From the Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Box 1112, Blindern, N-0317 Oslo, Norway
Received for publication, November 8, 2002, and in revised form, February 17, 2003
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ABSTRACT |
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Regulation of Src kinase activity is tightly
coupled to the phosphorylation status of the C-terminal regulatory
tyrosine Tyr527, which, when phosphorylated by Csk,
represses Src. Here, we demonstrate that activation of Csk through a
prostaglandin E2-cAMP-protein kinase A (PKA) pathway
inhibits Src. This inhibitory pathway is operative in
detergent-resistant membrane fractions where cAMP-elevating agents
activate Csk, resulting in a concomitant decrease in Src activity. The
inhibitory effect on Src depends on a detergent-resistant membrane-anchored Csk and co-localization of all components of the
inhibitory pathway in membrane microdomains. Furthermore, epidermal
growth factor-induced activation of Src and phosphorylation of the Src
substrates Cbl and focal adhesion kinase are inhibited by activation of
the cAMP-PKA-Csk pathway. We propose a novel mechanism whereby G
protein-coupled receptors inhibit Src signaling by activation of Csk in
a cAMP-PKA-dependent manner.
Src family kinases
(SFKs)1 are involved in a
variety of signal-transducing events leading to diverse cellular
processes such as migration, adhesion, proliferation, differentiation,
and survival (1). In mammals, eight different SFKs have been described
(2). Each member has a distinct unique domain, but all SFKs have in common that they contain acylation site(s) for membrane localization and single SH3, SH2, and protein-tyrosine kinase domains (3). In
addition, SFKs contain two critical tyrosine residues (corresponding to
Tyr527 and Tyr416 in Src) important for overall
regulation of SFK activity. When phosphorylated by the ubiquitously
expressed C-terminal Src kinase, Csk, the C-terminal tyrosine
(Tyr527) interacts with the SH2 domain, resulting in
repression of the catalytic activity (4-9). The other regulatory
tyrosine residue (Tyr416) is located in the activation
loop, and autophosphorylation of this site is required for full
catalytic activity. Thus, induction of Src kinase activity involves
sequential displacement of the SH2 domain from phosphorylated
Tyr527 and/or dephosphorylation of Tyr527 (by
PTPases such as RPTP Due to single or double acylations, SFKs are associated with the inner
leaflet of the plasma membrane and typically partition into
detergent-resistant membranes (DRMs), which is suggested as an overall
term including both lipid rafts and caveolae (13). Data indicate that
lipid rafts and caveolae function as platforms for the
signal-transducing machinery (14) and serve for example to position
kinases and their substrates in close proximity for signal transduction
events to occur rapidly upon the appropriate signal. The functional
significance of lipid rafts as signaling units has best been
characterized downstream of the T cell receptor, where the integrity of
lipid rafts has been shown essential for proper signaling (15).
Several signal transduction pathways induce SFK activity, including
immunoreceptors, cytokine receptors, G protein-coupled receptors
(GPCRs), integrins, and receptor tyrosine kinases (e.g. the
EGF and the PDGF receptors (EGFRs and PDGFRs, respectively)) (1). Both
EGFRs and PDGFRs are activated by dimerization followed by
trans-autophosphorylation, giving rise to docking sites for a variety
of signaling molecules such as phosphatidylinositol 3-kinase,
phospholipase C DRM-associated SFKs are negatively regulated through C-terminal
phosphorylation catalyzed by a DRM-associated pool of Csk (20-23). Csk
is recruited to membrane microdomains via binding of its SH2 domain to
phosphorylated Tyr317 in the phosphoprotein Cbp/PAG, which
is ubiquitously expressed and partitions exclusively into the DRM
fraction (20, 21). A similar SH2-phosphotyrosine-mediated interaction
has recently been demonstrated between Csk and Tyr14 in
caveolin, which is an important constituent of caveolae (24). In
addition to spatial regulation, different mechanisms for modulation of
Csk kinase activity have been described. First, Csk kinase activity is
induced 2-4-fold by binding of the Csk SH2 domain to Cbp/PAG (25).
Second, G PKA, Csk, and SFKs are present in all cell types. Therefore, we
hypothesized that PKA-mediated Csk activation could regulate C-terminal
phosphorylation and activity of other SFKs in addition to Lck. Here, we
demonstrate that an adenylyl cyclase (AC)-PKA-Csk inhibitory pathway
down-regulates the kinase activity of Src in both human embryonic
kidney (HEK293) cells and NIH-3T3 fibroblasts, suggesting the presence
of a general principle for PKA-mediated inhibition of SFKs through
activation of Csk. We further suggest that the PKA-Csk-mediated Src
down-regulation plays an important role in modulating signaling
downstream of Src.
Reagents and Antibodies--
EGF, prostaglandin E2,
and methyl- Cell Culture and Transfections--
The human leukemia T cell
line Jurkat TAg, a derivative of the Jurkat cell line stably
transfected with the SV40 large T antigen, was kept in logarithmic
growth in RPMI medium supplemented with 10% fetal calf serum and
antibiotics. HEK293 (human embryonic kidney cells, purchased from ATCC
(Manassas, VA), catalog no. CRL-1573) and mouse NIH-3T3 and
NIH-3T3-A14 cells (mouse NIH-3T3 cells expressing insulin receptor,
kindly provided by Boudewijn Burgering, University Medical Center of
Utrecht, The Netherlands) were kept in logarithmic growth in
minimal essential medium supplemented with glutamine, sodium pyruvate,
nonessential amino acids, 10% fetal calf serum, and antibiotics
(penicillin and streptomycin) and split by trypsination at less than
80% confluence. When indicated, cells were serum-starved for 12-16 h
before cell stimulation. For transfections, Jurkat TAg cells (2 × 107) in 0.4 ml of Opti-MEM were mixed with 10 µg of each
DNA construct in electroporation cuvettes with a 0.4-cm electrode gap
(Bio-Rad) and subjected to an electric field of 250 V/cm with
960-microfarad capacitance. The cells were expanded in complete medium
and harvested 20 h post-transfection. Transfection of HEK293,
NIH-3T3, and NIH-3T3-A14 cells were performed with LipofectAMINE
according to the manufacturer's instruction (1:2.5 DNA/LipofectAMINE
in Opti-MEM). After incubation for 5 h at 37 °C, the
transfection solution was removed, and regular medium was added. Cells
were harvested by trypsination 20-24 h later.
Purification of DRMs--
Isolation of detergent-resistant
membranes was performed as described in detail elsewhere (27). Briefly,
cells were homogenized in 1 ml of an ice-cold standard lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM
EDTA, 0.7% Triton X-100 with 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, and 50 mM sodium fluoride) by 10 pestle
strokes in a Dounce homogenizer, loaded at the bottom of a 40 to 5%
sucrose gradient (in 25 mM MES, pH 6.5, 5 mM
EDTA, 150 mM NaCl) and ultracentrifuged at 200,000 × g for 20 h at 4 °C. Fractions (0.4 ml) were
collected from the top. In the presence of ATP (1 mM) and
MgCl2 (15 mM), DRM fractions were stimulated
with or without forskolin (100 µM, 10 min if not
otherwise stated) or prostaglandin E2 (100 µM, 2 min if not otherwise stated) at 30 °C;
thereafter, either cAMP measurements or immunoprecipitations were
conducted. Mixed DRM fractions subjected to immunoprecipitations were
always solubilized with 50 mM
n-octyl- Stimulation of Cells and Immunoprecipitation--
Cells were
stimulated with forskolin (100 µM, 10 min if not
otherwise stated) or prostaglandin E2 (100 µM, 2 min if not otherwise stated) in the absence and
presence of EGF (100 ng/ml) at 37 °C and then disrupted in ice-cold
standard lysis buffer containing n-octyl- Src Kinase Assay--
Src was immunoprecipitated by using
monoclonal Src antibodies (clone GD11; Upstate Biotechnology) and
washed three times in standard lysis buffer, and then precipitates were
divided into three or four. Immunoblot analysis was either conducted on
precipitates directly using phosphotyrosine-specific antibodies toward
Tyr527 in Src or nonphospho-416 in Src or monoclonal
Src antibodies or washed three times in a buffer containing Hepes (50 mM, pH 7.4) and MgCl2 (5 mM) and
then assayed for autophosphorylation in the presence of 1 mM ATP at 30 °C for 10 min. Reactions were stopped by
the addition of SDS sample buffer, and immunoblot analysis was
performed by using phosphotyrosine-specific antibodies toward Tyr416 in Src. When Src phosphotransferase activity was
measured toward poly(Glu,Tyr) 4:1 (Sigma) as a substrate, the protocol
was as for a Csk kinase assay.
Csk Kinase Assay--
Csk was immunoprecipitated with polyclonal
antibodies and washed three times in standard lysis buffer. After three
additional washes in Hepes buffer (50 mM, pH 7.4) with 50 MgCl2, the Csk phosphotransferase activity was measured as
incorporation of [32P]phosphate into the synthetic
polyamino acid poly(Glu,Tyr) 4:1 (Sigma). A standard protocol was
followed (28) with reaction volumes of 50 µl containing 50 mM Hepes buffer, pH 7.4, 5 mM
MgCl2, 200 µM [ Cyclic AMP Assay--
A standard cAMP assay (cAMP kit
from PerkinElmer Life Sciences; catalog no. SP004) was performed
in accordance with the manufacturer's instructions.
Protein Measurements--
Proteins were quantified by the method
of Bradford (29) using Generation of Constructs--
The different Csk constructs
employed have been described elsewhere (22). The plasmids encoding wild
type Src or Src-Y527F were purchased from Upstate Biotechnology
(catalog nos. 21-114 and 21-115). The Src-S17A mutant was generated by
site-directed mutagenesis (QuikChange; Stratagene) of Src wild type,
and the resulting construct was sequenced (GATC Biotech).
The cAMP-PKA Pathway Negatively Regulates Src--
We have
previously shown that cAMP/PKA, through phosphorylation
dependent activation of Csk, specifically interfere with and inhibit Lck-mediated signaling in T cells (23). To test the hypothesis
that a general pathway from GPCRs via cAMP and PKA to Csk negatively
regulates different SFKs, we studied HEK293 cells. These cells are of
epithelial origin (proximal tubules in kidney) and express the SFK Src.
Treatment of HEK293 cells with the cAMP-elevating agent forskolin and
subsequent kinase assay with immunoprecipitated Src revealed strongly
reduced ability to autophosphorylate on Tyr416
(densitometric scanning analysis: 4.8 ± 1.5-fold decrease,
average ± S.E. n = 3), whereas there was a
concomitant increase in phosphorylation of Tyr527 (1.6 ± 0.1-fold increase, average ± S.E., n = 3)
(Fig. 1A). Similar results
were obtained when immunoprecipitated Src not subjected to the
autophosphorylation assay was analyzed for Tyr(P)416
(1.5 ± 0.2-fold decrease with forskolin, average ± S.E.,
n = 3) or Tyr(P)527 (1.9 ± 0.1-fold
increase with forskolin, average ± S.E., n = 3)
levels (data not shown). The phosphotransferase activity of Src toward
the synthetic polyamino acid poly(Glu,Tyr) was also lowered upon
forskolin treatment of HEK cells (Fig. 1B). This points
toward a role for cAMP in down-regulation of the kinase activity of
Src. Furthermore, the physiological cAMP-elevating agent prostaglandin
E2 (PGE2) had an inhibitory effect on Src similar to that of forskolin (Fig. 1C). Pretreatment of
cells with the PKA inhibitor H89 blocked this effect, implicating PKA in cAMP-mediated regulation of Src (Fig. 1C). Similar
results were also obtained in NIH-3T3-A14 cells (fibroblasts) (Fig.
1D) and in HEK293 cells treated with forskolin in the
presence or absence of another PKA inhibitor, KT5720 (100 nM, 15 min, data not shown). Taken together, these results
suggest that cAMP is acting through PKA via Csk to inhibit Src in
these cell types.
The cAMP-PKA Inhibitory Pathway Is Functional in DRM Fractions and
Lowers Src Kinase Activity in Response to cAMP-elevating
Agents--
Since all SFKs have lipid modifications involved in
membrane targeting, we wanted to investigate the inhibitory effect of cAMP on Src in the DRM fractions of HEK293 cells. Upon sucrose gradient
fractionation of HEK293 cells, less than 0.5% of total cellular
protein was found in the DRM fractions (fractions 2-5, Fig.
2A) compared with the soluble
fractions (fractions 9-12). Nevertheless, upon the addition of Mg-ATP
to the different fractions to reconstitute AC activity,
forskolin-induced cAMP production peaked in the DRM fractions (Fig.
2B). Similarly, PGE2 also induced cAMP
production in DRM fractions (data not shown), indicating the presence
of both PGE2 receptors and AC in membrane microdomains. Furthermore, small amounts of PKA C and RI Expression of Dominant Negative Csk Disrupts Regulation of Src by
cAMP--
Csk phosphorylates Tyr527 in Src, and the
observation that cAMP induces phosphorylation of this site indicates a
role for Csk. Therefore, we next studied the effect of increased cAMP
on Csk activity in DRMs from HEK293 cells. Stimulation of
Mg-ATP-reconstituted DRM fractions with forskolin or PGE2
led to a 2-2.5-fold increase in Csk kinase activity (Fig.
3A). This is consistent with
previous findings in T cells (23). In order to identify a role for Csk in cAMP-induced Src inhibition, we overexpressed kinase-deficient Csk-SH3-SH2, an interfering mutant that can displace endogenous Csk
from its anchor protein Cbp/PAG in lipid rafts (22). When expressed in
HEK293 cells, this mutant was properly recruited into DRMs (Fig.
3B). Interestingly, expression of this mutant caused
spontaneous hyperphosphorylation of Tyr416 in
DRM-associated Src (Fig. 3C) and abolished the inhibitory effect of cAMP on Src kinase activity (Fig. 3D). In
conclusion, these results show that cAMP/PKA-mediated activation of Csk
down-regulates Src kinase activity in DRMs of HEK293 cells.
PKA Does Not Directly Phosphorylate Src in DRMs--
Previous
studies have indicated that Src can be phosphorylated on
Ser17 by PKA, but no physiological role for this
phosphorylation was demonstrated (3). However, it was recently reported
that direct phosphorylation of Ser17 in Src by PKA
up-regulates Src kinase activity (30). To assess direct regulation of
Src versus indirect PKA-mediated regulation via Csk in DRMs,
we overexpressed either wild type Src or mutant Src-S17A in HEK293
cells. The contribution of endogenous Src in these experiments was
negligible due to very low expression levels compared with those of the
transfected constructs (Fig.
4A, upper panel). Both wild type Src and Src-S17A partitioned into DRM
fractions, and compared with control transfected cells (empty vector),
the levels of these proteins in DRMs exceeded endogenous Src protein levels severalfold (Fig. 4A, lower
panels). Although forskolin treatment reduced
Tyr416 phosphorylation of both DRM-associated wild type Src
and Src-S17A, no changes in DRM association were observed (Fig.
4B). Furthermore, wild type Src and Src-S17A
immunoprecipitated from DRM fractions stimulated with forskolin
revealed comparable reductions in Tyr416 phosphorylation,
suggesting that phosphorylation of Ser17 does not appear to
regulate Src activity (Fig. 4C). This is also consistent
with previous findings (31). Treatment with forskolin did not change
the phosphorylation status of Ser17 of immunoprecipitated
wild type Src, as indicated after immunoblotting with a phosphospecific
PKA-substrate antibody (Fig. 4C). However, PKA was clearly
activated by forskolin in these experiments, since whole cell lysates
analyzed with the phospho-specific PKA-substrate antibody showed
increased phosphorylation compared with unstimulated cells (data not
shown). Additionally, no significant differences were seen in
phosphorylation status of Tyr416 in wild type Src compared
with Src-S17A in Jurkat TAg cells stimulated with forskolin (Fig.
4D). This indicates that in T cells, although normally not
expressing Src, direct phosphorylation of Ser17 of Src is
not likely to be involved in regulation of Src kinase activity.
Constitutive phosphorylation of Ser17 in Src to ~60%
stoichiometry in vivo has been reported (3). Since our
results showed basal Ser17 phosphorylation that was not
altered by forskolin-induced PKA activation (Fig. 4C), we
speculated that PKA-mediated Ser17 phosphorylation was not
physiologically relevant in this cellular setting. To fully study the
impact of the PKA-mediated Ser17 phosphorylation, we
incubated immunoprecipitated wild type Src or Src-S17A with increasing
amounts of highly active recombinant PKA C subunit in the presence of
Mg-ATP. Neither in wild type Src nor in Src-S17A did PKA change
Ser17 phosphorylation, and autophosphorylation assays
revealed that Tyr416 phosphorylation was unaffected (Fig.
4E). PKA was clearly active, since it autophosphorylated in
the same assay and was able to phosphorylate and activate recombinant
Csk in a separate assay (data not shown). Thus, in the absence of the
inhibitory Csk, PKA neither increased phosphorylation of
Ser17 in Src nor affected the activity of Src or Src-S17A.
In conclusion, DRM-associated Src is not directly phosphorylated by PKA
in response to elevated cAMP in the cells examined, and the inhibitory
effect of cAMP on Src in DRMs is therefore not mediated directly by PKA but rather via an indirect pathway involving Csk.
The cAMP-PKA-Csk Pathway Regulates EGFR Signaling through
Src--
Since cAMP indirectly inhibits Src kinase activity, we next
wanted to assess the impact of this inhibitory pathway in a setting where Src normally would be activated. The EGFR, which partitions into
DRMs (Fig. 5A) (17), activates
Src upon stimulation with EGF. We therefore wanted to investigate
whether the AC-PKA-Csk pathway could intersect EGF-induced Src
activation. Upon stimulation of HEK293 cells with EGF,
Tyr416 phosphorylation of both DRM-associated Src and total
cellular Src was increased. (Fig. 5, B and C,
respectively). Similar results were also obtained with whole cell
lysates from NIH-3T3-A14 cells (data not shown). Interestingly,
EGF- induced activation of Src was significantly inhibited when cells
were pretreated with forskolin (Fig. 5C). This could not be
explained by lowered tyrosine phosphorylation of EGFR, since its
phosphorylation status was unaffected by forskolin pretreatment in this
system (Fig. 5D). However, overexpression of dominant
negative Csk-SH3-SH2 fully restored EGF-induced Src activation even in
the presence of forskolin (Fig. 5E). Thus, in a
physiological setting cAMP can interfere with receptor tyrosine kinase-induced Src activation in a Csk-dependent
manner.
Cyclic AMP Inhibits EGF-induced Src Signaling toward Cbl and
FAK--
Since cAMP inhibits EGF-induced Src activation, we next
investigated whether this could be reflected in reduced activation of
proteins downstream of Src. Cbl is phosphorylated by Src upon EGFR
activation (32) (our own observations in HEK293 and NIH-3T3-A14 cells;
data not shown) and thereby contributes to receptor internalization (18). Both in HEK293 cells and in A14 cells, EGF-induced tyrosine phosphorylation of Cbl was clearly inhibited when cells were pretreated with forskolin (Fig. 6, A and
B). Another Src substrate, the focal adhesion kinase (FAK),
was also inducibly tyrosine-phosphorylated upon EGF stimulation of
NIH-3T3 transfected with wild-type Src, whereas forskolin pretreatment
inhibited this phosphorylation (Fig. 6C). In contrast, the
inhibitory effects of forskolin on EGF-induced FAK phosphorylation were
abolished in NIH-3T3 cells transfected with Src-Y527F, which cannot be
regulated by Csk (Fig. 6C), suggesting that the inhibitory
effect of cAMP is dependent on Tyr527 in Src. Last,
EGF-induced phosphorylation of Stat3 mediated through Src was also
inhibited by cAMP (not shown). Altogether, this indicates that the
cAMP/PKA pathway, through activation of Csk, intersects growth
factor-induced signaling mediated by Src in DRMs.
Cyclic AMP exerts inhibitory effects in many cell types and is
known to interfere with mitogenic signaling from growth factors (33).
Most of the studies published so far describing the inhibitory effect
of cAMP have focused on the inhibition of growth factor-induced extracellular signal-regulated kinase signaling by cAMP, which is
likely to be mediated by direct phosphorylation of Raf-1 by PKA (34,
35). Furthermore, we recently described a proximal inhibitory pathway
in T cell lipid rafts whereby cAMP, through PKA type I, activates Csk
by phosphorylation of Ser364 in Csk, leading to inhibition
of Lck and of T cell activation through the T cell receptor complex
(23). Here we demonstrate a new inhibitory pathway for regulation of
growth factor-induced signaling, in which GPCRs that signal through
cAMP-PKA activate Csk, which in turn directly inhibits Src activity.
Several distinct mechanisms of Src activation exist. The activity of
Src is tightly coupled to the phosphorylation status of the C-terminal
negatively regulatory Tyr527 (11, 36, 37), and a plausible
model for activation of Src includes initial dephosphorylation of
Tyr527. Furthermore, Src contains several other
phosphorylation sites that have modulatory effects on Src kinase
activity. For instance, increased Src activity at the onset of mitosis
correlates with increased phosphorylation of serine (Ser72)
and threonine (Thr34 and Thr46) residues in the
unique domain of Src, probably catalyzed by Cdc2-cyclin
complexes (38-41). However, since neutralizing mutations of these
residues do not abolish this activation of Src, one might speculate
that these phosphorylations modulate the effects of other regulatory
mechanisms acting on Src. Furthermore, Ser17
phosphorylation by PKA is reported to occur in vitro
(reviewed in Ref. 3) and to directly activate Src in NIH-3T3 cells
(30). However, this site does not appear to have physiological impact, since (i) Ser17 appears to be constitutively phosphorylated
in HEK293 cells (this report); (ii) we do not observe activation of Src
in response to cAMP in HEK293 cells, not even when Csk is displaced and
the inhibitory effect of PKA on Src via Csk is abolished (this report); (iii) we are not able to show PKA activation of Src in NIH-3T3 cells
(this report) as reported in Ref. 30. In addition, the Src SH3 and SH2
domains contain other phosphorylation sites that can modulate activity,
either by changing domain interactions or Src structure. Finally,
phosphorylation-independent activation of Src can occur via
G Notwithstanding the above-discussed modulating mechanisms, C-terminal
phosphorylation seems to be a crucial master switch in regulation of
Src, and factors regulating Csk activity and localization would
consequently be of physiological importance (43). In this study, we
show that the majority of Src in HEK293 cells partitions into the DRM
fraction, and given the established role of lipid rafts and caveolae in
signal transduction, this probably reflects a physiologically relevant
pool of Src involved in signal transduction. Thus, mechanisms
regulating the activity of DRM-associated Csk and the total amount of
Csk in DRMs are expected to affect Src signaling through caveolae/lipid
rafts. Indeed, csk knockout cells reveal a phenotype with
increased basal Tyr416 phosphorylation of Src and hardly
detectable Tyr527 phosphorylation (11, 12). In accordance
with this, we show that total amount of Csk associated with DRMs is
important for Src regulation, since profound effects on basal Src
kinase activity are observed by overexpression of dominant negative
Csk, which competes endogenous Csk from its DRM association provided by
Cbp/PAG. We also show that PKA-mediated phosphorylation and activation of endogenous DRM-associated Csk modulates Src kinase activity. This
provides a model for a novel mechanism of cAMP-mediated inhibition of
Src signaling by modulation of Csk activity. Importantly, this inhibitory pathway is able to interfere with growth factor-induced Src
signaling, demonstrating its physiological relevance. Growth factor
receptors such as the EGFR partition into DRMs, and our observations
suggest that EGF stimulation results in activation of DRM-associated
Src. Furthermore, physiological activation of Src upon EGF stimulation
was clearly inhibited by pretreatment with cAMP- elevating agents.
Previously, this has been explained by cAMP/PKA-mediated inhibition of
EGF-induced tyrosine phosphorylation of the EGFR, resulting in reduced
activation of Src (44). However, in the cell line studied here, no
changes in tyrosine phosphorylation of the EGFR were observed,
indicating that cAMP did not affect EGFR activation. In contrast,
overexpression of dominant negative Csk abolished the inhibitory effect
of cAMP on EGF-induced Src activation, which supports a role for Csk in
this respect. Finally, the inhibitory effect of increased cAMP on
EGF-induced phosphorylation of Cbl and FAK was profound. Regulation of
EGF-induced phosphorylation of Stat3 was also observed (not shown).
Altogether, this indicates that PKA through activation of Csk not only
regulates basal activity of DRM-associated Src but potentially also
intersects Src-mediated signaling in a physiological setting (model
presented in Fig. 7). This also seems to
be a general principle, since the same effects were seen in different
cell types such as HEK293 and NIH-3T3 cells.
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ABSTRACT
INTRODUCTION
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DISCUSSION
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) (10) and subsequent autophosphorylation of
Tyr416. The importance of Csk-mediated regulation of
Tyr527 is illustrated by the fact that disruption of the
csk gene is embryonically lethal, and studies of cell lines
established from these animals revealed that although Src expression
levels were reduced, relative Src kinase activity was increased
5-15-fold (11, 12). This was associated with very low levels of
Tyr527 phosphorylation in Src, whereas the phosphorylation
of Tyr416 was highly increased (11).
, Grb2/Sos, SHC, and Cbl. Prior to ligand binding,
40-60% of the EGFRs associate with DRMs, but rapidly after ligand
binding, the receptors migrate out of DRMs and into clathrin-coated
pits, where receptor-mediated endocytosis will occur (16, 17). Cbl
plays a dual role in the sorting and degradation process of the
receptors. First, Cbl binds to and ubiquitinylates the phosphorylated
receptors and thus sorts them for degradation by the proteasome.
Second, Src-mediated tyrosine phosphorylation of Cbl induces
conformational changes in Cbl. This facilitates binding of the
CIN85-endophilin complex and subsequent endocytosis and lysosomal
degradation of the activated receptors (18, 19).
can also increase Csk kinase activity 2-fold, probably
via interaction with the catalytic domain (26). Last, Csk kinase
activity can be regulated via covalent modification as well; in T
cells, we recently demonstrated a 2-4-fold induction of Csk
phosphotransferase activity after protein kinase A (PKA)-mediated phosphorylation of Ser364 in Csk, with a concomitant
increase in C-terminal inhibitory phosphorylation of lipid
raft-associated Lck (23).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin were purchased from Sigma;
n-octyl-
-D-glucoside was from U.S.
Biochemical Corp.; and H89 and forskolin were from Calbiochem.
Anti-Tyr(P) monoclonal antibody (4G10; Upstate Biotechnology,
Inc., Lake Placid, NY), anti-Csk, anti-LAT, anti-Grb2, anti-HA, and
antibodies toward PKA subunits RI
and C were as before (22, 23). The
phosphotyrosine-specific antibodies toward Tyr416 and
Tyr527 in Src were from BIOSOURCE
International (catalog no. 44-660 and 44-662, respectively), whereas
monoclonal anti-Src antibody was from Upstate Biotechnology (clone
GD11). Anti-caveolin (catalog no. C37120-150/610407) was from
Transduction Laboratories, and both anti-Cbl and anti-EGFR were from
Santa Cruz Biotechnology, Inc. (catalog no. sc-170 and sc-120,
respectively), and anti-FAK was from Upstate Biotechnology (catalog no.
05-537). PKA-substrate phosphospecific antibody (abbreviated
anti-RXX(PS/PT), where pS and pT represent
phosphoserine and phosphothreonine, respectively) was purchased from
Cell Signaling Technology and is reactive toward phosphorylated
threonine with arginine in position
3 or toward phosphoserine
with arginine in position
2 or
3. The antibody that recognizes Src
only when it is not phosphorylated on Tyr416
(nonphospho-416) was also purchased from Cell Signaling Technology (catalog no. 2102). Anti-Cbp/PAG was kindly provided by Dr.
Václav Horejsí (Institute of Molecular Genetics AS CR,
Prague, Czech Republic).
-D-glucoside.
-D-glucoside (50 mM) and
subjected to immunoprecipitation with the indicated antibodies. After
incubation at 4 °C for a minimum of 4 h, protein A/G-Sepharose
(Santa Cruz Biotechnology) was added, and the incubation continued for
1 h. Immune complexes were washed three times in lysis buffer and
subjected to Western blot analysis.
-32P]ATP
(0.15 Ci/mmol), 200 µg/ml poly(Glu,Tyr), and
immunoprecipitated Csk. The reactions were incubated at 30 °C for 12 min. Equal amounts of immunoprecipitated Csk in each kinase reaction
were verified by immunoblotting.
-globulin as a standard.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
Cyclic AMP-mediated activation of PKA
inhibits Src kinase activity in HEK293 and NIH-3T3-A14 cells.
A, cAMP inhibits Src activity. HEK293 cells were incubated
with or without forskolin (7 min) and then disrupted in standard lysis
buffer. Immunoprecipitated Src was divided into four aliquots, three of
which were immunoblotted with antibodies against either
nonphosphotyrosine 416 of Src, phosphotyrosine 527 of Src, or total
Src. The fourth aliquot was reconstituted with Mg-ATP and subjected to
an autophosphorylation kinase assay. Subsequently, the level of Src
Tyr416 phosphorylation was assessed using a phosphospecific
antibody. Densitometric scanning analyses of Western blots (normalized
for the amount of Src) are shown for both phosphorylated
Tyr416 (4.8 ± 1.5-fold decrease, average ± S.E., n = 3) and Tyr527 (1.6 ± 0.1-fold increase, average ± S.E., n = 3).
B, phosphotransferase activity of Src is lowered upon
forskolin treatment. HEK293 cells were stimulated with or without
forskolin as in A; thereafter, the phosphotransferase
activity of immunoprecipitated Src was assessed using poly(Glu,Tyr) as
a substrate. C, PGE2-induced inhibition of Src
is blocked by inhibition of PKA. HEK293 cells were pretreated with or
without the PKA inhibitor H89 (10 µM, 20 min) and then
stimulated with or without prostaglandin E2. After lysis of
cells, as in A, whole cell lysates were subjected to
immunoblotting with the indicated antibodies. Lysates corresponding to
equal amounts of cells were loaded in each lane. D,
forskolin-induced inhibition of Src is abolished by inhibition of PKA.
NIH-3T3-A14 cells were treated with forskolin (100 µM)
for the indicated times (min) with or without H89 (10 µM,
20 min), lysed, and subjected to immunoblotting with the indicated
antibodies. PY416, Tyr(P)416; PY527,
Tyr(P)527; Frsk, forskolin.
subunits as well as Csk
were found in DRM fractions of HEK293 cells, whereas substantial amounts of Src partitioned into these fractions (Fig. 2C).
Since the PKA-Csk-Lck pathway in T cells has been found localized to lipid rafts, we wanted to see if a similar pattern of regulation existed for Src in DRMs of HEK293 cells. Interestingly, forskolin treatment of HEK293 DRMs reconstituted with Mg-ATP was sufficient to
down-regulate the activity of Src associated with these fractions, as
indicated by increased Tyr527 phosphorylation (Fig.
2D), reduced Tyr416 autophosphorylation (Fig.
2D), and lowered phosphotransferase activity toward
poly(Glu,Tyr) (Fig. 2E). Similar results were also obtained
with lipid rafts from Jurkat TAg T-cells transfected with wild type Src
(Fig. 2D) and with DRMs from NIH-3T3 fibroblasts (Fig.
2E). Disruption of plasma membrane organization by
cholesterol depletion with methyl-
-cyclodextrin lowered the amounts
of Src, Csk, and PKA C subunit present in DRMs (Fig. 2F) but
not in soluble fractions (data not shown). As a result, disruption of
membrane microdomain integrity by methyl-
-cyclodextrin completely
abolished forskolin-induced Tyr527 phosphorylation of Src
(Fig. 2G). Altogether, these data show that all of the
components necessary for mediating the inhibitory effect of cAMP on Src
are present in DRMs and need to be properly organized to be fully
operative.
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Fig. 2.
All components of the GPCR-AC-PKA-Csk
inhibitory pathway are present in DRM fractions and lower Src kinase
activity in response to cAMP-elevating agents. A, only
about 0.5% of total cellular proteins are present in DRM fractions.
Unstimulated HEK293 cells were fractionated in a sucrose gradient, and
protein content of each fraction was determined by the method of
Bradford. B, AC activity is up-concentrated in DRM
fractions. Sucrose gradient-derived protein fractions reconstituted
with Mg-ATP were stimulated with forskolin (3 min), and cAMP production
in each fraction was determined. C, all components involved
in inhibition of Src are present in DRMs. Sucrose gradient-derived
protein fractions were analyzed by immunoblotting using indicated
antibodies. D, in DRMs of HEK293 and Jurkat TAg T cells,
cAMP inhibits Src activity. DRMs were isolated, mixed (fractions 2-5),
reconstituted with Mg-ATP, and treated with or without forskolin.
Subsequently, endogenous Src (HEK293) or transfected Src (Jurkat TAg)
were immunoprecipitated and subjected to autophosphorylation assays as
in Fig. 1A and detected with antibody against
Tyr(P)416 (PY416) of Src or directly analyzed
for Tyr527 (PY527) phosphorylation.
E, in DRMs from HEK293 or NIH-3T3 cells, cAMP inhibits Src
phosphotransferase activity. Isolated DRMs from HEK293 or NIH-3T3 cells
were treated as in D; thereafter, phosphotransferase
activity of immunoprecipitated (IP) Src toward poly(Gly,Tyr)
was assessed. F, the components of the inhibitory pathway do
not partition into DRMs after disruption with methyl- -cyclodextrin.
HEK293 cells were treated with or without methyl-
-cyclodextrin,
lysed, and subjected to sucrose gradient fractionation. The
DRM-containing fractions (2-5) from untreated or treated
cells were loaded on the same gel and analyzed with the indicated
antibodies. G, inhibition of Src occurs only in intact
membranes. HEK293 cells were preincubated in the presence or absence of
methyl-
-cyclodextrin (15 mM, 30 min, 37 °C) and
stimulated with forskolin for the indicated periods of time. For each
time point, equal amounts of cells were withdrawn, and cell lysates
were immunoblotted with the indicated antibodies.
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Fig. 3.
Cyclic AMP down-regulates Src kinase activity
in a Csk-dependent manner. A, cAMP
stimulates Csk activity. DRMs (fractions 2-5) from unstimulated HEK293
cells were reconstituted with Mg-ATP and subsequently stimulated with
either forskolin or PGE2. Thereafter, kinase activity of
immunoprecipitated Csk was assessed. Triplicate measurements are shown
(mean ± S.D.), data are representative of six independent
reactions. B, wild type Csk and kinase-deficient Csk
partition into DRMs. HEK293 cells were transfected with either
HA-tagged wild type Csk (Csk-wt) or HA-tagged
kinase-deficient Csk (Csk-SH3-SH2), lysed, and subjected to
sucrose gradient fractionation, followed by immunoblotting using HA
antibodies. C, overexpression of dominant negative,
kinase-deficient Csk results in hyperphosphorylation of
Tyr416 in endogenous Src. HEK293 cells transfected with
empty vector, wild type Csk (Csk-wt), or kinase-deficient
Csk (Csk-SH3-SH2) were subjected to sucrose gradient
fractionation. Activation status of Src immunoprecipitated from DRMs
was determined using phosphospecific antibodies against
Tyr416. D, kinase-deficient Csk abolishes
forskolin-induced inhibition of Src. HEK293 cells transfected with
empty vector or kinase-deficient Csk (Csk-SH3-SH2) were
stimulated with forskolin for the indicated times. Subsequently, equal
amounts of whole cell lysates (WCL) were subjected to
immunoblotting with antibodies toward phosphotyrosine 416 in Src.
PY416, Tyr(P)416.
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Fig. 4.
Cyclic AMP-induced phosphorylation of
Ser17 in Src does not affect Src kinase activity.
A, expression levels of different transfected Src constructs
exceed endogenous Src severalfold both in whole cell lysates
(WCL) and in DRM fractions. HEK293 cells transfected with
vector control or plasmids encoding wild type Src (Src-wt)
or mutant Src-S17A were lysed and either directly analyzed by
immunoblotting using Src antibodies or subjected to sucrose gradient
fractionation and then immunoblotted (equal exposure times for the
three lower panels). B, DRM
localization of Src is unchanged upon forskolin treatment. HEK293 cells
transfected with either wild type Src or mutant Src-S17A were treated
with forskolin, lysed, and sucrose gradient-fractionated. DRM fractions
were mixed and directly subjected to immunoblotting with the indicated
antibodies. C, phosphorylation on Ser17 has no
effect on Src activation. HEK293 cells transfected with either wild
type Src or mutant Src-S17A were treated with forskolin, lysed, and
sucrose gradient-fractionated. Thereafter, DRM fractions were mixed,
Src-immunoprecipitated, and analyzed with antibodies toward
phosphotyrosine 416 in Src, with a PKA substrate phosphospecific
antibody (anti-RXXPS/PT) or with antibody toward total Src,
respectively. D, cAMP reduces Tyr416
phosphorylation of transfected Src in Jurkat TAg cells. Wild type Src
or mutant Src-S17A were overexpressed in Jurkat TAg cells and
stimulated with forskolin, and equal amounts of cell lysates were
analyzed for phosphotyrosine 416 in Src. E, no effect of
recombinant PKA on Src activity. Immunoprecipitated Src from HEK293
cells transfected with wild type Src or mutant Src-S17A was allowed to
autophosphorylate in the presence of Mg-ATP and increasing amounts of
recombinant PKA catalytic subunit (0, 1, or 5 ng of active catalytic
subunit/µl, respectively). Thereafter, reactions were immunoblotted
with antibodies (Ab) against either phosphorylated
Tyr416 in Src or anti-phospho-PKA substrate or total Src,
respectively.
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Fig. 5.
Cyclic AMP inhibits EGF-induced Src
activation but not EGF-induced EGFR tyrosine phosphorylation.
A, EGFR partitions into HEK293 DRMs. Sucrose gradient
fractionation from unstimulated HEK293 cells were immunoblotted with an
antibody against EGFR. B, stimulation of EGFR increases Src
activity. HEK293 cells were stimulated with EGF for the indicated
times, disrupted in standard lysis buffer, and subjected to sucrose
gradient fractionation. DRM fractions (fractions 2-5) were mixed, and
immunoprecipitated Src from these mixtures was then analyzed with the
indicated antibodies. C, forskolin reduces EGF-induced Src
activation. Serum-starved HEK293 cells were preincubated with or
without forskolin (5 min) and then stimulated with EGF for the
indicated times or incubated with forskolin alone. Whole cell lysates
(WCL) were analyzed for activation with the indicated
antibodies. D, increased cAMP does not affect EGF-induced
EGFR phosphorylation. Serum-starved HEK293 cells were stimulated and
lysed, and EGFR was immunoprecipitated and analyzed by Western blotting
using antibodies against phosphotyrosine (clone 4G10) or the EGFR,
respectively. E, EGF-mediated Src signaling is inhibited by
cAMP in a Csk-dependent manner. HEK293 cells transfected
with either mock DNA or HA-tagged wild type Csk or mutant
kinase-deficient Csk (Csk-SH3-SH2) were EGF-stimulated for
the indicated times with or without a 5-min pretreatment of forskolin.
Equal amounts of cells were lysed and analyzed for phosphorylation of
Tyr416 in Src (upper three
panels). Expression control of transfected cells is also
shown (lower panel).
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Fig. 6.
EGF-mediated Src phosphorylation of Cbl and
FAK is inhibited by increased cAMP. A, cAMP reduces
Src-mediated Cbl phosphorylation in HEK293 cells. Serum-starved HEK293
cells were EGF-stimulated for the indicated times with or without
forskolin pretreatment (5 min). After lysis, Cbl was immunoprecipitated
(IP) and analyzed for phosphotyrosine content or total
amount of Cbl. B, cAMP reduces Src-mediated Cbl
phosphorylation in NIH-3T3-A14 cells. Serum-starved A14 cells were
treated and analyzed as in B. C,
Tyr527 in Src is essential for the inhibitory effect of
cAMP on EGF-induced FAK phosphorylation. NIH-3T3 cells were transfected
with plasmids encoding either wild-type Src or Src-Y527F. The next day,
cells were pretreated with or without forskolin and then stimulated
with EGF for the indicated periods of time. Thereafter, the
phosphotyrosine content in immunoprecipitated FAK was assessed.
anti-PY, anti-phosphotyrosine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s or G
i binding to the catalytic domain of Src, thereby inducing conformational changes that favor
autophosphorylation of Tyr416 and subsequent Src activation
(42). Thus, several distinct mechanisms for Src regulation exist,
although it remains largely unknown what is the contribution of the
different regulatory mechanisms in a given physiological setting.
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Fig. 7.
Model for a GPCR-cAMP-PKA-Csk pathway that
intersects Src-mediated signaling. EGF-induced Src
activation and phosphorylation of the Src substrates Cbl, FAK, and
Stat3 are inhibited by a GPCR-cAMP-PKA-Csk pathway localized in
membrane microdomains. Increased cAMP activates Csk through
PKA-mediated phosphorylation. Subsequently, Csk directly phosphorylates
the C-terminal inhibitory tyrosine in Src, thereby inhibiting Src
signaling.
The fact that cAMP/PKA can inhibit EGF-induced Cbl phosphorylation may have some important implications for EGFR turnover. Upon stimulation, DRM-associated EGFRs move out of DRMs and are internalized via clathrin-coated pits. Cbl directly binds to the phosphorylated EGFRs and becomes tyrosine-phosphorylated by Src (32). Recently, a Cbl-CIN85-endophilin complex was reported to play an important role in EGFR internalization, and tyrosine phosphorylation of Cbl is necessary for its interaction with CIN85 (18). Therefore, it is tempting to speculate that the novel inhibitory cAMP pathway we describe in this report would affect EGFR turnover. Further studies will be necessary to address this issue.
In summary, we report the presence of a novel inhibitory pathway in
DRMs, whereby cAMP/PKA through activation of Csk intersects Src-mediated signaling. Since PKA, Csk, and SFKs are expressed in all
cell types, this pathway may represent a general principle for
regulation of SFKs.
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ACKNOWLEDGEMENT |
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We are grateful for the technical assistance of Guri Opsahl.
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FOOTNOTES |
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* This work was supported by the Norwegian Cancer Society, the Program for Advanced Studies in Medicine, the Norwegian Research Council, Anders Jahre's Foundation, and Novo Nordisk Research Foundation.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.
Both authors contributed equally to this work.
§ To whom correspondence and reprint requests should be addressed. Tel.: 47-22851454; Fax: 47-22851497; E-mail: kjetil.tasken@basalmed.uio.no.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M211426200
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ABBREVIATIONS |
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The abbreviations used are: SFK, Src family kinase; SH2 and SH3, Src homology 2 and 3, respectively; PGE2, prostaglandin E2; GPCR, G protein-coupled receptor; AC, adenylyl cyclase; PKA, protein kinase A; PAG, phosphoprotein associated with glycosphingolipid-enriched membrane domains; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; DRM, detergent-resistant membrane; HA, hemagglutinin epitope; HEK293 cells, human embryonic kidney 293 cells; FAK, focal adhesion kinase; PTPase, protein-tyrosine phosphatase; MES, 4-morpholineethanesulfonic acid.
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