specific interactions with membrane or
effector proteins, and were therefore only indirectly involved in
signal transduction. This inference was based on studies in which
leader sequences (N-myristoylation or an actual
transmembrane domain (41)) were used to target non-farnesylated
Ha-Ras(C186S) proteins to the plasma membrane. Such chimeric proteins
possessed quite good activity in transformation of NIH 3T3 cells,
demonstrating that the biological function of transformation did not
specifically need farnesyl. However, one group reported that the
membrane binding provided by the N-myristoyl group
re-established palmitoylation of the two C-terminal Cys-181 and Cys-184
residues (23). That finding remains unconfirmed, as conflicting results
have been reported (11), but is potentially important because it
implies that acylation may have a direct impact on biological function.
Two additional observations have also implicated the C-terminal lipids
in roles beyond membrane anchoring. First, N-myristoylation of a cellular form of non-prenylated Ha-Ras(C186S) potentiated the low
transforming activity of the normal protein almost 1000-fold (26). This
implies that this particular N-terminal surrogate membrane targeting
system releases cellular Ha-Ras from a restraint that usually keeps
such oncogenic activity in check.
Second, some studies have inferred that Ha-Ras lipids, particularly
farnesyl, interact directly with proteins, rather than, or in addition
to, a membrane bilayer. A farnesyl has recently been reported to be
required for interaction of Ha-Ras with a plasma membrane protein (14).
A farnesyl-dependent interaction between Ha-Ras and the
zinc finger (cysteine-rich domain) of Raf-1 kinase has also been
suggested to occur (15, 42). In these instances, the non-farnesylated
forms that were used also lacked palmitate, so the contribution or
requirement of the acyl group to these interactions remains to be
answered. In the yeast Ras2 protein, farnesyl, but not palmitate, is
reported to be required for Ras-mediated signal transduction, where it
plays an essential, possibly direct role in promoting protein/protein
interactions between yeast Ras and a regulatory protein of adenylyl
cyclase, its downstream effector (20). All these results imply that
both C-terminal lipids may have important roles in Ha-Ras effector interaction and function that await exploration.
The strong differentiating and transforming activity of Ext61L was
somewhat surprising because previous studies had found that mutant Ras
proteins that were poorly membrane-bound were also poorly transforming
(10, 11). The potency of Ext61L could not be explained by abnormally
stable palmitoylation of the membrane-bound form or by a greater amount
of protein in membranes. Even when lesser amounts of
membrane-associated protein were present, Ext61L still caused a more
rapid and exceptional differentiation of PC12 cells than 61L protein.
The morphology of NIH 3T3 cells expressing Ext61L is also
distinguishable from that of cells expressing authentic 61L. These
unusual morphologies suggest that cytoskeletal pathways are perturbed.
Studies are underway to determine if Ext61L interacts with and
activates the same pathways as Ha-Ras61L with the native C terminus and
lipids or if the introduction of the basic residues has altered or
added effector proteins to the repertoire of Ha-Ras partners. The
change in lipidation of the Ext61L protein may also influence the
(sub)membrane localization of the protein and thus indirectly
affect its access to regulatory or effector proteins.
The possibility that interactions with effectors or membranes may have
been changed in the ExtRas proteins is supported by the unexpected
activity of ExtWT. Non-activated or wild-type forms of Ha-Ras do not
normally induce differentiation of PC12 cells (34, 43), yet the ExtWT
protein produced robust neurite extension. More important, the vigorous
biological activity of the ExtWT protein in PC12 cells did not seem to
be caused by decreased GTPase activity or altered interactions with
guanine nucleotide exchange factors or GTPase-activating proteins,
which regulate Ha-Ras/GTP binding, as the ExtWT protein was GDP-bound
to the same extent as the normal cellular form.
The ExtRas proteins appear to be a new type of activated Ha-Ras because
activation has occurred 1) by a mechanism that does not involve changes
in GTP binding or hydrolysis and 2) through alteration of a domain
previously not considered to be directly involved in signal
transduction. The exaggerated activity of the Ext61L protein further
emphasizes that mutation of the C terminus can have a direct
(stimulatory) effect on two types of Ha-Ras biological activity. These
results suggest an expanded model for Ras activation: that functional
interactions of the ExtRas proteins may be controlled, in addition to
the GTP-sensitive switch regions of the protein, by the C-terminal
domain. Studies with the ExtRas proteins will be valuable for
determining how this activation arises and if Ha-Ras with its natural
lipid modifications also utilizes the C-terminal domain and its
attached lipids in an active role in Ras-mediated signal transduction.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Post-translational processing of Ext61L.
A, shown are the amino acid sequences of the C termini of
the Ha-Ras61L and Ext61L proteins. B, NIH 3T3 cells
transfected with Ext61L or empty vector (mock) were
metabolically labeled with [35S]methionine/cysteine for
18 h in the presence or absence of 50 µM compactin.
The cell lysate was then separated into crude soluble (S)
and particulate (P) fractions. Labeled proteins were
immunoprecipitated from each fraction, resolved by SDS-PAGE, and
detected by fluorography. Endogenous Ha-Ras proteins are indicated by
the arrowhead. C, parallel cultures of COS cells
expressing Ha-Ras61L (lane 1) or Ext61L (lane 2)
were metabolically labeled with [3H]mevalonic acid for
18 h in the presence of 50 µM compactin.
Immunoprecipitates were resolved by SDS-PAGE and transferred to
polyvinylidene difluoride membrane, and radiolabel was detected by
fluorographic exposure for 14 days.
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Fig. 2.
Ext61L binds membranes rapidly and is located
on plasma membranes of PC12 cells. A, COS-1 cells
transfected with empty vector (Mock) or expressing the
Ext61L protein were labeled with [35S]methionine/cysteine
for 10 min and then either lysed immediately (0-min chase) or incubated
in nonradioactive medium for an additional 30 min. The resulting
lysates were separated into soluble (S) and particulate
(P) fractions; immunoprecipitates were formed with Ha-Ras
antibody; and proteins were resolved by SDS-PAGE. Labeled proteins were
detected by fluorography. The culture used to prepare the sample for
the 30-min chase point for Ext61L had fewer cells present than the
cultures used for the 0-min chase points. The arrowhead
designates endogenous Ha-Ras; the arrow indicates the Ext61L
protein. B, PC12 cells were plated on serum-coated
coverslips and transfected with 1 µg of Ext61L DNA. After 2 days, the
cells were fixed and permeabilized and then treated with anti-Ras
monoclonal antibody Y13-172 and fluorescein isothiocyanate-conjugated
secondary antibody. Cells were viewed by confocal fluorescence
microscopy. Scale bar = 40 µm. Displayed is a
representative cell with multiple membrane projections and outgrowths
frequently observed in PC12 cells expressing Ext61L. More than 100 cells that possessed neurite extensions were surveyed, and all
possessed similar plasma membrane-specific distribution of Ext61L. The
signal from endogenous Ha-Ras in untransfected cells was too low to be
detected (see Fig. 5a for morphology).
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Fig. 3.
Ionic and hydrophobic contributions to
membrane binding of Ext61L. A, untransfected and transfected
COS cells were separated into crude soluble (S1) and
particulate fractions. Particulate proteins were further separated by
two sequential incubations in 0.5 M NaCl and centrifugation
to generate salt-released proteins (S2 and S3).
Proteins that were not released by the salt washes were extracted by
suspension in a buffer containing SDS and Nonidet P-40 and separated a
final time into detergent-soluble (S4) and -insoluble (P4,
not shown) fractions. Ha-Ras proteins were immunoprecipitated from each
fraction, resolved by SDS-PAGE, and detected by immunoblotting. The
arrowhead designates endogenous Ha-Ras. B, at 4 days post-transfection, PC12 cells transfected with 1 µg of
Ha-Ras61L, 50 ng of Ext61L, 1 µg of Ext61L(C181S/C184S/C186S), or 1 µg of ExtWT(C181S/C184S/C186S) DNA were separated into cytosolic
(S) and particulate fractions. The particulate fraction was
further subjected to Triton X-114 partitioning. All fractions were
precipitated in cold acetone and resuspended in 100 µl of
electrophoresis sample buffer, and equal portions of the cytosolic
(S), aqueous (A), or detergent (D)
phase were resolved by SDS-PAGE. Ha-Ras proteins were detected by
immunoblotting. Ext61L is indicated by the arrow. The
arrowhead marks the mature form of 61L in the detergent
fraction, and the asterisk marks the precursor form of 61L
in the cytosolic fraction.
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Fig. 4.
Ext61L is palmitoylated and displays normal
palmitate turnover rates. G418-selected NIH 3T3 cell lines
expressing either Ha-Ras61L or Ext61L proteins were labeled for 4 h with [3H]palmitate and then incubated for the indicated
times in nonradioactive medium containing 200 µM
nonradioactive palmitate. The Ext61L proteins were immunoprecipitated
and resolved by SDS-PAGE, and incorporated [3H]palmitate
was detected by fluorography. The Ras proteins on the membrane were
then detected by immunoblotting (data not shown) to allow
[3H]palmitate incorporation to be normalized for protein
recovery.
Transformation of NIH 3T3 cells by ExtRas proteins
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Fig. 5.
ExtRas proteins cause neurite outgrowth in
PC12 cells. PC12 cells were transfected with the indicated amounts
of various Ras DNAs: a, parental cells; b, 10 µg of p21WT; c, 1 µg of Ext61L(C181S/C184S/C186S);
d, 1 µg of ExtWT(C181S/C184S/C186S); e, 1 µg
of Ha-Ras61L; f, 10 ng of Ext61L; g, 200 ng of
ExtWT. Cells were photographed using phase-contrast optics 4 days
post-transfection. Scale bars = 50 µm. On day 2, when
outgrowths could be clearly assigned to an individual cell body, cell
images were captured on computer, and extensions on 50 transfected
cells expressing either Ha-Ras61L or Ext61L were measured. A
successfully transfected cell was defined as a cell having a flattened
and adherent cell body and at least one visible outgrowth.
Cotransfections of Ext61L and -galactosidase expression plasmids
showed that >70% of the cells were transfected and that >95% of
these transfected cells possessed neurites. Ext61L cells had an average
of 3.4 extensions/cell, and 84% of these extensions were longer than
100 µm. For cells expressing Ha-Ras61L, the average number of
outgrowths was 2.0, but only 21% of these were longer than 100 µm.
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Fig. 6.
Identification of the nucleotide bound to
ExtRas proteins. Transfected PC12 cells were radiolabeled with
32Pi for 4 h, and Ha-Ras was collected by
immunoprecipitation. Protein-associated radioactive GTP and GDP were
released, separated by thin-layer chromatography, and detected by
autoradiography. The data were quantitated by densitometry, and the
ratio of GTP to the total of GTP plus GDP was determined. Lane
1, Ha-Ras61L; lane 2, Ext61L;
lane 3, ExtWT.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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The technical assistance of Jenny Dedek, Allison Friedlein, and Kelly Welch is gratefully acknowledged. We thank Philip Haydon for help with the confocal immunofluorescence studies, J. Pate Skene for the PC12 cells, Pat Casey for critical reading of the manuscript, and especially Robert Deschenes for helpful comments and continuing interest.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant CA51890 and by the Roy J. Carver Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry, Biophysics, and Molecular Biology, 3212 Molecular Biology Bldg., Iowa State University, Ames, IA 50011. Tel.: 515-294-6125; Fax:
515-294-0453; E-mail: jbuss{at}iastate.edu.
The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.
2 M. A. Booden, unpublished data.
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REFERENCES |
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