The Role of cAMP-dependent Signaling in
Receptor-recognized Forms of
2-Macroglobulin-induced Cellular Proliferation*
Uma Kant
Misra
,
Gamal
Akabani§, and
Salvatore Vincent
Pizzo¶
From the Department of Pathology and
§ Department of Radiology, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, April 12, 2002, and in revised form, July 2, 2002
 |
ABSTRACT |
Ligation of 
2-macroglobulin
receptors by receptor-recognized forms of
2-macroglobulin (2M*) activates various
signaling cascades and promotes cell proliferation. It also elevates
cAMP in murine peritoneal macrophages. We now report that a significant elevation of cAMP-response element-binding protein (CREB) occurs in
2M*-stimulated cells, and this effect is potentiated by
isobutylmethylxanthine, dibutyryl-cAMP, or forskolin. An
2M* concentration-dependent rapid increase
in phosphorylated CREB at Ser133 also occurred, a necessary
event in its activation. Inhibition of Ca2+/calmodulin
kinase, protein kinases A and C, tyrosine kinases, ribosomal S6 kinase,
farnesyl transferase, extracellular signal-regulated kinases 1/2,
phosphatidylinositol 3-kinase, or p38 mitogen-activated protein kinase
markedly reduce 2M*-induced phosphorylation of CREB,
indicating a role for the p21ras-dependent and
phosphatidylinositol 3-kinase signaling pathways in regulating
CREB activation by 2M*. Finally, silencing the CREB gene
by transfecting cells with a homologous gene sequence double-stranded RNA drastically reduced the expression of CREB and blocked the ability of 2M* to promote macrophage
cell division. We conclude that cAMP-dependent signal
transduction as well as other signaling cascades are essential for
2M*-induced cell proliferation.
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INTRODUCTION |
2-Macroglobulin
(2M)1 is part
of a large superfamily that includes proteinase inhibitors and
complement components (1). 2M is a homotetramer, and,
like C3 and C4, each subunit contains a 
-cysteinyl-
-glutamyl
thiolester (2, 3). Upon reaction of 2M with proteinases,
the thiolesters rupture, and the molecule undergoes a large
conformational change (2, 3). This exposes a cryptic determinant
located in the carboxyl-terminal domain of each subunit, which
constitutes the receptor recognition site (2, 3). Direct reaction of
the thiolesters with small nucleophiles, such as NH3 or
CH3NH2 also triggers exposure of the receptor
recognition sites (2, 3). 2M* binds to the low density
lipoprotein receptor-related protein and to the 2M
signaling receptor (2MSR), which appears to consist of a
coreceptor complexed to lipoprotein receptor-related protein (4-11).
Binding of 2M* to 2MSR activates a
pertussis toxin-insensitive phospholipase C, which hydrolyzes membrane
phosphoinositides, generating two second messengers, inositol
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).
IP3 raises cytosolic free Ca2+,
[Ca2+]i, by releasing Ca2+
sequestered in the endoplasmic reticulum, thus triggering the onset of
several Ca2+-dependent signaling cascades
(4-13). DAG, on the other hand, activates protein kinase C (PKC), thus
triggering the activation of phosphorylation-dependent
signaling components. Ligation of 2MSR induces DNA and
protein synthesis, which is Ca2+-dependent and
requires participation of activated tyrosine kinases, p21ras-dependent MAPK, and PI 3-kinase signaling
cascades (4-18, 12-17). Treatment of macrophages with
2M* also causes a 2-2.5-fold increase in cell number
(10).
cAMP-response element-binding protein (CREB) is a nuclear transcription
factor which is a downstream target of cAMP signaling (18, 19). Protein
kinase A (PKA) phosphorylates CREB at Ser-133 within the
kinase-inducible domain (18, 19). This increases its transcriptional
activity by promoting its association with CREB-binding protein,
leading to activation of the transcriptional machinery. CREB also can
be phosphorylated at Ser-133 by multiple signaling mechanisms including
ERK 1/2, PKC, Ca2+/calmodulin-dependent protein
kinases, p38 MAPK, and ribosomal S6 kinase (p70s6k) (18-27). MAPKs
activate CREB kinase (p90s6k), which in turn phosphorylates and
activates CREB. To elucidate the role of cAMP signaling in cellular
physiology and homeostasis, several studies have used genetic
manipulations in intact animals and cell systems. These include gene
knockout and gene overexpression. In the last several years, the use of
posttranscriptional gene silencing and RNA interference techniques have
been employed to block protein expression in a variety of in
vitro systems (28-36). The techniques of RNA interference employ
sequence-specific post-translational gene silencing in animals and
plants initiated by double-stranded RNA that is homologous in sequence
to the silenced gene (28-36). The mediators of sequence-specific
messenger RNA degradation are 21-23-nt small interfering RNA fragments
generated by ribonuclease III cleavage from longer double-stranded RNAs
(dsRNAs) (31, 32). To date, these techniques have not been employed
with primary macrophages.
2M* binding to macrophages significantly raises cAMP
levels (12); therefore, we examined the role of CREB in
2M*-induced macrophage proliferation. We studied
phosphorylation of CREB and the protein kinases involved in its
phosphorylation, and analyzed Ras family members in murine macrophages
treated with either 2M* or cAMP-elevating agents. We
report in the current study that treatment of macrophages with
2M* elevated the levels of CREB as well as
phosphorylated CREB and caused a 1.5-2-fold increase in macrophage
cell number at 24 h of incubation. These effects were potentiated
by dibutyryl-cAMP, IBMX, or forskolin. The maximal phosphorylation of
CREB in 2M*-treated cells occurred at ~10-20 min of
incubation. 2M* elevated phosphorylation of ERK 1/2 and other MAPKs as well as Rap-1, Raf-1, Raf-B, and p70s6k protein levels.
These effects were potentiated by dibutyryl-cAMP or forskolin treatment
of cells. Pharmacological intervention with agents that affect various
protein kinases affected phosphorylation of CREB, [3H]thymidine incorporation, and cellular growth. To
elucidate further the role of CREB, the target of cAMP signaling, in
the proliferation of 2M*-stimulated peritoneal
macrophages, we have transfected macrophages with dsRNA homologous in
sequence to the CREB gene and have measured various parameters of cell
macrophage proliferation. To our knowledge, this is the first use of
RNA interference in a primary cell line; we find that silencing of the
CREB gene in these 2M*-stimulated cells drastically
reduces cell proliferation. We thus show here that the mitogenic and
cell proliferative responses of murine peritoneal macrophages treated
with 2M* are primarily mediated by cAMP and
cAMP-dependent signaling cascades.
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EXPERIMENTAL PROCEDURES |
Materials--
Culture media were from Invitrogen.
Dibutyryl-cAMP, fatty acid-free bovine serum albumin (BSA), and
actinomycin D were from Sigma. Forskolin, IBMX, PD98059, SB203580,
wortmannin, LY294002, chelerythrin, genistein, rapamycin,
manumycin A, H-89, KN-62, and cycloheximide were procured from Biomol
(Plymouth Meeting, PA). [3H]thymidine (specific activity,
71.5 Ci/mmol) was from American Radiochemicals, Inc. (St. Louis, MO).
Antibodies against CREB, Rap-1, Raf-1, Raf-B, p70s6k, Grb2, Sos
1/2, and Shc were from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Antibodies against CREB phosphorylated at Ser-133 and
phosphorylated ERK 1/2, p38 MAPK, and JNK were procured from Cell
Signaling Technology, Inc. (Beverly, MA). Antibodies against
thymidylate synthase were procured from Zymed Laboratories, Inc. (South
San Francisco, CA). LipofectAMINE was procured from Invitrogen.
2M* was prepared as described previously (4-7). Other
reagents of the highest available grade were procured locally.
Determination of CREB by Western Blotting in Macrophages Treated
with 2M* and cAMP-elevating Agents--
This protocol
has been described in detail elsewhere (4, 5, 12). In brief,
macrophages (2 × 106 cells/well) were incubated
overnight in RPMI 1640 medium containing 0.2% fatty acid-free BSA. The
cells were washed twice with HHBSS, and a volume of medium was added,
followed by the additions of 2M* (100 pM),
dibutyryl-cAMP (1 mM), forskolin (20 µM), and
IBMX (100 µM), either alone or in combination with
2M* in separate experiments. The cells were incubated
for 20 min at 37 °C in a humidified CO2 (5%) incubator.
The reaction was terminated by aspirating the medium. The monolayers
were washed once with cold HHBSS, and the cells were lysed in lysis
buffer containing 20 mM Tris·HCl (pH 8.6), 0.1 M NaCl, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 0.5% Nonidet P40 for 10 min on ice (11). The
DNA strands were broken by passing the lysate though a 27-gauge needle
and syringe several times. The lysate was centrifuged at 800 × g for 5 min at 4 °C to remove cell debris. The
supernatants were transferred to clean tubes, and their protein contents were determined (37). Equal amounts of all lysate proteins were used for electrophoresis according to Laemmli et al.
(38). Proteins from the gel (10%) were transferred to Hybond P®
membrane (Amersham Biosciences) and immunoblotted with antibody against CREB (1:2000) according to the manufacturer's instructions. CREB protein bands on the membrane were visualized by ECF (Amersham Biosciences) and quantified using a Storm® 860 PhosphorImager (Amersham Biosciences). Experiments were performed to determine the effects of modulating of 2M*-induced CREB expression
by actinomycin D (5 µg/ml, 10 min), a transcriptional inhibitor, or
BAPTA/AM (10 µM/30 min), a chelator of intracellular
Ca2+. For these studies, compounds were added to separate
wells that were incubated for the specified time period at 37 °C
before adding 2M* (100 pM). Other details of
quantifying CREB were as described above.
Quantification of CREB Phosphorylated at Ser-133 by Western
Blotting in Macrophages Treated with 2M* and
cAMP-elevating Agents--
Phosphorylation of CREB protein at Ser-133
was measured using a specific antibody that detects CREB phosphorylated
at this residue. Macrophages (2 × 106 cells/well)
were incubated overnight in RPMI 1640 medium with 0.2% fatty acid-free
BSA. The cells were washed twice with HHBSS, and a volume of medium was
added, followed by the additions of 2M* (100 pM), dibutryrl-cAMP (1 mM), forskolin (20 µM), or IBMX (100 µM), either alone or in
combination with 2M* in separate experiments. The cells
were incubated for 20 min at 37 °C in a humidified incubator with
CO2 (5%). Other details of cell lysis, electrophoresis,
and membrane transfer were as described above. The membrane was
immunoblotted with antibody against phosphorylated CREB (1:3000
dilution), and phosphorylated CREB spots on the membranes were
visualized by ECF and quantified by a PhosphorImager. In experiments
where the effect of time of incubation of cells with 2M*
on CREB phosphorylation at Ser-133 was examined, the cells (2 × 106 cells/well) were treated with 2M* (100 pM) and incubated as above. At the specified times, the
reaction was terminated by aspirating the medium, and the cells were
washed with cold HHBSS and treated with lysis buffer as described
above. Other details of quantifying phosphorylated CREB are described above.
Modulation of CREB Phosphorylation by Protein Kinases in
Macrophages Treated with 2M* and cAMP-elevating
Agents--
CREB is the immediate downstream target protein of PKA,
which phosphorylates it at Ser-133; however, CREB phosphorylation at
Ser-133 is also brought about by other kinases as noted above (18-27).
We have assessed the involvement of these kinases in the phosphorylation of CREB at Ser-133 in agonist-stimulated cells by using
pharmacological interventions. Macrophages (2 × 106
cells/well) were incubated overnight in RPMI 1640 medium with 0.2%
fatty acid-free BSA. The cells were washed twice with HHBSS, and a
volume of medium was added, followed by the additions of the following
in respective wells: H-89, a PKA inhibitor (10 µM/90 min)
(39); KN-62, a specific inhibitor of Ca2+/calmodulin
kinases (1 µM/15 min) (40); PD98059, a specific inhibitor
of ERK 1/2 (50 µM/90 min) (41); genistein, a specific inhibitor of tyrosine kinases (40 µM/2 h) (42); SB
203580, a specific inhibitor of p38 MAPK (15 µM/15 min)
(43); wortmannin, a specific inhibitor of PI 3-kinase (30 nM/30 min) (44); manumycin A, a specific inhibitor of
farnesyl transferase (10 µM/60 min) (45); and
chelerythrin, a specific inhibitor of PKC (200 nM/15 min)
(46). The cells were incubated for the specified time at 37 °C in an
incubator. At the end of the incubation, 2M* (100 pM), dibutyryl-cAMP (1 mM), forskolin (20 µM), or IBMX (100 µM), either alone or in
combination with 2M*, were added to separate wells, and
the incubation continued for an additional 20 min as above. Other
details of cell lysis, electrophoresis, and membrane transfer were as
described above. The membrane was immunoblotted with antibody against
phosphorylated CREB (1:3000 dilution), and phosphorylated CREB was
visualized by ECF and quantified by a PhosphorImager.
Measurement of [3H]Thymidine Uptake by Macrophages
Exposed to Forskolin--
Murine peritoneal macrophages (4 × 105 cells/well) in 48-well plates, harvested as above, were
allowed to adhere for 2 h in RPMI 1640 medium containing 0.2%
fatty acid-free BSA, penicillin, streptomycin, and glutamine at
37 °C in a humidified CO2 (5%) incubator. The
monolayers were washed twice with HHBSS, and a volume of RPMI medium
was added, followed by the addition of [3H]thymidine. To
the respective wells, 2M* (100 pM) or
forskolin (20 µM) either alone or together were added. In
experiments where the effect of KN-62 (1 µM/15 min),
rapamycin (50 nM/15 min), PD98059 (50 µM/90
min), SB203580 (15 µM/30 min), manumycin A (10 µM/60 min), genistein (20 µM/16 h),
wortmannin (30 nM/30 min), LY294003 (25 µM/15
min), chelerythrin (200 nM/15 min), actinomycin D (5 µg/ml/10 min), cycloheximide (10 µg/ml/10 min), or BAPTA/AM (10 µM/30 min) were studied, these were added to their
respective wells, and cells were incubated for the specified time
before adding 2M* or forskolin. The cells were incubated
overnight in a humidified CO2 (5%) incubator. The
incubations were terminated by aspirating the medium and washing
macrophages twice first with 5% trichloroacetic acid (15 min/40 °C)
and then three times with HHBSS. The monolayers were lysed with 1 N NaOH, and an aliquot was used for liquid scintillation
counting and protein estimation (8, 10, 17).
Determination of Macrophage Cell Number--
Since increased DNA
synthesis is generally associated with an increase in total
cellularity, the number of macrophages present 0, 24, and 48 h
after exposure to 2M* (100 pM) or forskolin
(20 µM) either alone or together was determined.
Peritoneal macrophages were harvested and allowed to adhere in
four-well plates in RPMI 1640 medium containing 5% fetal bovine serum
for 2 h as described above. The adhered cells were carefully
scraped, centrifuged at 1200 rpm for 5 min, and suspended in a volume
of RPMI 1640 medium containing 0.2% fatty acid-free BSA, and 1-ml
aliquots (3 × 105 cells) were pipetted into 15-ml
siliconized polypropylene tubes. To the respective tubes,
2M* (100 pM), forskolin (20 µM), or 2M* (100 pM) with
forskolin (20 µM) was added, and the contents were gently
mixed and incubated for 24 and 48 h as above. After the specified
period of incubation, an aliquot was removed from each tube, trypan
blue was added, and the contents were gently shaken during incubation
for 2 min. A 10-µl aliquot was then employed for counting the number
of cells in a hemocytometer. The cell numbers were corrected for dead
cells. Changes in the morphology and macrophage number before and after
treatment with 2M*, forskolin, or 2M*
with forskolin at 24 and 48 h were determined by phase-contrast microscopy. For these studies, an equal number of macrophages adhered
for 2 h were pipetted into six-well plates and incubated as above.
After the specified periods of incubation with the agents, the cells
were examined under a phase-contrast microscope (20×) (10, 47).
Western Blotting of Phosphorylated ERK 1/2, p38 MAPK,
and JNK in Macrophages Stimulated with 2M* and
Forskolin--
Freshly harvested peritoneal macrophages in RPMI 1640 medium containing glutamine, penicillin, streptomycin, and 5% fetal bovine serum were allowed to adhere in six-well plates (3 × 106 cells/well) for 2 h as above. The monolayers were
washed twice with HHBSS; a volume of RPMI 1640 medium containing 0.2%
fatty acid-free BSA was added; and the cells were treated with
2M* (100 pM/20 min), forskolin (20 µM/20 min), or 2M* with forskolin. The
incubations were terminated by aspirating the medium. The lysis of
cells, their electrophoresis, and Western blotting with respective
antibodies against phosphorylated ERK 1/2, p38 MAPK, or JNK were
performed according to the manufacturer's instructions. In each case,
an equal amount of protein was used for electrophoresis. The detection
of phosphorylated proteins by ECF and quantification of their
distribution were performed as above (10, 47).
Western Blotting of Grb2, Sos, and Shc Proteins in Macrophages
Exposed to 2M* and Forskolin--
These studies were
performed as described above. The detection of Grb2, Sos 1/2, and Shc
by ECF and quantification of their distribution were performed
by PhosphorImager.
Western Blotting of p70s6k, Rap-1, Raf-1, and Raf-B Proteins in
Macrophages Exposed to 2M* and Forskolin--
These
studies were performed as described above. The detection of p70s6k,
Raf-1, Rap-1, and Raf-B by ECF and quantification of their
distribution were performed by PhosphorImager.
Western Blotting of c-Fos Protein in Macrophages Treated
with 2M* and Forskolin--
These studies were
performed as described above. The detection of a c-Fos protein by ECF
and quantification of their distribution were performed by PhosphorImager.
Chemical Synthesis of dsRNA Homologous in Sequence to the Target
CREB Gene Sequence--
The chemical synthesis of dsRNA homologous to
the target mouse CREB gene sequence nucleotides 324-344
(5'-AAGAGACAACAGAGAATGATA-3'; SWISS-PROT, entry name ATFB MOUSE,
primary accession number 035451) was performed by Ambion (sequence ID
173; Austin, TX). For making dsRNA, the sense
(5'-GAGACAACAGAGAAUGAUtt-3') and antisense
(5'-UAUCAUUCUGUUGUCUCtt-3') oligonucleotides were annealed
according to the manufacturer's instructions. Throughout the entire
period of experimentation, handling of reagents was performed in an
RNase-free environment Briefly, equal amounts of sense and antisense
oligonucleotides were mixed and heated at 90 °C for 1 min and then
for 1 h at 37 °C in an incubator. The dsRNA preparation was
stored at 
20 °C before use.
Transfection of Murine Peritoneal Macrophages, Stimulation with
2M*, and Western Blotting of CREB and Thymidylate
Synthase Proteins--
TG-elicited murine peritoneal (1 × 106 cells/well in a six-well plate) were lavaged as above
and allowed to adhere for 2 h in RPMI 1640 medium containing 10%
fetal bovine serum, penicillin (12.5 units/ml), streptomycin (6.5 µg/ml), and 2 mM glutamine at 37 °C in a
CO2 (5%) humidified incubator at 37 °C. The nonadherent cells were aspirated, monolayers were washed twice with HHBSS, 2 ml of
DMEM containing 10% fetal bovine serum and the above antibiotics was
added, and cells were incubated as above for 16 h. For each transfection, 2 µg of dsRNA was diluted into 100 µl of serum-free DMEM in a tube. In another tube, 10 µl of LipofectAMINE was diluted into 100 µl of serum-free medium. The two solutions were combined, mixed gently, and incubated for 45 min at room temperature followed by
the addition of 800 µl of serum-free and antibiotic-free medium to
each tube. The monolayers were washed twice with serum-free DMEM,
layered in each well with 1 ml of LipofectAMINE-DMEM (10 µl/ml) or
lipid-dsRNA mixtures, containing different amounts of dsRNA, gently
mixed, and incubated for 5 h at 37 °C in a humidified CO2 incubator. At the end of the incubation, 1 ml of
antibiotic-free DMEM containing 10% fetal bovine serum was added to
each well, and cells were incubated for 16 h as above. Microscopic
observation of the monolayers did not show evidence of toxicity. The
medium was replaced with DMEM containing antibiotics and 10% fetal
bovine serum 24 h after the start of transfection. The monolayers
were washed with the above DMEM once, and a volume of the same medium was added to monolayers, followed by the addition of 100 pM
2M*. The cells were incubated for 15 min as above. The
reaction was terminated by aspirating the medium, and cells were lysed
in lysis buffer as above. The lysate was electrophoresed; the
protein was transferred to membrane and CREB, phosphorylated CREB, and
thymidylate synthase proteins on the membrane detected by Western
blotting with the respective antibodies in separate experiments; and
protein was quantified by a Storm® PhosphorImager as detailed above.
Measurements of Cell Proliferation of Transfected Cells
Stimulated with 2M*--
The details of cell culture
(5 × 105 cells/well in a six-well plate),
transfection, and stimulation of transfected cells with 2M* were identical to those described above. At the
specified periods of incubation at 37 °C in a humidified
CO2 (5%) incubator, the cells were examined under a
phase-contrast microscope (20×) for changes in cell morphology. The
cells were detached with trypsin-EDTA (0.5%) and centrifuged, the
pellet was washed with DMEM, and cells were suspended in the same
medium. An aliquot was removed from each tube, trypan blue was added,
and the contents were gently shaken during incubation for 2 min. A
10-µl aliquot was used for counting the number of cells in a
hemocytometer. The cell numbers were corrected for dead cells (10).
| |
RESULTS |
2M* and cAMP-elevating Agents Increase CREB in
Macrophages--
CREB is a member of a family of factors that regulate
transcription by binding to sequences in gene promoters (18, 19). CREB
is a downstream target once cAMP is elevated, and it becomes functionally activated upon phosphorylation at Ser-133 (18, 19).
2M* treatment of macrophages caused a 2-3-fold increase in CREB (Fig. 1). An additional increase
in CREB levels occurred when macrophages were stimulated with
2M* after pretreatment with dibutyryl-cAMP, IBMX, or
forskolin (Fig. 1). Chelation of intracellular Ca2+ with
BAPTA/AM or inhibition of transcription with actinomycin D markedly
attenuated 2M*-induced increase in CREB (Fig. 1).
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Fig. 1.
Levels of CREB protein in macrophages
stimulated with cAMP-elevating agents. CREB protein levels were
determined by Western blotting and quantified by a PhosphorImager as
described under "Experimental Procedures." Bar
1, buffer; bar 2, 2M*
(100 pM/20 min); bar 3,
dibutyryl-cAMP (1 mM/20 min); bar 4,
dibutyryl-cAMP and then 2M*; bar
5, IBMX (100 µM/20 min); bar
6, IBMX (100 µM/20 min) then
2M* (100 pM); bar 7,
forskolin (20 µM/20 min); bar 8,
forskolin and then 2M*; bar
9, BAPTA/AM (10 µM/30 min) and then
2M*; bar 10, actinomycin D (5 µg/ml/10 min) and then 2M*. A representative Western
blot is shown at the bottom of the corresponding
bar graph. Values are mean ± S.E. from
three or four independent experiments performed in triplicate and are
expressed as arbitrary units.
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Ligation of 2MSR with 2M* Increases
CREB Phosphorylation--
We next demonstrated that treatment of
macrophages with 2M* promoted phosphorylation of Ser-133
in CREB. The effect of stimulating macrophages with increasing
concentrations of 2M* (0-20 nM) on the
levels of phosphorylated CREB is shown in Fig.
2B. The maximal phosphorylation of CREB occurred at 2M* concentrations
of 50-100 pM (Fig. 2B). Stimulation of
macrophages with 2M* after pretreatment with
dibutyryl-cAMP, IBMX, or forskolin elevated the levels of CREB
phosphorylated at Ser-133 significantly when compared with buffer-treated cells (Fig. 3).
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Fig. 2.
Effect of
2M* concentration and time of
incubation on the formation of CREB phosphorylated at Ser-133 in
macrophages. The levels of phosphorylated CREB were determined by
Western blotting and quantified by a PhosphorImager. A,
effect of time of incubation with 2M* (100 pM) on phosphorylated CREB (CREB-P) formation.
B, effect of concentration of 2M* on
phosphorylation of CREB. Representative corresponding Western blots are
shown below the respective line
graphs. Values are mean ± S.E. from three independent
experiments performed in triplicate and are expressed as arbitrary
units.
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Fig. 3.
Levels of phosphorylated CREB in macrophages
stimulated with different cAMP-elevating agents. The levels of
phosphorylated CREB were determined by Western blotting using
antibodies against Ser-133-phosphorylated CREB and quantified by a
PhosphorImager. Bar 1, buffer; bar
2, 2M* (100 pM); bar
3, dibutyryl-cAMP (1 mM); bar
4, dibutyryl-cAMP and then 2M*;
bar 5, IBMX (100 µM);
bar 6, IBMX and then 2M*;
bar 7, forskolin (20 µM);
bar 8, forskolin and then 2M*. A
representative Western blot is shown at the bottom of the
corresponding bar graph. Values are mean ± S.E. from 3-4 independent experiments performed in triplicate and are
expressed as arbitrary units.
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Modulation of CREB Phosphorylation at Ser-133--
The maximal
increase in cAMP levels in 2M*-treated cells was
observed between 15 and 20 min after 2M* treatment (12); however, maximal CREB phosphorylation also occurred between 10 and 15 min (Fig. 2A). These data suggest that in addition to PKA, other protein kinases activated via
IP3-dependent signaling cascades functioning
before the levels of cAMP are elevated are involved in the
phosphorylation of CREB (18-27). We therefore next studied the
modulation of CREB phosphorylation. Chelation of
intracellular Ca2+ with BAPTA/AM prevented agonist-induced
CREB phosphorylation (Fig.
4A). Treatment of macrophages
with chelerythrin, a specific inhibitor of PKC, with H-89, a specific
inhibitor of PKA, or with KN-62, a specific inhibitor of
Ca2+/calmodulin kinase, before stimulation with
2M* nearly abolished 2M*-induced CREB
phosphorylation at Ser-133 (Fig. 4A). Likewise, treatment of
macrophages with PD98059, a specific inhibitor of ERK 1/2, with
SB203580, a specific inhibitor of p38 MAPK, with genistein, a specific
inhibitor of tyrosine kinases, or manumycin A, a specific inhibitor of
farnesyl transferase, before stimulation with 2M*
significantly inhibited CREB phosphorylation compared with
buffer-stimulated cells (Fig. 4B).
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Fig. 4.
Effect of various protein kinase inhibitors
on 2M*-induced levels of
Ser-133-phosphorylated CREB in macrophages. The levels of
phosphorylated CREB were determined by Western blotting using
antibodies against Ser-133-phosphorylated CREB and quantified by a
PhosphorImager. Representative corresponding Western blots are shown
below their respective bar graphs.
A, bar 1, buffer; bar
2, 2M* (100 pM/20 min);
bar 3, chelerythrin (200 nM/15 min)
and then 2M*; bar 4, H-89 (10 µM/90 min) and then 2M*; bar
5, KN-62 (1 µM/1 h) and then
2M*; bar 6, BAPTA/AM (10 µM/30 min) and then 2M*; bar
7, okadaic acid (50 nM/15 min) and then
2M*. B, bar 1, buffer;
bar 2, 2M* (100 pM);
bar 3, PD98059 (50 µM/90 min) and
then 2M*; bar 4, SB 203580 (25 µM/30 min) and then 2M*; bar
5, wortmannin (30 nM/30 min) and then
2M*; bar 6, LY294002 (15 µM/15 min) and then 2M*; bar
7, genistein (20 µM/16 h) and then
2M*; bar 8, manumycin A (15 µM/60 min) then 2M*. Values are means ± S.E. from three independent experiments performed in triplicate
expressed as arbitrary units.
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2M* and Forskolin Treatment of Cells Elevates the
Levels of Phosphorylated ERK 1/2, p38 MAPK, JNK, and p70s6k
Protein--
Both 2M* and forskolin raised levels of
phosphorylated ERK 1/2 (Fig.
5A), phosphorylated p38 MAPK
(Fig. 5B), and phosphorylated JNK (Fig. 5C) by
about 1.5-2-fold. Cell stimulation with 2M* or
forskolin also raised the levels of ribosomal kinase p70s6k (Fig.
5D).
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Fig. 5.
Effect of
2M* and forskolin on the levels of
signaling cascade components downstream from Ras. See
"Experimental Procedures" for details. The components were detected
by Western blotting and quantified by a PhosphorImager. A,
changes in the levels of phosphorylated ERK 1/2. Bars
1, buffer; bar 2, 2M*
(100 pM); bar 3, forskolin (20 µM); bar 4, forskolin and then
2M*. B, changes in the levels of
phosphorylated p38 MAPK. Bars 1, buffer;
bar 2, 2M* (100 pM);
bar 3, forskolin (20 µM);
bar 4, forskolin and then 2M*.
C, changes in the levels of phosphorylated JNK.
Bar 1, buffer; bar 2,
2M* (100 pM); bar 3,
forskolin (20 µM); bar 4, forskolin
and then 2M*. D, changes in the levels of
p70s6k protein. Bar 1, buffer; bar
2, 2M* (100 pM); bar
3, forskolin (20 µM); bar
4, forskolin and then 2M*. The values are
expressed in arbitrary units and are mean ± S.E. from three
individual experiments performed in triplicate.
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2M* and Forskolin Elevate
[3H]Thymidine Uptake into DNA--
We studied the
contribution of cAMP signaling to 2M*-induced cell
proliferation and DNA synthesis by quantifying the incorporation of
[3H]thymidine into DNA. We also determined macrophage
cell number and studied the morphology of cells treated as above and
incubated for 24 and 48 h under identical conditions. We compared
these effects with those induced by forskolin, an established
cAMP-elevating agent (Figs.
6-8).
2M* stimulation of macrophages, like forskolin, increased [3H]thymidine uptake by about 2-fold as
compared with buffer-treated cells (Fig. 6A). When
macrophages were stimulated with both 2M* and forskolin,
the [3H]thymidine uptake was nearly additive (Fig.
6A). The [3H]thymidine incorporation into DNA
of macrophages stimulated with 2M* and forskolin was
significantly reduced by pretreating the cells with KN-62, an inhibitor
of Ca2+/calmodulin kinase (40); rapamycin, an inhibitor of
p70s6k (48); PD98059, an inhibitor of ERK 1/2; SB 203580, an inhibitor
of p38 MAPK; manumycin A, an inhibitor of farnesyl transferase required for membrane attachment of Ras; genistein, an inhibitor of tyrosine kinases, chelerythrin, an inhibitor of PKC; BAPTA/AM, a chelator of
intracellular Ca2+; actinomycin D; and cycloheximide (Fig.
6B). These results suggest that cAMP-dependent
signaling as well as the p21ras- and PI
3-kinase-dependent pathways (9, 10, 12) are involved in
2M*-induced macrophage proliferation.
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Fig. 6.
Effect of cAMP-elevating agents on
[3H]thymidine incorporation into DNA and its modulation
by inhibitors of protein kinases. Experimental details are
described under "Experimental Procedures." A,
bar 1, buffer; bar 2,
2M* (100 pM); bar 3,
dibutyryl-cAMP (1 mM); bar 4,
dibutyryl-cAMP and then 2M*; bar
5, forskolin (20 µM); bar
6, forskolin and then 2M*; bar
7, H-89 (10 µM/2 h) and then forskolin;
bar 8, KN-62 (1 µM/1 h) and then
forskolin. B, bar 1, buffer;
bar 2, forskolin (20 µM/25 min);
bar 3, rapamycin (100 nM/20 min) and
then forskolin; bar 4, wortmannin (30 nM/30 min) and then forskolin; bar 5,
PD98059 (50 µM/90 min) and then forskolin; bar
6, BAPTA/AM (10 µM/30 min) and then forskolin;
bar 7, chelerythrin (200 nM/20 min)
and then forskolin; bar 8, genistein (20 µM/16 h) and then forskolin; bar 9,
actinomycin D (5 µg/ml/20 min) and then forskolin; bar
10, cycloheximide (10 µg/ml/20 min) and then forskolin;
bar 11, SB203580 (20 mM/30 min) and
then forskolin; bar 12, manumycin A (20 µM/60 min) and then forskolin. The values are means ± S.E. from two independent experiments performed in quadruplicate and
are expressed as fmol of [3H]thymidine uptake/mg of
protein. [3H]Thymidine incorporation in macrophages
treated with various inhibitors only was either equal to the basal
uptake or slightly lower.
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Fig. 7.
Effect of
2M* and forskolin on macrophage
proliferation. To an equal number of macrophages in suspension in
respective culture tubes were added buffer (A);
2M* (100 pM) (B); forskolin (20 mM) (C), and forskolin and then
2M* (D). The cells were incubated, and cell
density and cellular morphology were photomicrographed at 24 and 48 h of incubation as described under "Experimental
Procedures."
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Fig. 8.
The effect of
2M* and forskolin on macrophage cell
number. Cells were incubated with buffer (bar 1),
2M* (100 pM) (bar 2),
forskolin (20 µM) (bar 3), and
forskolin and then 2M* (bar 4) for
0 h (open); 24 h (closed), and 48 h (stippled). At the respective periods of incubation, an
aliquot of cell suspension was counted for cell numbers by
hemocytometer.
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[3H]Thymidine uptake may indicate enhanced DNA synthesis,
but there are potential mechanisms of enhanced uptake independent of
new synthesis of nucleic acids. We therefore also studied the effect of
2M*, dibutyryl-cAMP, and forskolin on cell morphology (Fig. 7) and macrophage cell number (Fig. 8 and Table
I) at 24 and 48 h of incubation.
Like [3H]thymidine uptake, macrophages treated with
2M* or forskolin showed a 1.5-2-fold increase in cell
numbers compared with buffer-stimulated cells at 24 h (Fig. 8 and
Table I). The decrease in cell numbers observed at 48 h of
incubation was largely due to cell death as evident by increased trypan
blue uptake (Fig. 7 and Table I). Pretreatment of cells treated with
H-89 or KN-62 inhibited 2M*- or forskolin-induced
increase in cell number (Table I). The 2M*- or
forskolin-treated macrophages showed increased numbers, were enlarged,
and exhibited a stellate morphology at 24 h compared with
buffer-treated macrophages, but by 48 h the cell number decreased drastically, and morphology also changed (Figs. 7 and 8 and Table I).
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Table I
Effect of cAMP-elevating agents on 2M*-induced macrophage
proliferation
Two studies were performed in triplicate.
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2M* and Forskolin Induce Expression of the c-fos
Gene--
Expression of c-fos is part of a mitogenic
response that is required for cell proliferation. Transcription of the
c-fos gene is regulated in part by CREB (18, 19). Increased
[Ca2+]i can activate c-fos
transcription through CREB phosphorylation. To understand the role of
cAMP signaling in early response gene expression, we quantified the
expression of the c-Fos protein by Western blotting in
macrophages stimulated with 2M* or forskolin (Fig.
9A). Both 2M*
and forskolin, which increased levels of phosphorylated CREB (Fig. 3),
also increased the levels of c-Fos protein by about 2-fold compared
with buffer-treated cells (Fig. 9A).
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Fig. 9.
Effect of
2M* and forskolin on the levels of
c-Fos, Grb2, Sos 1/2, and Shc in macrophages. A, c-Fos;
B, Grb2; C, Sos 1/2; D, Shc proteins
in macrophages. The proteins were detected by Western blotting and
quantified by a PhosphorImager as described under "Experimental
Procedures." All panels, bar
1, buffer; bar 2, 2M*
(100 pM); bar 3, forskolin (20 µM); bar 4, forskolin and then
2M*. The corresponding gel blots are shown at the
bottom of the respective bar graphs.
The values are expressed in arbitrary units and are the means ± S.E. from two or three independent experiments performed in
triplicate.
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2M* and Forskolin Elevate the Levels of Grb2, Sos,
Shc, and the Small G Protein Rap-1 in Macrophages--
Receptor
tyrosine kinases propagate intracellular signals by coupling to
multiple signal transduction pathways. Many of these pathways are
mediated by interactions with SH2 and SH3 domain-containing proteins
(49-51). Molecules implicated in signal transduction pathways containing the SH2 domain include phospholipase C, PI 3-kinase, and
GTPase-activating proteins of Ras (51-53). Ras plays a central role in
signaling a variety of cellular responses including cell proliferation
and differentiation (51, 53-56). Ras is connected to receptor tyrosine
kinase through adaptor protein Grb2, containing two SH3 domains and one
SH2 domain, and Sos, a guanine nucleotide exchange factor (51, 53-56).
The SH2 domain of Grb2 provides a site for interaction with
tyrosine-phosphorylated proteins, and Sos functions as an activator of
Ras (53-57). Another SH2 domain-containing docking protein,
Shc, primarily a cytosolic protein that becomes tyrosine-phosphorylated and translocates to membranes in response to
growth factors, associates with Grb2/Sos (58-61). Shc, therefore, could provide an alternative mechanism of coupling to Ras and may
amplify or modulate the signaling input from receptor tyrosine kinases
to Ras. To understand the mechanism of cAMP-induced proliferation of
macrophages, under our experimental conditions, we assayed the levels
of Grb2, Sos, Shc, Raf-1, Rap-1, and Raf-B by Western blotting (Figs. 9
and 10). 2M* and
forskolin either alone or in combination increased the levels of Grb2,
Sos, and Shc (Fig. 9, B-D). We have shown earlier that
exposure of peritoneal macrophages to 2M* elevated the
levels of RAS·GT32P by about 2-2.5-fold (16). In
the next series of experiments, we quantified the levels of signaling
components downstream to Ras, namely Raf-1, Rap-1, and Raf-B (Fig. 10,
A-C). Treatment of macrophages with 2M*
raised the levels of Raf-1, whereas forskolin treatment either alone or
with 2M* decreased Raf-1 expression (Fig. 10,
A and B). Since Ras and Raf-1 are physically
associated, one possible explanation for the decreased levels of Raf-1
in the forskolin group may lie in the decreased stability of Raf-1 due
its uncoupling from Ras as a result of PKA phosphorylation of Raf-1
(55, 62-67). Treatment of macrophages with 2M* raised the levels of Rap-1 and Raf-B by about 1.5-2-fold, but forskolin either alone or with 2M* raised the levels of Rap-1 by
about 4-5.5-fold (Fig. 10, B and C). These
results show that 2M*-induced macrophage proliferation
utilizes predominantly MAPK activation through Ras/Raf-1 signaling,
whereas forskolin-induced cell proliferation utilizes largely
Rap-1/Raf-B signaling for MAPK activation. Thus, 2M*
utilizes both the
IP3/Ca2+-dependent signaling (early
phase) as well as cAMP-dependent signaling (late phase) to
achieve mitogenesis and cell proliferation.
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Fig. 10.
Effect of
2M* and forskolin on the levels of
Raf-1, Rap-1, and Raf-B in macrophages. A, Raf-1;
B, Rap-1; C, Raf-B. The proteins were detected by
Western blotting and quantified by a PhosphorImager as described under
"Experimental Procedures." All panels,
bar 1, buffer; bar 2,
2M* (100 pM); bar 3,
forskolin (20 µM); bar 4, forskolin
and then 2M*. The representative corresponding gel blots
are shown at the bottom of the respective bar
graphs. The values are expressed in arbitrary units and are
the means ± S.E. from two or three independent experiments
performed in triplicate.
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Transfection of Cells with dsRNA Homologous in Sequence to CREB
Gene Blocks 2M*-induced Cell Proliferation--
At both
concentrations of dsRNA (10 and 50 µg/ml) employed, expression of the
CREB gene was significantly inhibited (70%) (Fig.
11), as was its phosphorylation (Fig.
12). Macrophages in which CREB gene
expression was silenced no longer were responsive to 2M*
stimulation with respect either to CREB level or its phosphorylation (Figs. 11 and 12). Similar results were observed when forskolin was
employed as a stimulant (data not shown). We next evaluated the role of cAMP-CREB signaling in 2M*-induced cell
proliferation in macrophages after silencing the CREB gene with
sequence-homologous dsRNA. Cell number was determined 24 h after
2M* treatment of transfected cells (Table
II). Transfection of cells with dsRNA (10 or 50 µg/ml) nearly abolished 2M*-induced cell
proliferation (Table II). Microscopic examination of the cells as well
as trypan blue uptake did not show toxic effects secondary to
transfection. Cells transfected with LipofectAMINE alone showed no
effects on cell morphology, cell shape, or spreading. In contrast,
cells transfected with the LipofectAMINE-dsRNA complex were largely round and showed no spreading (Fig.
13). These changes in cell morphology
are similar to those observed in other cell types transfected with
dsRNA (29-36). These results conclusively demonstrate that CREB and
cAMP signaling are of crucial importance in 2M*-induced proliferation of murine peritoneal macrophages.
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Fig. 11.
Effect of silencing CREB gene expression by
transfection with dsRNA on CREB expression in
2M*-stimulated cells. Experimental
details are described under "Experimental Procedures."
Bar 1, LipofectAMINE plus buffer; bar
2, LipofectAMINE plus 2M* (100 pM); bar 3, LipofectAMINE plus dsRNA
complex (10 µg/ml) plus 2M*; bar
4, LipofectAMINE plus dsRNA complex (50 µg/ml) plus
2M* (100 pM). The proteins were detected by
Western blotting and quantitated by a PhosphorImager as described
above. The corresponding gel blots are shown at the bottom
of the respective bar graphs. The values are
expressed in arbitrary units and are mean ± S.E. from two
experiments performed in triplicate.
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Fig. 12.
Effect of silencing the CREB gene by
transfection with dsRNA homologous in sequence to the target gene upon
phosphorylation of CREB protein (CREB-P) in
2M*-stimulated cells. Experimental
details are described under "Experimental Procedures."
Bar 1, LipofectAMINE; bar
2, LipofectAMINE plus 2M* (100 pM); bar 3, LipofectAMINE plus dsRNA
complex (10 µg/ml) plus 2M*; bar 4,
LipofectAMINE plus dsRNA complex (50 µg/ml) plus 2M*
(100 pM). The proteins were detected by Western blotting
and quantitated by a PhosphorImager as described above. The
representative corresponding gel blots are shown at the
bottom of the respective bar graphs.
The values are expressed in arbitrary units and are mean ± S.E.
from two experiments performed in triplicate.
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Table II
Effect of LipofectAMINE/LipofectAMINE-RNA complex on
2M*-stimulated macrophage cell number
Two studies were performed in triplicate.
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Fig. 13.
Morphological changes in macrophages before
and 24 h after transfection with dsRNA. A,
macrophages before transfection; B, macrophages transfected
with 50 µg of dsRNA for 24 h and then stimulated with
2M* (100 pM for 24 h). The
images shown are representative of two independent
experiments.
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Silencing the CREB Gene Blocks Up-regulation of Thymidylate
Synthase Induced by 2M*--
To further examine the
cell-proliferative role that CREB plays in macrophages stimulated with
2M*, we quantified the levels of thymidylate synthetase,
a critical enzyme involved in DNA synthesis under these experimental
manipulations (Fig. 14). An appreciable amount of thymidylate synthetase protein is observed in macrophage, and
2M* stimulation nearly doubles the amount of thymidylate synthase protein. However, thymidylate synthase protein is not up-regulated in 2M*-treated macrophages upon silencing
of the CREB gene with sequence-homologous dsRNA. Thymidylate synthase is clearly an important enzyme in regulating the intracellular thymidine pool necessary to provide precursors for DNA synthesis. These
results further suggest that CREB signaling is involved in
2M*-induced mitogenesis and cell proliferation.
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Fig. 14.
Effect of silencing the CREB gene by
transfection with dsRNA on thymidylate synthase protein in
2M*-stimulated cells. Experimental
details are described under "Experimental Procedures."
Bar 1, LipofectAMINE (10 µl/ml) plus buffer;
bar 2, LipofectAMINE (10 µl/ml) plus
2M* (100 pM); bar 3,
LipofectAMINE plus dsRNA complex (10 µg/ml) plus
2M*; bar 4, LipofectAMINE plus
dsRNA complex (50 mg/ml) plus 2M* (100 pM).
The proteins were detected by Western blotting and quantitated by a
PhosphorImager as described above. The corresponding gel blots are
shown at the bottom of the respective bar
graphs. The values are expressed in arbitrary units and are
the means ± S.E. from two experiments performed in
triplicate.
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Silencing of the CREB Gene with Sequence-homologous dsRNA Inhibits
2M*-induced c-Fos Expression--
In order to
investigate the relationship between CREB and c-Fos expression,
we examined the effect of silencing the CREB gene on the expression of
c-Fos in 2M* stimulated-macrophages. dsRNA treatment of macrophages profoundly reduced the expression of c-Fos
protein in 2M*-stimulated cells (Fig.
15). These results suggest that CREB
modulates c-Fos-mediated cellular events.
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Fig. 15.
Effect of silencing the
CREB gene by transfection with dsRNA on c-Fos protein in
2M*-stimulated cells. Experimental
details are described under "Experimental Procedures."
Bar 1, LipofectAMINE (10 µg/ml) and buffer;
bar 2, LipofectAMINE (10 µl/ml) plus
2M* (100 pM); bar 3,
LipofectAMINE plus dsRNA complex (10 µg/ml) plus
2M*; bar 4, LipofectAMINE plus
dsRNA complex (50 µg/ml) plus 2M*. The proteins were
detected by Western blotting and quantitated by a PhosphorImager as
described above. The corresponding gel blots are shown at the
bottom of the respective bar graphs.
The values are expressed in arbitrary units and are means from two
experiments performed in triplicate.
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DISCUSSION |
We have studied the role of cAMP-dependent signaling
pathways in 2M*-induced macrophage proliferation. The
binding of 2M* to its receptors causes a significant
increase in CREB expression and phosphorylation of CREB at Ser-133.
2M*-induced phosphorylation of CREB was reduced by
inhibitors of PKA, PKC, Ca2+/calmodulin kinase, ERK 1/2,
p38 MAPK, tyrosine kinases, PI 3-kinase, and p70s6k as well as by
BAPTA/AM, actinomycin D, and cycloheximide. Binding of
2M* to macrophages elevated the levels of phosphorylated ERK 1/2, p38 MAPK, JNK, and p70s6k, comparable with levels induced by
forskolin. 2M* and forskolin both increased the uptake
of [3H]thymidine by macrophages as well as cell number.
Like CREB phosphorylation, [3H]thymidine uptake was
reduced by inhibitors of PKA, PKC, Ca2+/calmodulin kinase,
ERK 1/2, p38 MAPK, tyrosine kinases, and PI 3-kinase, p70s6k, or
BAPTA/AM, actinomycin D, and cycloheximide. Both 2M* and
forskolin elevated the levels of the docking proteins Grb2 and Shc and
the guanine nucleotide exchange factor Sos. Both 2M* and
forskolin significantly raised the levels of Rap-1 and Raf-B either alone or in combination, whereas only 2M*
elevated the levels of Raf-1. These results demonstrate that
2M* triggers both IP3- and
cAMP-dependent pathways, culminating in enhanced mitogenesis and increased cell proliferation. By contrast, forskolin is
known to act only through elevating cAMP (59-64). These observations are schematically depicted in Fig.
16.
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Fig. 16.
A schematic representation of the
involvement of signaling cascades and CREB in
2M*-dependent macrophage
regulation.
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Beginning, in 1993, we reported that binding of 2M* to
cells including macrophages activated signaling cascades (4, 5, 8, 10,
12, 14). These signaling events are mediated by 2M*
binding to 2MSR, which consists of lipoprotein
receptor-related protein in complex with a coreceptor
(11)2; moreover, this
signaling pathway requires a number of adapter proteins (68, 69). Based
on these and other observations, we hypothesized that
2MSR functions like a growth factor receptor and that
2M* functions as a growth factor (10). Binding of 2M* to 2MSR induces tyrosine
phosphorylation of phospholipase C (14), which is induced by the
tyrosine phosphorylation of 2MSR (70, 71).
Tyrosine-phosphorylated receptor recruits docking protein Grb2 and Shc
and guanine nucleotide exchange factor Sos (49-51). The
Grb2-Sos or Grb2-Sos-Shc complex activates membrane binding and
formation of Ras·GTP (51, 53-56), activation of Raf-1 by PKC (55),
and phosphorylation of downstream MEK and MAPKs (55). The activated
MAPKs translocate to nuclei and phosphorylate several genes involved in
mitogenesis and cell proliferation. In addition, 2M*
binding to 2MSR activates membrane phosphatidylinositol 4,5-bisphosphate hydrolysis by
phosphatidylinositol-dependent phospholipase C, which
raises [Ca2+]i, and DAG membrane PKC as well as
several other Ca2+-dependent protein kinases
are activated, culminating ultimately in the onset of several
intracellular signaling cascades and cellular<
TOP
ABSTRACT
INTRODUCTION
