From the Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, Scotland, United Kingdom
Received for publication, November 27, 2002, and in revised form, March 4, 2003
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ABSTRACT |
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The inhibitory Mitogenic stimuli initiate cell proliferation via different
classes of cell surface receptors that include growth factor receptor tyrosine kinase receptors and G-protein-coupled receptors
(GPCRs).1 This involves
stimulation of the p42/p44 mitogen-activated protein kinase (p42/p44
MAPK) pathway (1, 2). In certain cases, growth factor- and GPCR
agonist-mediated stimulation of the p42/p44 MAPK pathway require the
G-protein-regulated aggregation of signaling molecules followed by
endocytosis of receptor signal complexes at clathrin-coated pits via a
dynamin II-dependent process (3). For instance,
isoproterenol, insulin-like growth factor-1, platelet-derived growth
factor, fibroblast growth factor, and nerve growth factor can sometimes
use G-proteins, G-protein-coupled receptor kinase 2 (GRK2), and
The phototransduction cascade involving rhodopsin (GPCR), GRK,
We have found that PDE In this paper, we have further explored the dynamic of the interaction
between PDE Materials--
All biochemicals were from Roche Applied Science,
whereas general chemicals were from Sigma. Cell culture supplies were
from Invitrogen. Anti-phospho-p42/p44 MAPK and anti-dynamin II
antibodies were from New England Biolabs. Anti-Grb-2 and anti-p42 MAPK
antibodies were from Transduction Laboratories (Lexington, KY).
Anti-Src and anti-GRK2 antibodies were from Santa Cruz Biotechnology.
pRK5-GRK2 cDNA plasmid construct was a kind gift from Professor R. Lefkowitz (Duke University). Anti-PDE Cell Culture--
HEK 293 cells were maintained in minimal
essential medium containing 10% (v/v) fetal calf serum. These cells
were placed in minimal essential medium for 24 h before
experimentation. ASM cells were maintained in Dulbecco's modified
Eagle's medium with 10% (v/v) fetal calf serum and 10% donor horse
serum. These cells were placed in Dulbecco's modified Eagle's medium
with 0.1% (v/v) fetal calf serum for 24 h before experimentation.
Transfection--
HEK 293 cells were transiently transfected
with vector or rod PDE Site-directed Mutagenesis--
To generate the Thr-62 (replaced
with Ala) mutant, a PCR was performed using mouse lung rod PDE HEK 293 Cell Lysates--
Stimulations of HEK 293 cells were
carried out at 37 °C in serum-free medium. After stimulation, medium
was removed from the monolayer cell and washed with ice-cold
phosphate-buffered saline and lysed in 1 ml of buffer containing 1×
phosphate-buffered saline, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium
deoxycholate, 0.1% (w/v) SDS, 1 mM sodium orthovanadate, 2 mM PMSF, leupeptin, pepstatin, and aprotinin (all protease
inhibitors were at 10 µg/ml). The lysates were passed through the
21-gauge needle to shear the DNA. The lysates were then incubated at
4 °C for 30 min. The cell lysate supernatant was then harvested by
centrifugation at 10,000 × g for 10 min at
4 °C.
Recombinant His6-tagged Rod PDE Magnetocapture Assay--
Ni-NTA magnetic agarose beads (Qiagen)
were resuspended by vortexing for 2 s. 500 µl of the recombinant
His6-tagged rod PDE Immunoprecipitation Assay--
The medium was removed, and cells
were lysed in ice-cold lysis buffer ((1 ml) containing 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 1% (v/v)
Nonidet P-40, 1% (w/v) deoxycholate, 0.1% (w/v) SDS, 10%
(v/v) glycerol, 1 mg/ml bovine serum albumin, 20 mM
Tris-HCl, 0.5 mM sodium orthovanadate, 0.2 mM
PMSF, leupeptin, pepstatin, and aprotinin (all protease inhibitors were
at 10 µg/ml, pH 8.0)) for immunoprecipitation. The cells were
harvested and centrifuged at 13,000 rpm for 5 min at 4 °C. The
concentration of cell lysate supernatant was determined and equalized
by performing Bradford colorimetric assay (0.5-1 mg/ml). Cell lysate
supernatant was electrophoresed in polyacrylamide gel as positive control.
For immunoprecipitation assay, cell lysate supernatant (500 µl) was
taken for immunoprecipitation with specific antibodies (2 µg of
antibodies and 100 µl of 1:1 protein A-Sepharose CL4B; 1:1 indicates
equal part of protein A-Sepharose and immunoprecipitation buffer).
After agitation for 2 h at 4 °C, the immune complex was collected by centrifugation at 13,000 rpm for 15 s at 4 °C.
Immunoprecipitates were washed twice with ice-cold buffer A (containing
10 mM Hepes, pH 7.0, 100 mM NaCl, 0.2 mM PMSF, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and
0.5% (v/v) Nonidet P-40) and once in buffer A without Nonidet P-40.
The immunoprecipitates were resuspended in boiling sample buffer
containing 62 mM Tris-HCl, pH 6.7, 1.25% (w/v) SDS, 10%
(v/v) glycerol, 3.75% (v/v) mercaptoethanol, and 0.05% (w/v) bromphenol blue. The samples were then subjected to SDS-PAGE and Western blotting.
Blotting--
Western blotting for phosphorylated p42/p44 MAPK,
c-Src, GRK2, Grb-2, dynamin II, and PDE p42/p44 MAPK Assays--
The phosphorylated forms of
p42/p44 MAPK were detected by Western blotting cell lysates with
anti-phospho-p42/p44 MAPK antibody. Anti-p42 MAPK antibody was used to
establish equal loading of p42 MAPK in each sample.
Quantification--
Quantification was by densitometry.
Interaction between PDE
Only a small fraction of PDE
Our findings are important because they are the first to define a role
for PDE Role of Thr-62 in PDE Short Cone PDE
We have therefore investigated the effect of the truncated recombinant
cone PDE
The increase in thrombin-dependent activation of p42/p44
MAPK induced by either rod or large cone PDE PDE
We conclude that the stimulation of cells with EGF might induce three
events. First, EGF promotes association of GRK2 with the c-Src-PDE Negative Feedback Regulation by p42/p44 MAPK--
The
20PVTPRKGPP28 site in rod PDE
To conclude, these findings highlight an important role for PDE subunit of the
retinal photoreceptor type 6 cGMP phosphodiesterase (PDE
) is
phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent
stimulation of p42/p44 mitogen-activated protein kinase in human
embryonic kidney 293 cells. We report here that PDE
is in a
pre-formed complex with c-Src and that stimulation of cells with
epidermal growth factor promotes the association of GRK2 with this
complex. c-Src has a critical role in the stimulation of the p42/p44
mitogen-activated protein kinase cascade by epidermal growth factor,
because c-Src inhibitors block the activation of this kinase by the
growth factor. Mutation of Thr-62 (to Ala) in PDE
produced a GRK2
phosphorylation-resistant mutant that was less effective in associating
with GRK2 in response to epidermal growth factor and did not potentiate
the stimulation of p42/p44 mitogen-activated protein kinase by this
growth factor. The transcript for a short splice variant version of
PDE
lacking the Thr-62 phosphorylation site is also expressed in
certain mammalian cells and, in common with the Thr-62 mutant, failed
to potentiate the stimulatory effect of epidermal growth factor on
p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to
Ala) in PDE
, which is a site for phosphorylation by p42/p44
mitogen-activated protein kinase, resulted in a prolonged activation of
p42/p44 mitogen-activated protein kinase by epidermal growth factor,
suggesting a role for this phosphorylation event in the negative
feedback control of PDE
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-arrestin I/II to regulate the p42/p44 MAPK pathway (4-11). GRK2 is
activated by G-protein
subunits and phosphorylates GPCRs, which
are, in certain cases, associated with growth factor receptors (10,
12). The phosphorylation of the GPCR promotes binding of
-arrestin,
which is required for dynamin II-dependent endocytosis of
the receptor-signal complex and subsequent activation of p42/p44 MAPK
(13). Thus, the expression of dominant-inhibitory
-arrestin I or
dynamin II mutants impairs insulin-like growth factor-1-,
-adrenergic-, and lysophosphatidic acid-dependent activation of p42/p44 MAPK (14-16). In the presence of these
inhibitory mutants, the p42/p44 MAPK signaling cascade proceeds only as
far as Raf activation (16).
-Arrestin is a clathrin adaptor that binds certain receptor complexes and targets them to clathrin-coated pits, whereas dynamin II is a GTPase involved in the "pinching off" of clathrin-coated endocytic vesicles containing the
receptor-signal complex (17). The hydrolysis of GTP by dynamin II is
believed to be catalytic for pinching off of endocytic vesicles
and subsequent relocalization of receptor signal complexes with
cytoplasmic MEK-1 and p42/p44 MAPK.
-arrestin, and RGS9G
5 (18) bears many similarities with signaling
by growth factors and G-protein-coupled receptors in other mammalian
cell systems. The phototransduction cascade involves cGMP
phosphodiesterases that are expressed in rod and cone photoreceptors (termed PDE6) as tetrameric proteins composed of two catalytic subunits
and two
subunits (PDE
). PDE
inhibits cGMP hydrolysis at the
catalytic sites. The two types of photoreceptor cells, rod and cone,
express different isoforms of PDE
. These proteins differ in their
extreme N-terminal regions, whereas the central polycationic and
C-terminal domains that are involved in the interaction with both PDE6
and transducin are almost identical.
has a wider role in mammalian cell biology
(19-22). Indeed, we have reported that rod PDE
is expressed in
lung, kidney, testes, liver, heart, airway, and pulmonary smooth muscle
and HEK 293 cells and is absent from these tissues in rod PDE
knockout mice. We have also identified a novel role for PDE
in
regulating the EGF- and thrombin-dependent activation of
the p42/p44 MAPK pathway in HEK 293 cells (21). We also found that GRK2
is required for the stimulatory effect of rod PDE
on both the EGF-
and thrombin-dependent activation of p42/p44 MAPK. Indeed, rod and cone PDE
are substrates for phosphorylation by GRK2. Moreover, a GRK2 phosphorylation-resistant (Thr-62 changed to Ala) rod
PDE
mutant failed to increase the EGF- or
thrombin-dependent activation of p42/p44 MAPK, and in fact
functioned as a dominant negative. We also presented evidence to show
that thrombin stimulates the formation of a complex between rod PDE
and dynamin II (21). This is significant because it is well established
that GTP hydrolysis by dynamin II promotes endocytosis of vesicles
containing receptor signal complexes that subsequently relocalize with
and activate cytoplasmic p42/p44 MAPK. Taken together, the data are
consistent with the phosphorylation of Thr-62 in rod PDE
by GRK2
being essential for interaction with dynamin II.
and GRK2. We show for the first time that PDE
is a
functional linker/regulator of both c-Src and GRK2. We also show that a
GRK2 phosphorylation-resistant PDE
mutant (Thr-62 PDE
mutant) is
less effective than the wild type protein in binding GRK2 in response
to EGF. The mutant appears to function as an endogenous dominant
negative by acting as a sink for c-Src. We also provide the first
evidence for a negative feedback mechanism involving p42/p44 MAPK that
regulates PDE
and appears to limit the duration of p42/p44 MAPK
activation in response to EGF.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
antibody to the C-terminal
domain of photoreceptor PDE
and which reacts with both rod and cone isoforms was a kind gift from Dr. R. Cote (University of New Hampshire).
or Thr-62 rod PDE
mutant pcDNA-6xHis
plasmid constructs or pRK5-GRK2 plasmid constructs. Cells at 90%
confluence were placed in minimal essential medium containing 2% (v/v)
fetal calf serum and transfected with 2 µg of plasmid construct
following complex formation with LipofectAMINETM 2000, according to the manufacturer's instructions. The cDNA-containing media were then removed following incubation for 24 h at 37 °C, and the cells were incubated for a further 24 h in serum-free medium prior to addition of epidermal growth factor. ASM cells were
transiently transfected with mouse lung rod (wild type and Thr-22
mutant) PDE
pcDNA-3.1 plasmid constructs in Dulbecco's modified
Eagle's medium with 2% (v/v) fetal calf serum for 24 h before experimentation.
pcDNA-3.1 plasmid construct with forward (5'-GGA AGG CCT GGG G(G)C
AGA TAT CAC CGT CAT C-3') and reverse primers (5'-GAT GAC GGT GAT ATC
TG(C) CCC CAG GCC TTC C-3') (Invitrogen). To generate the Thr-22
(replaced with Ala) mutant, a PCR was performed using mouse lung rod
PDE
pcDNA-3.1 plasmid construct with forward (5'-GGA GGA CCA GTC
GCC CCC AGG AAA G-3') and reverse primers (5'-C TTT CCT GGG GGC GAC TGG
TCC TCC-3') (Invitrogen). Both of these sets of primers were used in a
QuikChange (Stratagene) PCR. The PCR conditions were per the QuikChange
manual with the following changes: 1 cycle at 95 °C for 30 s
followed by 12 cycles at 95 °C for 30 s, 55 °C for 1 min,
and 68 °C for 11 min. The reaction was digested with
DpnI, and a 1-µl aliquot was transformed into Top10 strain
Escherichia coli (Invitrogen). Plasmid preparations were
obtained from the resultant colonies and screened for the correct
mutation by sequencing. The mutated insert was subcloned from
pcDNA-3.1 by digesting with BamHI and HindIII
and was also inserted in-frame into pTrcHis-B (Invitrogen).
--
The open
reading frame of mouse lung rod PDE
was subcloned into an expression
vector pTrcHis (Invitrogen). The vector was transformed into TOP10F'
E. coli strain. The E. coli was grown in 2 ml of
Luria Broth (LB) containing 10% (w/v) tryptone, 5% (w/v) yeast
extract, and 10% (w/v) NaCl supplemented with 50 µg/ml ampicillin at
37 °C overnight. The overnight E. coli culture was then
diluted 50-fold with 100 ml of LB medium containing 50 µg/ml ampicillin and grown for an additional 2.5 h, until the
A600 of the culture was 0.6-0.8.
Isopropyl-
-D-thiogalactoside was then added to a final
concentration of 1 mM for induction of the culture at
30 °C. After a suitable period of induction (3 h), the cells were
harvested by centrifugation (A-18C rotor Centrikon T-42K, 15 min at
7,400 rpm at 4 °C) and resuspended in lysis buffer containing 20 mM Na2HPO4, pH 7.8. Each sample was
then lysed by 3 times ultrasonication followed by 3 times freeze-thaw
cycles in lysis buffer. All buffers above contained protease inhibitors
(20 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM
benzamidine, and 0.1 mM PMSF) to inhibit proteolytic reaction in the sample. The lysed cells were then centrifuged at 13,000 rpm/10 min at 4 °C (Sigma laboratory centrifuge IK15) to remove the
insoluble cell debris. The supernatant was stored at
70 °C.
was immediately added to 50 µl of
the 5% (w/v) Ni-NTA magnetic agarose beads suspension. The suspension
was incubated on an end-over-end shaker for 1 h at 4 °C. This
was to allow efficient binding of the His-tagged rod PDE
to the
Ni-NTA magnetic agarose beads. After 1 h, the microcentrifuge
tubes containing the complexes were placed on a 12-tube magnetic
separator for 1 min, and the supernatant was removed with a pipette.
The magnetic beads-His-tagged rod PDE
complexes were washed with
wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH 8.0) for 3 times at 4 °C, and the wash buffer was removed by placing the
microcentrifuge tubes on the magnetic separator for 1 min. The HEK 293 cell lysate supernatant was then added to the magnetic beads. The
suspension was incubated on an end-over-end shaker for 1 h at
4 °C. After 1 h, the complexes were washed with wash buffer
once, and the wash buffer was removed as described above. The potential
interaction proteins with His-tagged rod PDE
were eluted with
elution buffer containing 50 mM
NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0, and collected for detection by
SDS-PAGE/Western blot.
was as described previously
(21). Immunoreactive proteins were visualized using enhanced
chemiluminescence detection kit.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, GRK2, and c-Src--
The ability of
GRK2 to phosphorylate PDE
in an EGF-dependent manner
prompted us to investigate whether these proteins exist in a complex in
HEK 293 cells. In addition, several reports (23) have shown that growth
factors activate c-Src and GRK2 and that these proteins regulate each
other in a bi-directional manner. Moreover, in previous studies (21) we
reported that the overexpression of GRK2 and/or PDE
increases the
activation of p42/p44 MAPK induced by EGF. Therefore, HEK 293 cells
were transfected with PDE
plasmid constructs and PDE
, c-Src, and
GRK2 immunoprecipitated with specific respective antibodies from
lysates of cells treated with and without EGF. We report here that
PDE
and c-Src exist in a pre-formed complex and that stimulation of
cells with EGF promotes association of GRK2 with the PDE
-c-Src
complex. This was based upon several lines of evidence. First, Fig.
1a shows that
recombinant overexpressed PDE
(14 kDa) and c-Src (60 kDa) are
co-immunoprecipitated with GRK2 (85 kDa) from lysates of HEK 293 cells
using anti-GRK2 antibodies. The amount of both c-Src and recombinant
PDE
co-immunoprecipitated with GRK2 was increased from cells treated
with EGF (fold stimulations in response to EGF: c-Src, 2.74 ± 1.26-fold; PDE
, 1.75 ± 0.05-fold, n = 3, p < 0.05 versus control). The
EGF-dependent increase in the amount of c-Src and PDE
retrieved in anti-GRK2 immunoprecipitates is entirely to be expected if
c-Src and PDE
are indeed pre-complexed, and EGF stimulation of cells
promotes their association with GRK2. The data obtained are therefore
entirely compatible with this model. Second, Fig. 1b shows
that PDE
and GRK2 were co-immunoprecipitated with c-Src using
anti-c-Src antibodies. The amount of PDE
co-immunoprecipitated was
not increased from cells treated with EGF. In contrast, the amount of
GRK2 associated with c-Src was increased from cells treated with EGF
(fold stimulations in response to EGF: GRK2, 1.85 ± 0.12-fold,
GRK2 plus recombinant overexpressed PDE
, 1.47 ± 0.21-fold,
n = 3, p < 0.05 versus
control). In addition, there is a 3.05 ± 0.64-fold increase
(n = 2) in the amount of GRK2 associated with c-Src in
cells overexpressing PDE
compared with cells expressing only
endogenous PDE
. The fact that the association of PDE
with c-Src
is not sensitive to EGF stimulation provides additional evidence that
these proteins are in a pre-formed complex that is strictly
EGF-independent. The increased amount of GRK2 in anti-c-Src immunoprecipitates isolated from cells treated with EGF provides additional evidence that it is the binding of GRK2 to the PDE
-c-Src complex that is, in fact, EGF-dependent. We also found that
Grb-2 (26 kDa) was co-immunoprecipitated with c-Src, indicating
association between these proteins. This association was also increased
in cells transfected with PDE
. Third, Fig. 1c shows that
c-Src and GRK2 were co-immunoprecipitated with PDE
from lysates. The
amount of c-Src co-immunoprecipitated with PDE
using anti-PDE
antibodies was not increased from cells treated with EGF. Again, this
is entirely in line with our interpretation that the c-Src-PDE
complex is pre-formed in an EGF-independent manner. The amount of GRK2 co-immunoprecipitated with PDE
was increased from cells treated with
EGF (fold stimulations in response to EGF: GRK2, 2.01 ± 0.82-fold, n = 3, p < 0.05 versus control), again indicating that it is the GRK2
binding step that is EGF-dependent. None of the proteins were co-immunoprecipitated when antibodies were omitted from the immunoprecipitation procedure (data not shown).
View larger version (23K):
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Fig. 1.
Interactions of rod PDE
with GRK2, c-Src, and Grb2 in HEK 293 cells. HEK 293 cells
were transiently transfected with vector, pRK5-GRK2, or rod PDE
pcDNA-6xHis plasmid constructs. The cells were then stimulated with
EGF (50 ng/ml) for 5 min. a, Western blots
(WB) showing co-immunoprecipitation of rod PDE
and c-Src
with GRK2 from control and EGF-stimulated transfected cells using
anti-GRK2 antibodies (Ab). b,
co-immunoprecipitation of GRK2, rod PDE
, and Grb2 with c-Src from
control and EGF-stimulated transfected cells using anti-c-Src antibodies. c,
co-immunoprecipitation of c-Src and GRK2 with rod PDE
from control
and EGF-stimulated transfected cells using anti-C-terminal PDE
antibodies. a-c, HEK 293 cell lysates (CL)
were used as positive control for the various proteins, run on the same
SDS-PAGE as the immunoprecipitates (IP). d,
Western blots showing the effect of recombinant rod PDE
and GRK2 on
the EGF-dependent activation of p42/p44 MAPK in transfected
cells. e, Western blots showing the effect of PP2 on
the EGF-dependent activation of p42/p44 MAPK in vector- and
rod PDE
-transfected cells. The cells were pretreated with c-Src
inhibitor, PP2 (10 µM, 15 min). f,
Western blot of anti-PDE
immunoprecipitates with
anti-phospho-p42/p44 MAPK antibodies. For d, blots were
also stripped and re-probed with anti-p42 MAPK antibodies to ensure
equal protein loading. These are representative results of an
experiment performed three times.
is present in the complex with c-Src
and GRK2 (see Fig. 1, a and b compared with
c). Thus, only a limited increase in the expression of this
protein is actually required to bind c-Src and GRK2 in order to support
EGF receptor signaling. This is corroborated by previous findings (21)
showing that PDE
is limiting for EGF receptor signaling to p42/p44
MAPK. The current findings are also compatible with the fact that cells are stimulated with a single agonist that might use only a relatively small fraction of the PDE
expressed. The fold increases in the EGF-dependent association of GRK2 with the c-Src-PDE
complex reported here are also in agreement with the potentiation of
EGF-stimulated p42/p44 MAPK activation by PDE
(see Fig.
1d). In addition to the recombinant form of the protein, we
have also reported previously (21) that endogenous PDE
participates
in regulating EGF receptor signaling to p42/p44 MAPK. This was
supported by data showing that the effect of endogenous PDE
can be
ablated by transfection of cells with PDE
antisense plasmid
construct (21). Endogenous PDE
, c-Src, and GRK2 clearly form a
complex in cells, as significant amounts of these proteins were
isolated in anti-Src immunoprecipitates from vector-transfected cells
(Fig. 1b). As with overexpressed recombinant PDE
, EGF
promoted the association of GRK2 with endogenous PDE
in this complex
(Fig. 1b).
as a functional linker between GRK2 and c-Src. Thus, PDE
may function to recruit GRK2 close to c-Src, whereupon there may be the
reciprocal activation of each kinase. This functional interaction
between PDE
, GRK2, and c-Src is important because the formation of
the complex appears to be required for EGF-dependent activation of p42/p44 MAPK. Thus, overexpression of PDE
or GRK2 increased EGF-dependent activation of p42/p44 MAPK (Fig.
1d, fold stimulations in response to EGF versus
vector-transfected cells: rod PDE
, 1.7 ± 0.1; GRK2, 1.8 ± 0.2, n = 3, p < 0.05 versus vector-transfected cells), whereas pretreatment of
cells with the c-Src inhibitor, PP2, ablated EGF-dependent
activation of p42/p44 MAPK (Fig. 1e). We have also obtained
additional evidence for a mechanistic link between c-Src-PDE
and
p42/p44 MAPK. We report here that the p42/p44 MAPK activated in
response to EGF associates with the c-Src-PDE
complex. Thus,
phosphorylated p42/p44 MAPK is present in anti-PDE
immunoprecipitates with PDE
, c-Src, and GRK2 from vector- and PDE
-transfected cells treated with EGF but not from control cells (Fig. 1f). These data strongly suggest that the inhibition
of the pool of PDE
associated c-Src by PP2 is directly responsible for the attenuation of the EGF-dependent activation of
p42/p44 MAPK by this compound. The results therefore further highlight the physiological significance of the PDE
-c-Src complex in
regulating this kinase pathway. These findings also suggest that the
PDE
-c-Src-GRK2 complex might undergo endocytosis and relocalization
with components of the p42/p44 MAPK pathway.
Interaction with c-Src and GRK2--
In
previous studies we reported that GRK2 phosphorylates PDE
on Thr-62.
Thus, mutagenesis of the Thr-62 (to Ala) produces a protein whose
phosphorylation by GRK2 is severely impeded (21). Mutagenesis of the
Thr-62 in PDE
has no impact on the folding of this protein, which
exists in solution as a polypeptide without tertiary structure. In
contrast with wild type PDE
, the Thr-62 mutant cannot support
EGF-dependent activation of p42/p44 MAPK and, indeed,
functions as a dominant negative to block the involvement of endogenous
PDE
in regulating p42/p44 MAPK signaling (Fig. 2a, fold stimulations of
p42/p44 MAPK in response to EGF versus vector-transfected
cells: rod PDE
, 1.6 ± 0.34; Thr-62 PDE
, 0.54 ± 0.02-fold, n = 3, p < 0.05 versus vector-transfected cells). We now show that the
mutant PDE
is not an efficient binding partner of GRK2 in cells
stimulated with EGF when compared with wild type PDE
(Fig.
2b). Consistent with this, we found that the amount of GRK2 associated with c-Src was reduced by 52 ± 30%
(n = 3, p < 0.05 versus
vector transfected cells) in EGF-stimulated cells overexpressing Thr-62
mutant PDE
compared with vector-transfected cells. This finding
indicates that the mutant might prevent GRK2 binding to endogenous
PDE
(Fig. 2b). How can this be achieved? One possibility
is that the Thr-62 mutant might act as a sink for c-Src, thereby
preventing interaction of c-Src with GRK2 via endogenous PDE
.
Consistent with this possibility is our finding that the Thr-62 mutant
was still capable of binding c-Src. Thus, the amount of wild type or
Thr-62 mutant PDE
co-immunoprecipitated with c-Src was similar (Fig.
2b). Mutation of Thr-62 in PDE
also reduced the
interaction between the c-Src and Grb-2 (Fig. 2b).
View larger version (34K):
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Fig. 2.
The role of GRK2 phosphorylation of rod
PDE in multiprotein complex formation in HEK
293 cells. HEK 293 cells were transiently transfected with vector
or rod PDE
or Thr-62 rod PDE
mutant pcDNA-6xHis plasmid
constructs. The cells were then stimulated with EGF (50 ng/ml) for 5 min. a, Western blots (WB) showing the
effect of wild type and Thr-62 PDE
on EGF-dependent
p42/p44 MAPK activation. Blots were also stripped and re-probed with
anti-p42 MAPK antibodies (Ab) to ensure equal protein
loading. b, co-immunoprecipitation of GRK2, rod PDE
,
and Grb2 with c-Src from control and EGF-stimulated transfected cells
using anti-c-Src antibodies. HEK 293 cell lysates (CL) were
used as positive control and run on the same SDS-PAGE as the
immunoprecipitates. These are representative results of an experiment
performed three times.
--
We have also detected transcript for a
short cone PDE
isoform, which has a 41-bp deletion (corresponding to
exon 3) resulting in a frame change (22). This deletion produces a new
"in-frame" stop codon resulting in an early termination to produce
a truncated protein (short cone PDE
) that lacks Thr-62. This protein
is predicted to have an identical N-terminal and polycationic
mid-region but a different C-terminal domain compared with the larger
version of cone PDE
.
on p42/p44 MAPK signaling. Overexpression of the truncated
cone PDE
reduced the EGF-dependent activation of p42/p44
MAPK (Fig. 3, upper panel,
fold stimulations of p42/p44 MAPK in response to EGF
versus vector-transfected cells: rod PDE
, 1.6 ± 0.34; short cone PDE
; 0.59 ± 0.27; rod PDE
plus short cone
PDE
; 1.15 ± 0.06, n = 3-6, p < 0.05 for rod PDE
plus short cone PDE
-transfected
versus rod PDE
-transfected cells).
View larger version (27K):
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Fig. 3.
The effect of the truncated cone
PDE on the EGF/thrombin-dependent
activation of p42/p44 MAPK. HEK 293 cells were transiently
transfected with vector or truncated cone PDE
and/or rod PDE
and/or cone PDE
pcDNA-3.1 plasmid constructs. The cells were
then stimulated with EGF (50 ng/ml, 5 min) or thrombin (0.03 unit/ml,
10 min). Western blots (WB) showing upper panel,
the EGF-stimulated p42/p44 MAPK activation in rod PDE
- and/or
truncated cone PDE
-transfected cells; lower panel,
thrombin-stimulated p42/p44 MAPK activation in rod PDE
- and/or cone
PDE
- and/or truncated cone PDE
-transfected cells. p42/p44 MAPK
activation was detected using anti-phospho-p42/p44 MAPK-specific
antibodies (Ab). Blots were also stripped and re-probed with
anti-p42 MAPK antibodies to ensure equal protein loading. These are
representative results of an experiment performed three times.
was also reduced in cells overexpressing the truncated cone PDE
(Fig. 3, lower
panel, fold stimulations of p42/p44 MAPK in response to thrombin
versus vector-transfected cells: rod PDE
, 2.3 ± 0.2; cone PDE
, 2.05 ± 0.18; short cone PDE
, 0.77 ± 0.15; short cone PDE
plus rod PDE
, 0.95 ± 0.03; short cone
PDE
plus cone PDE
, 0.84 ± 0.12, n = 3-6,
p < 0.05 for rod or cone PDE
plus short cone
PDE
-transfected versus rod or cone PDE
-transfected
cells). Thus, the truncated cone PDE
may also act as an endogenous
dominant negative modulator of EGF- and thrombin-dependent
stimulation of the p42/p44 MAPK pathway.
-PDE
Interaction--
One of the structural determinants
in PDE
that may be important for association with c-Src is an
SH3-binding site. PDE
contains an SH3 consensus binding site
20PVTPRKGPP28, which is identical with the
corresponding region in the cone isoform with the exception that valine
at amino acid position 21 is replaced by threonine. This site may
therefore interact with an SH3 domain in c-Src. In previous studies
(21) we reported that thrombin also promotes the association of PDE
with dynamin II. This might also involve interaction of dynamin II
SH3-binding site via an intermediate double SH3 domain containing
protein (e.g. Grb-2) with
20PVTPRKGPP28 in PDE
. Thus, PDE
might
bind several proteins via SH3 interaction. Indeed, along with c-Src and
GRK2, dynamin II is involved in endocytic processes that are required
for certain GPCR/growth factor-dependent activations of
p42/p44 MAPK (4-11). However, PDE
contains only one SH3 domain
binding site. It is therefore possible that PDE
might form either
dimers or tetramers, thereby increasing the number of SH3 domain
binding sites from 1 to 4. Indeed, it is well established that PDE
forms dimers via hydrophobic interaction, and indeed, two molecules of
PDE
bind to PDE6 in rod photoreceptors (18). To assess this
possibility, we immobilized His-tagged PDE
on a nickel-agarose
matrix in order to capture endogenous PDE
-c-Src-Grk2 complexes
present in lysates from HEK 293 cells. Fig.
4a shows that GRK2 (85 kDa),
c-Src (60 kDa), dynamin II (112 kDa), and Grb-2 (26 kDa) in lysates
from control cells were all trapped by the His-tagged PDE
-agarose
matrix and eluted with imidazole. The amount of each of these proteins
binding to the His-tagged PDE
-agarose matrix was increased from
lysates of cells treated with EGF (fold stimulations in response to EGF
versus control cells: GRK2, 1.73 ± 0.51-fold; dynamin
II, 1.53 ± 0.18-fold; Grb-2, 1.47 ± 0.14-fold; c-Src,
1.45 ± 0.11-fold, n = 3, p < 0.05 for all versus control). PDE
was also eluted with
imidazole from the nickel matrix (Fig. 4b). The fold
increases in the binding of the various proteins are all similar. This
is entirely consistent with the possibility that EGF treatment of cells
might promote the binding of the entire complex of proteins to PDE
immobilized on the nickel matrix. Taken together, these findings
suggest that EGF may increase the affinity of PDE
for PDE
-
protein complexes.
View larger version (23K):
[in a new window]
Fig. 4.
The EGF-dependent effect on the
interaction between immobilized PDE and
PDE
-dynamin II-GRK2-c-Src-Grb-2
complexes. Recombinant His-tagged bacterially expressed rod PDE
was immobilized on Ni-NTA magnetic agarose beads by preincubating the
bacterial lysates with magnetic beads. HEK 293 cells were treated with
or without EGF (50 ng/ml) for 5 min. HEK 293 cell lysates were then
incubated with Ni-NTA magnetic agarose beads-His-tagged rod PDE
. HEK
293 cell lysates (both control and EGF-stimulated) were separated by
SDS-PAGE and serve as positive control to determine the migration of
specific proteins. The protein complexes were eluted with 250 mM imidazole elution buffer. a, Western
blotting (WB) of imidazole eluates with antibodies
(Ab) to dynamin II, GRK2, c-Src, and Grb2;
b, Western blotting of imidazole eluates with
anti-C-terminal rod PDE
antibodies. These are representative results
of an experiment performed three times.
complex. Second, EGF promotes GRK2-catalyzed phosphorylation of PDE
;
and third, the growth factor may increase the affinity of PDE
for
PDE
. This may promote formation of a dimeric or tetrameric PDE
platform upon which other proteins involved in endocytic signaling to
p42/p44 MAPK, such as dynamin II, can associate.
also contains
a consensus site for phosphorylation by p42/p44 MAPK
(20PVTP23). Indeed, we found that this site was
phosphorylated by p42/p44 MAPK. Paglia and colleagues (24) have
formerly reported stoichiometric phosphorylation of PDE
by p42 MAPK.
Therefore, we have mutated Thr-22 to establish its effect on
PDE
-mediated regulation of p42/p44 MAPK. For this purpose we used
cultured airway smooth muscle cells. These cells contain abundant
amounts of PDE
(18), such that the protein is saturating for
EGF-stimulated p42/p44 MAPK activation (data not shown). Transfection
of these cells with Thr-22 mutant PDE
-pcDNA3.1 plasmid
construct led to a prolonged activation of p42/p44 MAPK by EGF compared
with vector (H)-transfected cells (Fig.
5). These data are compatible with the
possibility that p42/p44 MAPK can phosphorylate PDE
at Thr-22 to
exert the feedback inhibition of PDE
-c-Src/GRK2 activity, thereby
limiting the duration of p42/p44 MAPK activation in response to EGF.
Presumably, mutagenesis of Thr-22 to Ala does not in itself disrupt
interaction between PDE
, c-Src, and GRK2, as this mutant is still
capable of supporting activation of p42/p44 MAPK in response to EGF.
Indeed, the mutant is more efficient compared with the endogenous wild type PDE
. In this case, the Thr-22 mutant presumably replaces endogenous wild type PDE
in the signaling pathway regulating p42/p44
MAPK.
View larger version (39K):
[in a new window]
Fig. 5.
The Thr-22 mutant PDE
functions to prolong EGF-dependent activation of
p42/p44 MAPK in airway smooth muscle cells. ASM cells were
transiently transfected with vector (H) or rod PDE
and/or Thr-22
mutant rod PDE
pcDNA-3.1 plasmid constructs. The cells were then
stimulated with EGF (50 ng/ml) for the indicated times. Western blots
showing the time course of EGF-stimulated p42/p44 MAPK activation in
vector or rod PDE
- or Thr-22 mutant rod PDE
-transfected cells.
p42/p44 MAPK activation was detected using anti-phospho-p42/p44 MAPK
specific antibodies. Blots were also stripped and re-probed with
anti-p42 MAPK antibodies to ensure equal protein loading. These are
representative results of an experiment performed three times.
in
transducing signals from GRK2/c-Src to p42/p44 MAPK. They also
demonstrate that PDE
functions as an important intermediate regulating this mitogenic signaling pathway.
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FOOTNOTES |
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* This work was supported by grants from The Wellcome 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. Tel.: 0141-552-4400 (ext. 2659); Fax: 0141-552-2562; E-mail: n.j.pyne@strath.ac.uk.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M212103200
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ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G-protein-coupled receptor; EGF, epidermal growth factor; GRK, G-protein coupled receptor kinase; Grb-2, growth factor receptor-binding protein; HEK, human embryonic kidney; MAPK, mitogen activated protein kinase; PDE, phosphodiesterase; PDGF, platelet-derived growth factor; SH3, Src homology 3; PMSF, phenylmethylsulfonyl fluoride; Ni-NTA, nickel-nitrilotriacetic acid.
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