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J. Biol. Chem., Vol. 278, Issue 31, 29164-29173, August 1, 2003
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From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, E-28049 Madrid, Spain
Received for publication, April 24, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-arrestin function and Src-mediated
phosphorylation are involved in targeting GRK2 for proteolysis. In this
report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2
turnover by the proteasome pathway. Modulation of MAPK activation alters the
degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics
phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a
direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated
modulation of wild-type GRK2 stability requires -arrestin function and
is facilitated by previous phosphorylation of GRK2 on tyrosine residues by
c-Src. Consistent with an important physiological role, interfering with this
GRK2 degradation process results in altered GPCR responsiveness. Our data
suggest that both c-Src and MAPK-mediated phosphorylation would contribute to
modulate GRK2 degradation, and put forward the existence of new feedback
mechanisms connecting MAPK cascades and GPCR signaling. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and 
subunits, both of which regulate a wide variety of
effector systems. In addition, agonist stimulation also leads to the
deactivation of GPCR signaling (desensitization) by triggering receptor
phosphorylation by specific G protein-coupled receptor kinases (GRKs) and
binding of the cytosolic proteins -arrestins to the phosphorylated
receptor (3,
4). GRK2 is a ubiquitous member
of the GRK family, which has been shown to modulate a variety of GPCRs
(5,
6).
The same regulatory molecules that contribute to receptor uncoupling from G
proteins also regulate GPCR endocytosis, intracellular trafficking, and
resensitization and participate in the modulation of mitogen-activated protein
kinase (MAPK) cascades by GPCR
(7). Thus, -arrestins
mediate the recruitment of clathrin and 2-adaptin to allow
for receptor internalization and also act as scaffold molecules by bridging
receptors with signaling proteins such as c-Src, thus facilitating the
activation of the ERK/MAPK cascade by GPCR
(8,
9). In addition, the isoforms
of -arrestins can directly interact with components of two different
MAPK cascades bringing these molecules into close proximity with the receptor
complex (10,
11). On the other hand, GRK2
has been shown to interact with the ARF modulator GIT, with
phosphatidylinositol 3'-OH kinase , and the G protein
q subunit
(12) and to phosphorylate
non-receptor substrates such as tubulin, synucleins, or phosducin
(13), thus also extending the
cellular functions of GRK2 beyond GPCR desensitization.
The key role that GRKs and -arrestins play in GPCR signaling and
modulation suggests that the overall activity of GPCRs would be strongly
dependent on the cellular complement and functionality of these proteins.
Consistent with this idea, the altered expression of GRK2 described in several
pathological conditions such as hypertension
(14), congestive heart failure
(15), or rheumatoid arthritis
(6) has been correlated with
impaired GPCR signaling in these situations.
GRK2 activity and subcellular distribution are tightly regulated by
interactions with G subunits, lipids, agonist-activated
receptors, anchoring proteins and calmodulin, or phosphorylation by other
kinases (reviewed in Refs.
16–18).
On the other hand, the mechanisms that govern GRK2 cellular levels and that
may explain its alterations in physiological and pathological conditions have
begun to be addressed recently. In this regard, we have reported recently
(19) that GRK2 is degraded by
the proteasome pathway and that kinase turnover was enhanced upon GPCR
stimulation. Moreover, we have shown that -arrestin function and c-Src
activity are involved in GRK2 proteolysis. Agonist-dependent binding of
-arrestin to GPCRs allows for the recruitment of c-Src, leading to
phosphorylation of GRK2 on tyrosine residues and its targeting for degradation
(20). However, the occurrence
of additional pathways that may control GRK2 stability was also suggested by
some of our data (20).
We and others (21, 22) have described that GPCR activation promotes the presence of active MAPK and GRK2 in the same multimolecular complex and that MAPK phosphorylates GRK2. In this report, we show that MAPK-mediated GRK2 phosphorylation triggers GRK2 degradation in a process that appears to be facilitated when GRK2 is previously tyrosine-phosphorylated. Our data indicate that both c-Src and MAPK pathways would contribute to modulate GRK2 protein stability by promoting proteasome-dependent GRK2 degradation, and put forward new functional regulatory relationships between MAPK cascades and GPCR signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PKC*), and MEK
(MEK*) were kindly provided by Dr. J. Moscat (Centro de Biología
Molecular, Madrid, Spain). The cDNA of constitutively active Ras (H-Ras V-12)
was obtained from Dr. M. Serrano (Centro Nacional de Biotecnología,
Madrid, Spain). The source of other expression plasmids and materials was
reported previously (19,
20). All other reagents were
of the highest grade commercially available. Site-directed Mutagenesis of GRK2—Specific GRK2 mutation at serine 670 to aspartic acid (GRK2-S670D) was performed by using a "bridge" PCR method. Thus, this mutant was generated by sequentially using the external primers upstream (nucleotides 1755–1785) and downstream (nucleotides 2199–2220) of the mutation in combination with the mutant oligonucleotides 5'-CCGCGCGAGCCCGTCGTCGAGCTGAGCAAG-3' and its pair 5'-GACGGGGTCGCGCGGCTTGTTCTTCATCTT-3'. The amplified product was subcloned into ApaI-XhoI sites in pcDNA3. The GRK2-S670A mutant was generated with the "Quick Change Site-directed mutagenesis kit" (Stratagene) using the following primers 5'-CCGCGCGCGCCCGTCGTGGAGCTGAGCAAG-3' and its pair 5'-GAGGGGCGCGCGCGGCTTGTTCTTCATCTT-3'. Finally, the double mutants GRK2-Y13F/Y86F/Y92F-S670A and GRK2-Y13F/Y86F/Y92F-S670D were generated by digestion of the fragment containing the specific mutation at serine 670 with XbaI-XhoI and subsequent subcloning in the mutant GRK2-Y13F/Y86F/Y92F engineered previously as described (20).
Cell Culture and Transfection—HEK-293 and COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified 5% CO2 atmosphere. Jurkat T cells were maintained in RPMI 1640 medium supplemented with 5% fetal bovine serum. Plasmid DNA (5–10 µg) was transiently transfected by using the LipofectAMINE reagent as described (20). Empty vector was added as needed to keep the total amount of DNA transfected constant. Expression of wild-type and mutant proteins was analyzed by immunoblot with specific antibodies to confirm similar expression levels.
Cell Treatment and Metabolic Labeling—Metabolic labeling and
pulse-chase experiments were performed as described
(20). In cells expressing
2AR, receptor activation was triggered by 10 µM
isoproterenol during chase periods in Dulbecco's modified Eagle's medium
supplemented with 20 mM HEPES, pH 7.5, and 1 mM ascorbic
acid. When indicated, receptor activation was blocked with the antagonist
betaxolol (10 µM; a gift from Synthelabo Reserche) added 30 min
before labeling and maintained during chase periods. In some experiments,
cells expressing transfected or endogenous 2ARs were
chronically exposed to isoproterenol (10 µM) for 3–5 h,
followed by extensive washing and incubation with medium for 30 min, and
subsequently challenged with LPA 10 µM for 5 min. The MEK kinase
inhibitors PD98059 (50 µM; Calbiochem) or UO126 (10
µM; Biomol) and the c-Src tyrosine kinase inhibitor PP2 (5
µM, Calbiochem) were added 90 min prior to metabolic labeling or
cell lysis. In some experiments, endogenous CXCR4 receptors in Jurkat cells
maintained in 1% serum for 18 h were activated with the chemokine SDF-1
(100 nM for 5 min, PeproTech). The proteasome inhibitors MG132 (10
µM; Biomol) and ALLN (50 µM; Sigma) were added 90
min before labeling and maintained during the chase period.
Immunoprecipitation and Western Blot Analysis—Cells were washed and lysed in RIPA buffer, and cellular extracts were immunoprecipitated with the specific GRK2 polyclonal antibody AbFP1 as reported (20). Immunoprecipitates were resolved in 10% SDS-PAGE and gels either subjected to fluorography (pulse-chase experiments) or transferred to nitrocellulose membranes to be probed with an anti-phosphotyrosine monoclonal antibody (PY99-horseradish peroxidase; Santa Cruz Biotechnology) and then reprobed after stripping with AbFP1. In some experiments, the presence of GRK2 phosphorylated on serine 670 in the immunoprecipitates was tested by using an specific anti-phosphoserine polyclonal antibody (pS670 anti-GRK2 antibody from BIOSOURCE International). MAPK activation was detected in cellular lysates by using a phospho-p42/p44 MAPK (Thr-202/Tyr-204) polyclonal antibody (1:500; New England Biolabs). The same blots were subsequently reprobed with a p42/p44 MAPK polyclonal antibody (Calbiochem) to normalize MAPK activation to whole MAPK protein levels. Lysate aliquots were taken to assess protein overexpression of the different wild-type and mutant constructs as described (20). When indicated, actin expression was determined by using a polyclonal antibody (Santa Cruz Biotechnology). Blots were developed using a chemiluminescent method (ECL, Amersham Biosciences). Band density was quantitated by laser densitometric analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2-adrenergic receptor (2AR) agonists,
different endogenous GPCRs and/or the basal activity of overexpressed
2AR (20) are
modulating GRK2 stability through a mechanism also involving MAPK stimulation.
Consistent with a role for basal 2AR activity in GRK2
turnover, the degradation of GRK2 in cells co-expressing 2AR
was clearly delayed upon treatment with the antagonist betaxolol (93 ±
4.5% of GRK2 remaining after 1 h of chase with betaxolol versus 51
± 10% without, data not shown).
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To explore further the involvement of MAPK in GRK2 degradation, we examined
whether the expression of constitutively active upstream activators of the
MAPK cascade would affect GRK2 turnover. HEK-293 cells were transiently
transfected with GRK2 and a mutant construct of Raf (Raf*) lacking an
NH2-terminal regulatory domain
(23), thus promoting a robust
MAPK activation without altering MAPK protein levels (data not shown). The
sole expression of this mutant notably increases GRK2 degradation as assessed
by pulse-chase experiments (31.6 ± 3.8% of kinase remaining after 1 h
compared with 51 ± 10% in control conditions)
(Fig. 2A). Moreover,
the expression of an active PKC mutant (PKC*) that stimulates MAPK
activity in a Raf-dependent manner
(24) also enhances the GRK2
degradation rate (32.5 ± 0.7% of kinase remaining after 1 h of chase)
(Fig. 2B). Similar
patterns of GRK2 decay were also detected by expressing a constitutively
active Ras mutant (data not shown). The augmented component of GRK2
proteolysis promoted by Raf* in HEK-293 cells was completely abolished by
co-expression of a dominant-negative ERK1 mutant
(Fig. 2C), thereby
confirming that the effect observed was mediated through MAPK. Overall, these
results pointed to a role for MAPK stimulation in GRK2 turnover.
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In order to validate the potential biological significance of this regulatory mechanism, we analyzed the role of MAPK activity in the degradation of endogenous GRK2 in Jurkat T cells. We have reported previously (20) that GRK2 was rapidly degraded by the proteasome pathway in these cells and that this process was enhanced by activation of the endogenous chemokine receptor CXCR4 and diminished by tyrosine kinase inhibitors. As shown in Fig. 3A, the presence of the MEK inhibitor PD098059, which strongly reduces MAPK activation (not shown), markedly impairs endogenous GRK2 degradation as assessed by pulse-chase experiments. Consistently, steady-state GRK2 levels detected by immunoblot analysis are increased in Jurkat cells in these conditions (Fig. 3B). Such increase is specific, because the amount of other cellular proteins remains unaltered (Fig. 3B, lower panel).
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The effect of MAPK on GRK2 degradation could be a consequence of the direct phosphorylation of GRK2 by MAPK or an indirect effect caused by MAPK-mediated phosphorylation of other proteins. In this regard, it has been reported previously that GRK2 is phosphorylated in vivo at the MAPK consensus phosphorylation site Ser-670 (22). To address the potential role of direct MAPK phosphorylation of GRK2 on its degradation, we generated mutants at Ser-670 to both mimic MAPK phosphorylation (S670D) or to prevent it (S670A). As shown in Fig. 4, the turnover of the S670D mutant transiently expressed in HEK-293 cells was clearly increased as compared with wild-type GRK2, and its degradation rate was very similar to that observed for GRK2 upon receptor stimulation (19) (Fig. 4) or in the presence of upstream activators of the MAPK pathway (30.5 ± 5.7% of GRK2-S670D protein remaining after 1 h of chase versus 32.5 ± 0.7% of wild-type protein in the presence of Raf*). Proteolysis of GRK2-S670D was blocked by specific inhibitors of the proteasome pathway (not shown) which also prevent degradation of wild-type GRK2 upon GPCR activation (19). These data are consistent with the notion that receptor occupancy leads to MAPK activation and that direct phosphorylation of GRK2 by MAPK triggers its degradation.
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Surprisingly, basal degradation of the GRK2-S670A mutant was not
significantly retarded when compared with that of wild-type GRK2
(Fig. 4) and takes place by the
proteasome pathway, because specific inhibitors efficiently blocked
proteolysis (not shown). However, the marked increase in wild-type GRK2
degradation that takes place in the presence of -adrenergic agonists is
not observed with the GRK2-S670A mutant
(Fig. 4). These results
suggested that GRK2 phosphorylation by MAPK plays a role in accelerating GRK2
degradation, although such covalent modification is not strictly required for
proteasomal targeting of the kinase, pointing to the occurrence of alternative
and independent pathways leading to GRK2 degradation.
We next analyzed in more detail the mechanisms underlying GRK2-S670A turnover. GPCR can sequentially activate c-Src and MAPK, and GRK2 can be phosphorylated by both kinases in an agonist-dependent way. Since we have recently shown (20) that c-Src-mediated phosphorylation is involved in GRK2 proteolysis, we explored the potential role of tyrosine phosphorylation on GRK2-S670A degradation. Thus, tyrosine residues critical for c-Src phosphorylation (20) were mutated to phenylalanine in the GRK2-S670A construct. Interestingly, the turnover of this combined mutant was severely impaired as compared with GRK2-S670A or wild-type GRK2 (81.6 ± 5.5% of protein remaining after 1 h of chase) (Fig. 5A) and is more similar to the pattern described for the Y13F/Y86F/Y92F mutant (84 ± 6% of labeled protein remaining at 1 h of chase). These data indicate that when the potential modulation of GRK2 degradation by MAPK-phosphorylation is abolished, the proteolysis takes place through a tyrosine phosphorylation-dependent mechanism, suggesting the involvement of different regulatory processes in GRK2 degradation. Interestingly, mutation of the residues critical for c-Src phosphorylation in the S670D mutant (GRK2-Y13F/Y86F/Y92F-S670D) has no effect on its turnover (Fig. 5B). Moreover, co-transfection with a catalytically inactive mutant of the kinase domain of c-Src (SH1-KD), which does block wild-type GRK2 degradation (20), does not alter the rate of degradation of the GRK2-S670D mutant (Fig. 5B). These results would suggest that once GRK2 is phosphorylated by MAPK, its degradation could take place independently of Src activity.
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Overall, these data suggest that c-Src and MAPK-mediated phosphorylation
target GRK2 to the proteasome pathway independently. Once GRK2 is
phosphorylated by MAPK, it proceeds for degradation, and tyrosine
phosphorylation is dispensable. If GRK2 cannot be phosphorylated by MAPK,
proteolysis takes place through a tyrosine phosphorylation-dependent pathway.
In fact, both wild-type GRK2 and S670A mutant are efficiently
tyrosine-phosphorylated upon 2AR stimulation (data not
shown). However, some kind of functional relationship appears to occur between
these two turnover mechanisms. Fig.
5C shows that the expression of constitutively active Raf
does not increase the degradation rate of the Y13F/Y86F/Y92F GRK2 mutant nor
does the presence of the MEK inhibitor PD98059 further retard the GRK2
Y13F/Y86F/Y92F turnover. These data suggest that, for wild-type GRK2, MAPK
phosphorylation might be preferentially modulating the stability of the
fraction of the GRK2 cellular pool previously phosphorylated on tyrosine
residues. Experiments of stimulation of endogenous CXCR4 receptors in Jurkat
cells are consistent with this sequential model. Activation of CXCR4 receptors
leads to both Src and MAPK stimulation, although activation of the latter is
not dependent on tyrosine kinase activity (data not shown). We observed that
CXCR4 stimulation promotes the rapid phosphorylation of endogenous GRK2 on
serine 670, as assessed with a specific antibody raised against this
phosphorylated epitope (Fig.
5D). Interestingly, the phosphorylation of this MAPK site
is not observed in the presence of the c-Src tyrosine kinase inhibitor PP2,
although MAPK activation is not abrogated under these conditions.
It is interesting to note that a given cellular context is needed for
efficient phosphorylation-mediated regulation of GRK2 stability by MAPK. In
this regard, we have reported previously
(19) that the kinase-dead
GRK2-K220R is barely degraded when expressed in cells. This is due to its
inability to promote the recruitment of -arrestin and associated
molecules such as c-Src to the vicinity of the receptor, because
-arrestin overexpression can "rescue" GRK2-K220R turnover
(20). Interestingly,
overexpression of Raf* does not promote GRK2-K220R proteolysis
(Fig. 6A), supporting
the hypothesis that additional factors are required for MAPK-induced GRK2
turnover. Therefore, we explored whether co-transfection of -arrestin1
with GRK2-K220R and Raf* had any effect on the degradation of this mutant. The
presence of Raf* causes a clear additional increase in the turnover of
GRK2-K220R promoted by -arrestin overexpression
(Fig. 6A, lower
panel). In other experiments, HEK-293 cells were co-transfected with Raf*
and wild-type GRK2 in the presence of different -arrestin1 mutants
(Fig. 6B). Expression
of wild-type -arrestin1 had no effect on the enhanced GRK2 degradation
promoted by Raf*, indicating that endogenous -arrestin levels in these
cells are sufficient to support GRK2 degradation as demonstrated previously
(20). On the contrary,
expression of -arrestin-S412D (which is unable to recruit c-Src, Ref.
18) blocked the degradation
induced by Raf*, whereas -arrestin-S412A (which binds efficiently to
Src) has the same effect as wild-type -arrestin.
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Overall, our results demonstrate that MAPK-mediated control of GRK2
turnover normally requires phosphorylation of GRK2 on tyrosine residues by
c-Src and the involvement of -arrestin as a protein adaptor. This is in
agreement with the ability of -arrestin to recruit c-Src
(18) and to assemble signaling
complexes involving Raf and Erk
(10), and possibly additional
proteins needed to promote GRK2 degradation (see
"Discussion").
The complex mechanisms of control of GRK2 stability suggest that such tight
modulation could have a role in the regulation of GPCR signaling. Therefore,
we explored whether modifications in GRK2 levels and turnover rate may alter
subsequent cellular responses to GPCR agonists. In a first set of experiments,
we found that chronic agonist challenge of endogenous AR in HEK-293
cells clearly down-regulates endogenous GRK2 levels (0.56 ± 0.1 of
cellular GRK2 complement after isoproterenol treatment as compared with
control cells incubated with vehicle, Fig.
7A). Interestingly, MAPK activation in response to LPA
was clearly enhanced in those cells with lower GRK2 levels as a result of
isoproterenol pretreatment (Fig.
7A, right panels, 4.2 ± 0.4-fold ERK2
activation over unstimulated conditions in pretreated cells versus
2.5 ± 0.5-fold activation in control cells, data from three independent
experiments with triplicate determinations). Therefore, a reduction in the
pool of GRK2 in an endogenous system appears to favor subsequent
responsiveness even to other GPCR. These results suggested that
agonist-stimulated degradation of GRK2 might contribute to preserve GPCR
responsiveness in physiological conditions characterized by sustained GPCR
stimulation. The importance of an adequate control of GRK2 turnover in GPCR
signaling is also supported by similar experiments performed in COS-7 cells
overexpressing 2AR and either wild-type GRK2 or the
GRK2-S670A mutant (Fig.
7B). As observed in the endogenous system, wild-type GRK2
protein levels declined upon chronic 2AR stimulation (0.63
± 0.08 of kinase levels as compared with control conditions). However,
no significant changes in GRK2-S670A protein expression were observed in
response to isoproterenol, consistent with its deficient agonist-induced
degradation. When subsequent activation of endogenous LPA receptors was
tested, MAPK activation was clearly impaired as compared with wild-type
conditions in pretreated cells expressing the GRK2-S670A mutant. In these
experimental conditions, we do not observe in wild-type GRK2-overexpressing
cells the additional increase in MAPK activation in response to LPA as
consequence of isoproterenol pretreatment that is apparent in an endogenous
system. However, the clear disparity in MAPK response between wild-type GRK2
and GRK2-S670A-expressing cells is likely due to stability differences,
because distal components of the MAPK cascade are not altered, as epidermal
growth factor promotes similar MAPK activation in cells expressing either
wild-type or mutant GRK2 (data not shown). Taken together, these observations
strongly suggest that changes in GRK2 turnover are important for the
fine-tuning of signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The control of GRK2 stability emerges as a complex process involving
different signaling pathways. We have reported recently
(20) that recruitment of
-arrestin and Src-mediated GRK2 phosphorylation on tyrosine residues are
critical signals that trigger GRK2 degradation through the proteasome pathway.
A key question is how Src and MAPK-dependent mechanisms contribute to GRK2
turnover. In order to address this issue, we have investigated how different
experimental conditions affect the turnover of a variety of GRK2 mutants.
These include proteins lacking residues critical for MAPK (GRK2-S670A) or
c-Src (GRK2 Y13F/Y86F/Y92F)-mediated phosphorylation, a mutant that mimics
permanent phosphorylation by MAPK (GRK2–670D), or combinations of those
(GRK2 Y13F/Y86F/Y92F-S670A; GRK2 Y13F/Y86F/Y92F-S670D).
It is worth noting that, although the agonist-induced turnover of the GRK2
S670A mutant is significantly retarded with respect to the wild-type kinase,
the basal degradation is not blocked. This would suggest that MAPK-mediated
modulation is relevant for agonist-promoted turnover, although additional
signals can trigger GRK2 proteolysis. The alternative pathway for GRK2-S670A
degradation appears to be dependent on tyrosine phosphorylation, because the
turnover of the combined Y13F/Y86F/Y92F-S670A mutant is severely impaired. On
the contrary, when permanent phosphorylation by MAPK is mimicked by the S670D
mutation, the protein rapidly proceeds to degradation, even if Src activity is
blocked or if tyrosine phosphorylation of the mutant is hampered by replacing
the critical tyrosine residues (GRK2-Y13F/Y86F/Y92F-S670D mutant). Overall,
these data suggest that either tyrosine or MAPK-dependent phosphorylation can
independently trigger GRK2 degradation by the proteasome pathway. However, our
results also indicate the occurrence of a "facilitatory" role of
GRK2 tyrosine phosphorylation for the subsequent MAPK-dependent control of
wild-type GRK2 turnover. The degradation of a GRK2 mutant unable to be
phosphorylated by Src, such as Y13F/Y86F/Y92F, is not enhanced nor inhibited
by activators or blockers of MAPK cascades, respectively. It could be
hypothesized that tyrosine-phosphorylated GRK2 is a better substrate for MAPK.
In fact, it has been reported that GRK2 must undergo some kind of
conformational change to be efficiently phosphorylated by MAPK
(22). In line with this, we
find that rapid agonist-induced phosphorylation of endogenous GRK2 in serine
670 is blocked in the presence of a c-Src inhibitor
(Fig. 5D).
Alternatively, or in addition, tyrosine-phosphorylated GRK2 could be present
in an adequate cellular context or multimolecular complex that facilitates the
subsequent interaction of GRK2 with MAPK and other molecules involved in the
degradation process. In this regard, we find that stimulation of the MAPK
cascade by active Raf* is unable to accelerate the slow GRK2-K220R turnover,
except if -arrestin is overexpressed (as in Ref.
20) thus pointing to the
involvement of -arrestin function also in MAPK-dependent modulation of
GRK2 stability. Moreover, the increased degradation of wild-type GRK2 detected
upon stimulation of MAPK cascades is blocked in the presence of a
-arrestin1 mutant (S412D) that is unable to recruit c-Src to the
receptor complex. These data underline the relevance of an adequate cellular
context for MAPK-mediated control of GRK2 stability.
It is worth noting that -arrestin function appears to play a central
role in both Src (20) and
MAPK-dependent GRK2 degradation processes. In addition to recruiting c-Src
(8) and thus allowing for GRK2
phosphorylation on tyrosine residues
(20,
26), -arrestin has been
reported to assemble key components of the MAPK cascade (Raf, MEK, and ERK) in
the vicinity of activated GPCR
(9,
10). The scaffold function of
-arrestin would contribute to the spatial and sequential organization of
the multimolecular complexes where the functional relationships among Src,
MAPK, and GRK2 could take place. Moreover, -arrestin has been reported
recently (27) to interact with
Mdm2 and other as yet unidentified ubiquitin ligases involved in
2AR and -arrestin ubiquitination. By recruiting these
or other ubiquitin-protein isopeptide ligases, -arrestin may also
contribute to link Src and/or MAPK-phosphorylated GRK2 to the degradation
machinery.
The scheme in Fig. 8 depicts
our current working model for the molecular mechanisms of GRK2 degradation
through the proteasome pathway. Receptor activation would promote the
sequential recruitment of GRK2, -arrestin, and c-Src to the GPCR
complex, resulting in tyrosine phosphorylation of GRK2
(26). This process can be
inhibited at several steps, leading to impaired GRK2 degradation. GRK2-K220R
would bind to the receptor but not promote the sequential binding of
-arrestin and c-Src (what can be rescued by -arrestin
overexpression); phosphorylation of GRK2 on tyrosine residues can be inhibited
by specifically blocking -arrestin/c-Src interaction with either
-arrestin S412D (8) or
the SH1KD constructs (28), by
pharmacological tyrosine kinase inhibitors (not shown in the scheme), or GRK2
mutants lacking critical residues for c-Src-mediated phosphorylation
(20). Tyrosine phosphorylation
of GRK2 would be sufficient for triggering kinase proteolysis through the
proteasome pathway. In addition, it would facilitate GRK2 phosphorylation by
MAPK and a subsequent, additional targeting route to the proteasome. Both
degradation pathways would contribute to GRK2 turnover upon GPCR stimulation,
consistent with the fact that this process is retarded in the presence of MAPK
cascade inhibitors or in the GRK2-S670A mutant. Although previous
phosphorylation by c-Src is normally required, mimicking GRK2 phosphorylation
by MAPK as in the S670D mutants would directly lead to rapid degradation
through the latter pathway independent of phosphorylation by c-Src. Such a
model for the control of GRK2 stability would allow for the fine regulation of
GRK2 turnover rates, integrating inputs such as GPCR stimulation and the
activity of cytosolic tyrosine kinases and MAPK cascades.
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An increasing number of reports show that protein phosphorylation functions as a regulatory label for triggering proteasomal degradation (29) of a variety of signaling proteins. Phosphorylation on tyrosine residues can trigger ubiquitination and degradation of the epidermal growth factor receptor, Src-kinases, E-cadherin or JAK-2 by promoting their interaction with specific ubiquitin-protein isopeptide ligase adaptors (30–33). Recent examples of MAPK phosphorylation-dependent proteasomal degradation include the inducible cAMP early repressor ICER, the TAL1/SC2 transcription factor, cell cycle inhibitors, or the upstream kinase Ste11 in yeast (34–37). The identification of proteins able to interact specifically with tyrosine or MAPK-phosphorylated GRK2 that could help target the kinase for degradation is currently underway in our laboratory.
The existence of these complex mechanisms of control of GRK2 stability
appears to play a relevant physiological role as a feedback mechanism that
would modulate receptor responsiveness, limiting the extent of desensitization
upon chronic receptor stimulation under physiological and pathological
conditions. As shown in this report, persistent 2AR
activation decreases GRK2 cellular levels and allows an enhanced MAPK
activation in response to other GPCR agonist such as LPA. However, when such a
normal decrease in GRK2 levels upon GPCR stimulation is impaired by
suppressing the MAPK pathway component in the GRK2-S670A mutant, subsequent
activation of the MAPK cascade by LPA is clearly reduced as compared with
wild-type conditions. These results further confirm the occurrence of a strong
functional relationship between GRK2 cellular levels and signaling to MAPK
cascades. Consistent with our data, decreased GRK2 cellular levels such as
those present in heterozygous GRK2+/– mice leads to enhanced
chemokine-mediated MAPK cascade
activation.2,3
On the contrary, recent reports indicate that increased GRK2 cellular levels
decrease -adrenergic activation of the ERK/MAPK cascade
(38,
39) or chemokine
receptor-mediated MAPK
stimulation.3
The existence of different pathways for the modulation of GRK2 stability
may help understand its altered cellular levels in different pathologies and
put forward new potential feedback mechanisms for regulating GPCR signaling.
Such control mechanisms might be particularly relevant in pathological
conditions characterized both by increased GPCR-mediated stimulation of Src
and MAPK cascades and by altered GRK2 cellular levels, such as in heart
failure, cardiac hypertrophy or hypertension (increased GRK2 levels), or
chronic inflammation (decreased kinase levels)
(20). It should be stressed,
however, that phosphorylation-dependent modulation of GRK2 stability is not
the only factor determining GRK2 cellular levels. A better knowledge of the
signals governing GRK2 gene transcription is needed to better understand how
this balance is altered in pathological situations characterized by GRK2
expression changes. It is also possible that such rapid processes of GRK2
turnover are not mainly intended to promote marked changes in steady-state
GRK2 cellular levels but to preferentially target for degradation the active
pool of GRK2 as a rapid feedback mechanism, as recently suggested for
MAPK-dependent Ste11 degradation
(35). Finally, in the light of
recent reports (40,
41) suggesting the possible
involvement of GRK2 and -arrestin in receptor tyrosine kinase regulation
and signaling, our finding of Src and MAPK phosphorylation-dependent
modulation of GRK2 proteolysis suggests new potential cross-talk mechanisms
between signaling pathways that deserve to be explored in the future.
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FOOTNOTES |
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Both authors contributed equally to this work.
Recipient of a postdoctoral contract of the I3P Programme of the Consejo
Superior de Investigaciones Científicas.
¶ To whom correspondence should be addressed: Centro de Biología Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Tel.: 34-91-397-48-65; Fax: 34-91-397-47-99; E-mail: fmayor{at}cbm.uam.es.
1 The abbreviations used are: GPCR, G protein-coupled receptors;
2AR, 2-adrenergic receptor; ERK,
extracellular signal-regulated kinase; LPA, lysophosphatidic acid; MAPK,
mitogen-activated protein kinase; MEK, MAPK kinase; PKC, protein kinase C;
GRKs, G protein-coupled receptor kinases.
2 A. Vroon, C. J. Heijnen, M. S. Lombardi, P. M. Cobelins, F. Mayor, M. G.
Caron, and A. Kavelaars, submitted for publication.
3 M. C. Jimenez, C. Murga, F. Mayor, and A. Aragay, manuscript in
preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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