Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and
Trang 1phosphorylation of the guanine nucleotide exchange
factor Ras-GRF1 in human embryonic kidney 293 cells
Jens Henrik Norum1, Trond Me´thi1, Raymond R Mattingly2and Finn Olav Levy1
1 Department of Pharmacology, University of Oslo, Norway
2 Department of Pharmacology, Wayne State University, Detroit, MI, USA
Introduction
Signals mediated through receptor tyrosine kinases [1]
and G-protein-coupled receptors (GPCRs) can induce
the activation of intracellular cascades such as the
mitogen-activated protein (MAP) kinase – also called
extracellular signal-regulated kinase (ERK) – cascade The serine⁄ threonine kinases ERK1 and ERK2 are activated by dual phosphorylation by the MAP kinase kinase, MEK, which becomes phosphorylated and activated by MEK kinases of the Raf family All three Raf isoforms [A-Raf, B-Raf and Raf-1 (C-Raf)]
Keywords
5-HT 7 , cAMP, ERK, GEF, serotonin
Correspondence
F O Levy, Department of Pharmacology,
University of Oslo, PO Box 1057 Blindern,
N-0316 Oslo, Norway
Fax: +47 22840202
Tel: +47 22840237 or +47 22840201
E-mail: f.o.levy@medisin.uio.no
(Received 2 December 2004, revised 1
February 2005, accepted 10 March 2005)
doi:10.1111/j.1742-4658.2005.04658.x
We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1⁄ 2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells Whereas Gi- and Gq-coupled receptors have been shown
to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+⁄ calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs -cou-pled receptors is not fully understood We report the endogenous expres-sion of Ras-GRF1 in HEK293 cells Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1
on Ser927 and of transfected mouse Ras-GRF1 on Ser916 Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1⁄ 2 phos-phorylation Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp⁄ Glu) phosphorylation did not alter these effects However, the deletion of amino acids 1–225, including the Ca2+⁄ calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1⁄ 2 phosphorylation Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the sero-tonin-induced ERK1⁄ 2 phosphorylation was Ca2+-dependent Therefore, both cAMP and Ca2+may contribute to the Ras-dependent ERK1⁄ 2 acti-vation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras
Abbreviations
5-HT, 5-hydroxytryptamine (serotonin); CaM, calmodulin; EGF, epidermal growth factor; Epac, exchange protein directly activated by cAMP; ERK, extracellular signal-regulated kinase; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; H89,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; HEK, human embryonic kidney; HRP, horseradish peroxidase; MAP, mitogen-activated protein; MEK, mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase; PKA, protein kinase A; Sos1, son of sevenless 1.
Trang 2expressed in mammalian cells may become activated
by members of the Ras family of small G proteins
The activity of Ras proteins is under tight control of
several classes of guanine nucleotide exchange factors
(GEFs) and GTPase-activating proteins Mammalian
Son of sevenless 1 (Sos1) is a ubiquitous Ras GEF and
activates Ras following the stimulation of receptor
tyrosine kinases, e.g the epidermal growth factor
(EGF) receptor [1] Various GPCRs can also induce
Ras activation via several classes of GEFs [2,3]
Activation of phospholipase C through Gq-coupled
receptors, with subsequent increased levels of
inositol-1,4,5-trisphosphate, diacylglycerol and free intracellular
Ca2+, can activate Sos1 through a cascade that
includes the proline-rich tyrosine kinase, Pyk2, Src and
Grb2 [4,5], as well as Ras GEFs of the Ras-GRP
(cal-DAG-GEF) family, through binding of Ca2+⁄
calmo-dulin (CaM) and diacylglycerol [6] Ras-GRF1, also
called CDC25Mm [7,8], is another major GEF with
activity towards Ras Ras-GRF1 mediates activation
of Ras subsequent to the stimulation of Gi- and Gq
-coupled receptors [8,9]
The main mechanism for the activation of
Ras-GRF1 is the binding of Ca2+⁄ CaM to the N-terminal
IQ motif [10] We have previously shown that the
treatment of NIH3T3 and COS-7 cells with carbachol
[9] and lysophosphatidic acid [11], activating both
Gq- and Gi-coupled receptors, induces the activation
and phosphorylation of Ras-GRF1 Furthermore,
Ras-GRF1 is also heavily phosphorylated upon
agon-ist activation of GPCRs, but the exact role of these
phosphorylations is not fully understood Protein
kin-ase A (PKA) is one of probably several kinkin-ases that
can induce the phosphorylation of Ras-GRF1 [12,13]
The residues Ser916 and Ser898 in the mouse and rat
sequences, respectively, are homologous PKA
phos-phorylation sites [14] Although forskolin-induced
phosphorylation of Ser916 is not sufficient to activate
wild-type GRF1, a recombinant version of
Ras-GRF1, with a mutated phosphorylation site
(Ser916-Ala), has been shown to have reduced activity
towards Ras both in vitro [12] and in an assay of
Ras-dependent outgrowth of neurites from PC12 cells
[14] These results indicate that even though
phos-phorylation of Ser916 may contribute to stimulation
of the Ras-GEF activity of Ras-GRF1,
cAMP-dependent phosphorylation alone is not sufficient to
activate Ras-GRF1
Ras-GRF1 is mainly expressed in brain tissue [15–
17], but expression of Ras-GRF1 mRNA has also been
reported in some other tissues and non-neural cell lines
[18] Several murine Ras-GRF1 cDNAs, encoding
proteins of different molecular mass (from 54 to
140 kDa), have been identified [17,19] The smaller iso-forms correspond to N-terminal deletions of the full-length 140 kDa protein The physiological role of the guanine nucleotide exchange activity of the truncated forms is not known as they are missing the Ca2+⁄ CaM-binding IQ domain that is involved in the activa-tion of Ras-GRF1
Stimulation of all the splice variants of the Gs-coupled serotonin receptor 5-hydroxytryptamine7 (5-HT7) increases intracellular levels of the second messenger cAMP [20], resulting in several intracellular effects, e.g activation of cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epacs), GEFs specific for Rap [21] In rat adrenal glo-merulosa cells, stimulation of the 5-HT7 receptor also induces the increased free intracellular Ca2+ concentra-tion ([Ca2+]i) through the low-voltage-activated T-type
Ca2+ channels [22,23] We have recently shown that serotonin treatment of human embryonic kidney (HEK)293 cells transiently expressing either one of the
Gs-coupled serotonin receptors 5-HT4(b) or 5-HT7(a) induces ERK1⁄ 2 phosphorylation [24] Although both Ras and Rap1 were activated, only Ras was involved in the pathway inducing ERK1⁄ 2 phosphorylation, which also involved Raf1 and MEK downstream of Ras How-ever, in PC12 cells, 5-HT7-mediated Ca2+-independent and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide dihydrochloride (H89)-insensitive ERK1⁄ 2 phosphorylation has been reported to be enhanced by the overexpression of Epac and mimicked by a cAMP analogue stimulating both PKA and Epac, but not by
an Epac-specific cAMP analogue [25] The differences in H89 sensitivity and possible signalling pathways involved may reflect cell-type variations in the ERK1⁄ 2 phosphorylation mediated through Gs-coupled sero-tonin receptors
The mechanism of Ras activation through Gs -cou-pled receptors is not fully understood In the present study, we show endogenous expression of the
Ca2+-dependent 140 kDa and shorter isoforms of Ras-GRF1 in HEK293 cells, as well as cAMP⁄ PKA-dependent phosphorylation of Ras-GRF1 associated with ERK1⁄ 2 phosphorylation following stimulation
of transfected 5-HT7 receptors However, mutating Ser916 of Ras-GRF1 to alanine, aspartic acid or gluta-mic acid did not alter the Ras-GRF1-induced ERK1⁄ 2 phosphorylation We confirm 5-HT7-mediated [Ca2+]i increase and show Ca2+ dependence of serotonin-induced ERK1⁄ 2 phosphorylation and a mandatory role of the Ca2+⁄ CaM-binding IQ domain in Ras-GRF1-stimulated ERK1⁄ 2 phosphorylation Thus, both cAMP and Ca2+ may contribute to Ras-depend-ent ERK1⁄ 2 activation following stimulation of the
Trang 35-HT7 receptor, by activating a guanine nucleotide
exchange factor with activity towards Ras
Results
HEK293 cells express the guanine nucleotide
exchange factor Ras-GRF1
The guanine nucleotide exchange factor Ras-GRF1 is
mainly expressed in neurones of the central nervous
system, although it has also been reported to be
expressed in some other tissues [18,26] To investigate
whether Ras-GRF1 plays a role in the activation of
Ras⁄ ERK signalling in HEK293 cells, we first used
immunoprecipitation analysis and RT-PCR to detect
whether Ras-GRF1 protein and mRNA, respectively,
were expressed in our HEK293 cells The proteins
im-munoprecipitated from HEK293 cell lysates, by using
a polyclonal antibody raised against a peptide mapping
to the C terminus of the rat Ras-GRF1 sequence, were
separated on SDS⁄ PAGE (6% gel) and visualized on
western blots probed with another polyclonal antibody
raised against the C terminus of the human Ras-GRF1
sequence A protein of 140 kDa was detected in
immunoprecipitates from HEK293 cells (Fig 1A), but
was not present in control immunoprecipitations In
whole-cell lysates from HEK293 cells, both full-length
140 kDa Ras-GRF1 and shorter isoforms, of 110, 95
and 60 kDa, were detected on western blots with
anti-(Ras-GRF1) Ig (Fig 2A, right panel) Preabsorb-ing the Ras-GRF1 antibody with a blockPreabsorb-ing peptide prevented the antibody from recognizing any of the Ras-GRF1 isoforms (data not shown)
cDNA to HEK293 cell mRNA was used as the sub-strate in PCR reactions, as described in the Experi-mental procedures The primer pairs specific for the
A
B
Fig 1 Human embryonic kidney (HEK)293 cells express Ras-GRF1.
(A) Paramagnetic beads coated with anti-(Ras-GRF1) Ig (# sc-224)
were used to immunoprecipitate Ras-GRF1 from the HEK293 cell
lysate The precipitated proteins were separated on 6% SDS ⁄ PAGE
and electroblotted over to poly(vinylidene difluoride) membranes
before probing with polyclonal Ras-GRF1 antibodies (# sc-863).
(B) cDNA produced from mRNA isolated from HEK293 cells was
used as the substrate in RT-PCR with primer pairs specific for
human Ras-GRF1 Primer pairs: lane 2, ON359 and ON360; lane 3,
ON357 and ON358; and lane 4, ON361 and ON360 The PCR
prod-ucts and a DNA size marker, lane 1, were separated on agarose
gels The expected sizes of the PCR products, in bp, are indicated
to the right.
A
B
C
D
Fig 2 Serotonin induces phosphorylation of Ras-GRF1 through the 5-hydroxytryptamine7(a)(5-HT7(a)) receptor, and HA-Ras-GRF1 indu-ces extracellular signal-regulated kinase (ERK)1 ⁄ 2 activation Human embryonic kidney (HEK)293 cells cotransfected with 5-HT7(a)receptor and empty or HA-Ras-GRF1 vector, as indicated, were treated with vehicle or 10 l M serotonin for the indicated peri-ods of time The control, C, was treated with vehicle (10 l M HCl) for 5 min Proteins were separated on 6% (A, B and D) or 10% (C) SDS ⁄ PAGE and electroblotted over to poly(vinylidene difluoride) membranes before probing with antibodies (A) Western blots were probed with phosphospecific Ras-GRF1 (pRas-GRF1; left panel) or anti-(Ras-GRF1) Igs (Ras-GRF1; right panel) to confirm equal load-ing (B) Western blot of cell lysates of HEK293 cells cotransfected with the 5-HT7(a)receptor and HA-Ras-GRF1 were incubated with anti-(pRas-GRF1) Ig (upper panel) or anti-(Ras-GRF1) Ig (lower panel)
to confirm equal loading (C) The same samples as in (A) and (B), but separated on 10% SDS ⁄ PAGE, were probed with phosphospe-cific ERK1 ⁄ 2 antibodies (pERK1 ⁄ 2; upper panel) and subsequently with ERK1 ⁄ 2 antibodies (ERK1 ⁄ 2; lower panel), to confirm equal loading (D) Non-transfected HEK293 cells were treated with or without 10 n M epidermal growth factor (EGF) for 5 min The pro-teins were separated, blotted and probed with antibodies as in (A).
Trang 4human Ras-GRF1 nucleotide sequence (NM_002891,
GI:24797098) gave PCR products of expected size
(Fig 1B) The primer sequences are located at the
5¢ end (ON361, ON360 and ON359) and in the middle
(ON357 and ON358) of the human Ras-GRF1
nucleo-tide sequence Sequencing of the purified PCR
prod-ucts confirmed sequence identity with cDNA encoding
the human 140 kDa Ras-GRF1 (data not shown)
Taken together, these mRNA and protein data
demon-strate that the full-length 140 kDa Ras-GRF1 protein
is endogenously expressed in the HEK293 cells used
for this study, and that truncated forms of Ras-GRF1
may also be present
Serotonin induces phosphorylation of Ras-GRF1
through 5-HT7receptors
The serine residue at position 916 in mouse Ras-GRF1
is a PKA phosphorylation site both in vitro [12] and
in vivo [14], and the corresponding human residue is
serine 927 We therefore used a polyclonal antibody
that was generated against a synthetic phosphopeptide
analogous to the Ser916 phosphorylation site, and
which has previously been shown to recognize mouse
and rat Ras-GRF1 when they are phosphorylated at
this residue [14], to test whether serotonin may
stimu-late phosphorylation of Ras-GRF1 in HEK293 cells
that express 5-HT7 receptors HEK293 cells transfected
with 5-HT7(a) receptors alone, or cotransfected with
the HA-tagged mouse Ras-GRF1 (HA-Ras-GRF1),
were treated with 10 lm serotonin for the indicated
periods of time (Fig 2) Serotonin treatment increased
the phosphorylation of the endogenous 140 kDa and
60 kDa isoforms of Ras-GRF1 (Fig 2A, left panel)
and of recombinant HA-Ras-GRF1 (Fig 2B, upper
panel) Phosphorylation of ERK1⁄ 2 in the same
sam-ples was fully induced after 3 min of treatment with
10 lm serotonin (Fig 2C) Furthermore, both basal
and serotonin-induced phosphorylation of ERK1⁄ 2
was increased in cells cotransfected with
HA-Ras-GRF1 and 5-HT7(a) receptor, compared to cells
trans-fected with receptor only (Fig 2C)
The EGF receptor induces activation of Ras
through a GEF, called Sos1, in a Ca2+-independent
manner Treatment of HEK293 cells with 10 nm EGF
for 5 min resulted in ERK1⁄ 2 phosphorylation
(Fig 6D) but not in phosphorylation of endogenous
Ras-GRF1 at the site recognized by the antibody
directed against Ras-GRF1 phosphorylated at Ser916⁄
927 (Fig 2D) This indicates that ERK1⁄ 2 activation
induced by EGF does not increase the
phosphoryla-tion of endogenously expressed Ras-GRF1 on Ser927
EGF has similarly been reported not to increase
phosphorylation of endogenous Ras-GRF1 in rat brain [9]
Serotonin-induced phosphorylation of Ras-GRF1
is dependent on cAMP and PKA Serotonin increases adenylyl cyclase activity in HEK293 cells expressing the human Gs-coupled sero-tonin receptor 5-HT7 [27] Forskolin increases aden-ylyl cyclase activity and induces the phosphorylation
of Ser916 in the mouse Ras-GRF1 sequence [12] and
of Ser898 in the rat sequence [14] To test whether serotonin stimulated Ras-GRF1 phosphorylation through PKA, HEK293 cells were cotransfected with 5-HT7(a) receptors, HA-Ras-GRF1 and either empty vector or the human phosphodiesterase hPDE4D2, which indirectly reduces PKA activity by reducing cAMP levels The serotonin-induced phosphorylation
of HA-Ras-GRF1 was essentially abolished and ERK1⁄ 2 phosphorylation was reduced in cells cotransfected with hPDE4D2 (Fig 3A) The phos-phorylation of overexpressed HA-Ras-GRF1 was also eliminated in cells incubated with 20 lm H89, an inhibitor of PKA but also of other kinases [28], for
25 min prior to treatment with 10 lm serotonin (Fig 3B) The serotonin-induced ERK1⁄ 2 phosphory-lation was concomitantly reduced, as expected based on the results of our previous publication [24] Cotrans-fection of HEK293 cells with the PKA inhibitor protein kinase inhibitor, in addition to 5-HT7(a) receptors and HA-RasGRF1, also reduced the serotonin-induced phosphorylation of recombinant HA-Ras-GRF1, as well as ERK1⁄ 2 phosphorylation (not shown) Phos-phorylation of the endogenously expressed 140 kDa and 60 kDa isoforms of Ras-GRF1 was increased following stimulation with serotonin The 60 kDa iso-form of Ras-GRF1 seems to be expressed at a higher level than the 140 kDa isoform The serotonin-induced increase in phosphorylation of both isoforms was reduced by the coexpression of hPDE4D2 with 5-HT7 receptors (Fig 3C) This was also the case for ERK1⁄ 2 phosphorylation (Fig 3D)
Phosphorylation of Ser916 is neither necessary nor sufficient for Ras-GRF1-mediated
phosphorylation of ERK1⁄ 2
To investigate the potential role of phosphorylation
at Ser916⁄ Ser927 of Ras-GRF1 in 5-HT7(a) receptor-dependent ERK1⁄ 2 activation, we compared the activities of wild-type Ras-GRF1 to proteins that had single amino acid substitutions at Ser916 We also used the mutants to verify the specificity of the
Trang 5phosphoRas-GRF1 antibody The antibody to
phos-phoSer916-Ras-GRF1 was developed against a
syn-thetic phosphopeptide corresponding to the residues
surrounding Ser916 of mouse Ras-GRF1 and had
previously been shown to be unreactive with a
Ras-GRF1 Ser916Ala mutant protein that was expressed
in COS-7 or PC12 cells [14] The antibody did not
recognize HA-Ras-GRF1 proteins mutated at the
Ser916 residue to alanine, aspartic acid or glutamic
acid and expressed in HEK293 cells (Fig 4A)
Inter-estingly, neither inhibiting phosphorylation of Ser916
by mutating the amino acid to alanine, nor
poten-tially mimicking it by mutation to aspartic acid or
glutamic acid, influenced the ability of recombinant
HA-Ras-GRF1 to induce phosphorylation of
ERK1⁄ 2 in HEK293 cells (Fig 4B) These results
suggest that the phosphorylation of Ras-GRF1 at
this residue may be neither necessary nor sufficient
to mediate stimulation of ERK1⁄ 2 activation in
HEK293 cells
An intact N-terminal region is required for Ras-GRF1 to potentiate ERK1/2 activation The role of calcium in the phosphorylation of ERK1⁄ 2 induced by Ras-GRF1 was addressed by cotransfecting HEK293 cells with 5-HT7(a) receptors and Ras-GRF1-D1-225 (i.e lacking the PH1-, coiled-coil and IQ domains) Cotransfection of HEK293 cells with this truncated form of Ras-GRF1 did not increase the basal
or serotonin-induced phosphorylation of ERK1⁄ 2 com-pared to cells transfected with the receptor only (Fig 4B) Serotonin treatment did, however, increase the phosphorylation of Ras-GRF1-D1-225 on Ser916 (Fig 4A)
Serotonin increases [Ca2+]ithrough 5-HT7 receptors
We have previously shown that the Gs-coupled sero-tonin receptors 5-HT4(b) and 5-HT7(a) induce
phos-A
B
C
D
Fig 3 Serotonin-induced Ras-GRF1 and ext-racellular signal-regulated kinase (ERK)1 ⁄ 2 phosphorylation is dependent on protein kin-ase A (PKA) ⁄ cAMP (A) Human embryonic kidney (HEK)293 cells cotransfected with the 5-hydroxytryptamine7(a)(5-HT7(a)) recep-tor, HA-Ras-GRF1, and either with or with-out hPDE4D2, were treated with 10 l M
5-HT for 5 min (B) HEK293 cells cotrans-fected with 5-HT7(a)receptor and HA-Ras-GRF1 were treated with or without 20 l M
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquin-olinesulfonamide dihydrochloride (H89) for
25 min prior to treatment with or without
10 l M serotonin for 5 min (C) HEK293 cells cotransfected with the 5-HT7(a)receptor and empty vector or hPDE4D2, as indicated, were treated with 10 l M serotonin for
5 min (D) The same samples as in (C) were assayed for ERK1 ⁄ 2 phosphorylation by SDS ⁄ PAGE (10% gel) and the western blot was probed with phosphospecific ERK1 ⁄ 2 antibodies (pERK1 ⁄ 2; upper panel) and then with ERK1 ⁄ 2 antibodies (ERK1 ⁄ 2; lower panel), to confirm equal loading The pro-teins were separated by SDS ⁄ PAGE (6% gel) for Ras-GRF1 and by SDS ⁄ PAGE (10% gel) for ERK1 ⁄ 2 and electroblotted to poly(vinylidene difluoride) membranes The membranes were probed with antibodies,
as indicated.
Trang 6phorylation of ERK1⁄ 2 through a Ras-dependent mechanism [24] The two other known human 5-HT7 receptor splice variants (5-HT7(b) and 5-HT7(d)) also induce phosphorylation of ERK1⁄ 2 through a Ras-dependent mechanism (data not shown) Therefore, in this respect we consider the different 5-HT7splice vari-ants to behave similarly when expressed in HEK293 cells HEK293 cells stably expressing the 5-HT7(b) receptor (KB1 cells) were used to determine whether serotonin can increase [Ca2+]i through human 5-HT7 receptors Treatment of the KB1 cells with 10 lm sero-tonin resulted in a rapid, transient increase in [Ca2+]i, with a maximum of 40–60% above the basal level, whereas there was no effect of vehicle (10 lm HCl; Fig 5) To establish that the effect was mediated through the 5-HT7(b) receptors, nontransfected HEK293 cells were subjected to the same treatment;
no effect of serotonin on [Ca2+]i was detected The serotonin-induced increase in [Ca2+]iwas abolished by the calcium influx inhibitor, carboxyamido-triazole (CAI) (20 lm), but not by vehicle control (dimethyl-sulfoxide) (Fig 5, inset) These results indicate that serotonin (10 lm) can increase [Ca2+]i through the human Gs-coupled 5-HT7 receptors in HEK293 cells The exact mechanism for the serotonin-mediated increase in Ca2+levels is not known
Phosphorylation of ERK1/2, mediated through 5-HT7receptors, is dependent on Ca2+
Transiently transfected HEK293 cells were incubated with CAI (20 lm) for 25 min prior to 5 min of
treat-A
B
Fig 4 Mutation of Ser916 of mouse Ras-GRF1 does not alter the
activation of extracellular signal-regulated kinase (ERK)1 ⁄ 2 but
dele-tion of amino acids 1–225 blocks the stimulatory effect of
Ras-GRF1 Human embryonic kidney (HEK)293 cells transfected with
the 5-hydroxytryptamine7(a)(5-HT7(a)) receptor and empty vector or
with GRF1, GRF1Ser916Ala, GRF1Ser916Asp,
Ras-GRF1Ser916Glu or Ras-GRF1-D1-225, were treated with or without
10 l M serotonin for 5 min (A) Western blots of SDS ⁄ PAGE (6%
gel) of lysates of cells, transfected as indicated, were probed with
anti-(pRas-GRF1) immunoglobulin (upper panel) and anti-HA-probe
immunoglobulin (lower panel), to confirm equal loading (B) Western
blots of SDS ⁄ PAGE (10% gel) of lysates of cells, transfected as
indicated, were probed with anti-pERK1⁄ 2 immunoglobulin (upper
panel) and anti-ERK1 ⁄ 2 immunoglobulin (lower panel), to confirm
equal loading.
Fig 5 Serotonin increases intracellular Ca 2+ concentration through 5-hydroxytryptamine7(b) (5-HT7(b)) receptors Non-transfected or stably transfected human embryonic kidney (HEK)293 cells expressing the 5-HT7(b)receptor, KB1 cells, were cultured, washed and loaded with
5 l M FURA-2-AM for 20 min The fluorescence intensity in single cells was recorded at 340 nm and 380 nm for up to 300 s on an inverted microscope The cells were treated with 10 l M serotonin 30 s subsequent to the start of the recordings, as indicated with an arrow Inset,
in addition to treatment with FURA-2-AM, as described above, the cells were treated with carboxyamido-triazole (CAI) (20 l M ) or vehicle con-trol (dimethylsulfoxide) for 25 min prior to treatment with 10 l M serotonin.
Trang 7ment with 10 lm serotonin Serotonin-induced
phos-phorylation of ERK1⁄ 2 was markedly reduced in the
presence of 20 lm CAI (Fig 6A) Serotonin-induced
phosphorylation of ERK1⁄ 2 was also reduced in
cells incubated with the Ca2+ chelator, BAPTA-AM
(40 lm), for 25 min prior to 5 min of treatment with
10 lm serotonin (Fig 6B) Increasing the free
intracel-lular levels of Ca2+by treatment of HEK293 cells with
thapsigargin induced phosphorylation of ERK1⁄ 2
(Fig 6C) Previously, CAI has been shown to inhibit
the thapsigargin-induced activation of ERK1⁄ 2 in
Rat1 cells [29] Thapsigargin-induced phosphorylation
of ERK1⁄ 2 in HEK293 cells was inhibited by pretreat-ment with 20 lm CAI for 25 min, demonstrating that CAI inhibited the calcium-mediated phosphorylation
of ERK1⁄ 2 under these conditions (Fig 6C)
To determine whether the effect of CAI on the sero-tonin-induced ERK1⁄ 2 phosphorylation was specific, HEK293 cells were treated with 20 lm CAI for 25 min prior to treatment with 10 nm EGF for 5 min EGF-induced phosphorylation of ERK1⁄ 2 was not influ-enced by the presence of CAI (Fig 6D), demonstrating that CAI does not have a general suppressive effect on the Ras-dependent activation of ERK1⁄ 2
Increased basal ERK1⁄ 2 phosphorylation in the presence of HA-Ras-GRF1 is reduced by CAI and RasN17
In HEK293 cells transfected with the 5-HT7(a) recep-tor, cotransfection with HA-Ras-GRF1 increased basal ERK1⁄ 2 phosphorylation (Fig 7A, lanes 5 and 6 vs lane 1) Serotonin-induced ERK1⁄ 2 phosphorylation
in these cotransfected cells was abolished by pretreat-ment with CAI (Fig 7A, lanes 5–12), as in cells
trans-A
B
C
D
Fig 6 Serotonin-induced extracellular signal-regulated kinase
(ERK)1 ⁄ 2 phosphorylation is dependent on Ca 2+ (A) Human
embry-onic kidney (HEK)293 cells transiently transfected with the
5-hy-droxytryptamine7(a)(5-HT7(a)) receptor were treated with or without
20 l M carboxyamido-triazole (CAI) for 25 min prior to treatment
with or without 10 l M serotonin for 5 min, as indicated (B)
HEK293 cells, transiently transfected with the 5-HT 7(a) receptor,
were treated with or without 40 l M BAPTA-AM for 25 min prior to
incubation for 5 min with or without 10 l M serotonin (C) and (D)
HEK293 cells were treated with or without 1 l M thapsigargin (C) or
10 n M epidermal growth factor (EGF) (D) for 5 min subsequent to
treatment with or without 20 l M CAI for 25 min, as indicated (A),
(B), (C) and (D) show representative western blots of proteins
sep-arated by SDS ⁄ PAGE (10% gel) and electroblotted over to
poly(vinylidene difluoride) membranes before probing with
antibod-ies, as indicated.
A
B
C
Fig 7 Phosphorylation of extracellular signal-regulated kinase (ERK)1 ⁄ 2, induced by recombinant HA-Ras-GRF1, is dependent on
Ca2+and Ras (A) Human embryonic kidney (HEK)293 cells cotrans-fected with the 5-hydroxytryptamine7(a) (5-HT7(a)) receptor and empty vector or HA-Ras-GRF1 were treated with or without 10 l M
serotonin for 5 min subsequent to treatment with 20 l M carbox-yamido-triazole (CAI) or vehicle for 25 min, as indicated (B) HEK293 cells transiently cotransfected with the 5-HT7(a) receptor and HA-Ras-GRF1 were treated with or without 20 l M CAI for 25 min prior to treatment with or without 10 l M serotonin for 5 min (C) HEK293 cells were cotransfected with 5-HT7(a)receptor and empty vector, HA-Ras-GRF1 or RasN17, as indicated The transfected cells were treated with or without 10 l M serotonin for 5 min (A), (B) and (C) show representative western blots of 10% (A and C) and 6% (B) SDS ⁄ PAGE, probed with antibodies as indicated.
Trang 8fected with the 5-HT7(a) receptor alone (Figs 6A and
7A) These results indicate that the
serotonin-stimula-ted ERK1⁄ 2 phosphorylation is Ca2+ dependent
There was also a slight inhibitory effect of CAI on the
increased basal phosphorylation of ERK1⁄ 2 observed
upon cotransfection with HA-Ras-GRF1 (Fig 7A)
On the other hand, the serotonin-induced
phosphory-lation of HA-Ras-GRF1 was not affected by CAI,
indicating that this phosphorylation is not Ca2+
dependent (Fig 7B)
To determine whether the increased ERK1⁄ 2
phos-phorylation in cells transfected with HA-Ras-GRF1
was mediated through Ras, HEK293 cells were
cotransfected with plasmids encoding the 5-HT7(a)
receptor, HA-Ras-GRF1 and a dominant-negative
construct of Ras, RasN17 RasN17 essentially
elimin-ated the increase in ERK1⁄ 2 phosphorylation (both
basal and serotonin-stimulated) induced by the
overex-pression of HA-Ras-GRF1 (Fig 7C), indicating that
the effect of Ras-GRF1 on basal and
serotonin-stimu-lated ERK1⁄ 2 phosphorylation is Ras-dependent
Discussion
We report the endogenous expression of several
isoforms of the guanine nucleotide exchange factor
Ras-GRF1 in HEK293 cells Serotonin treatment of
HEK293 cells, transiently transfected with the Gs
-cou-pled 5-HT7 receptors, induced cAMP⁄ PKA-dependent
phosphorylation of endogenous Ras-GRF1 at Ser927
and recombinant mouse HA-tagged Ras-GRF1 at
Ser916 However, mutation of the Ser916 PKA
phos-phorylation site did not alter the increased basal or
serotonin-induced ERK1⁄ 2 phosphorylation induced
by the overexpression of HA-Ras-GRF1 A truncated
version of Ras-GRF1, lacking the Ca2+⁄ CaM-binding
IQ domain, did not increase the basal or
serotonin-induced ERK1⁄ 2 phosphorylation The ERK1 ⁄ 2
phos-phorylation was inhibited in the presence of the
calcium influx inhibitor, CAI
The endogenous expression of 5-HT6 and 5-HT7
receptors has been reported in some HEK293 cells
[30] However, in the current study, serotonin
treat-ment of nontransfected HEK293 cells did not result in
ERK1⁄ 2 phosphorylation or increased [Ca2+]i (data
not shown), indicating that the HEK293 cells used did
not show endogenous expression of functional 5-HT7
or other Gs-coupled serotonin receptors
Ras-GRF1 contains several protein motifs that are
presumably involved in numerous regulatory
mecha-nisms Binding of Ca2+⁄ CaM to the N-terminal IQ
motif is considered to be the main mechanism for
Ras-GRF1 activation [10] Upon stimulation of
GPCRs Ras-GRF1 becomes phosphorylated on several sites, with incompletely understood effects The Ser916 residue of mouse Ras-GRF1 becomes phosphorylated
by PKA in vivo and in vitro [12] This phosphorylation
is insufficient for activation but may enhance the activ-ity of Ras-GRF1 towards Ras [12,14] The phospho-specific antibody that selectively recognizes mouse and rat Ras-GRF1, which are phosphorylated at Ser916⁄ 898, respectively, also recognizes human phos-phorylated Ras-GRF1 The sequence surrounding Ser927 in human Ras-GRF1 is homologous to that surrounding Ser916 in mouse Ras-GRF1, with three amino acid substitutions In addition, several other putative phosphorylation sites have been identified in Ras-GRF1 Baouz and colleagues, for example, did not find Ser916 as an in vitro PKA phosphorylation site [13], but rather identified Ser745 and Ser822 as the two most heavily phosphorylated residues However, compared with the human Ser927 sequence, the sequences surrounding these two serine residues do not align as well with the mouse Ser916 sequence There-fore, the phosphospecific antibody developed against mouse phosphoSer916-Ras-GRF1 probably recognizes human Ras-GRF1 phosphorylated at Ser927 The anti-body is highly specific for the phosphorylated residue,
as mutations of Ser916 (in the mouse sequence) to alanine, aspartic acid or glutamic acid were not recog-nized by the antibody Our finding, that reactivity of the endogenous Ras-GRF1 in HEK293 cells to the phospho-Ras-GRF1 antibody is stimulated by the acti-vation of 5-HT7 receptors, is also consistent with the selective recognition of human Ras-GRF1 by this anti-body when Ras-GRF1 is phosphorylated at Ser927 The serotonin-induced phosphorylation of both endogenous and recombinant Ras-GRF1 shows that Ras-GRF1 is modified by stimulation with serotonin, but is not direct evidence that Ras-GRF1 contributes
to the serotonin-induced activation of Ras and ERK1⁄ 2 Pretreatment with H89 eliminated the sero-tonin-induced phosphorylation of Ras-GRF1 at Ser916⁄ 927 Transfection with the human phosphodi-esterase PDE4D2 also reduced the serotonin-induced Ras-GRF1 phosphorylation In both cases, the sero-tonin-induced ERK1⁄ 2 phosphorylation was lowered concomitant with the reduced Ras-GRF1 phosphoryla-tion, but ERK1⁄ 2 phosphorylation was only partially reduced compared to the more substantial reduction of Ras-GRF1 phosphorylation
Neither preventing PKA-mediated phosphorylation
of mouse Ras-GRF1 Ser916 by mutating this residue
to alanine nor mutating the residue to either aspartic
or glutamic acid to potentially mimic the phosphoryla-tion, influenced the increased basal or
Trang 9serotonin-induced ERK1⁄ 2 phosphorylation Taken together,
these data indicate that the PKA-mediated
phosphory-lation of Ser916 of mouse Ras-GRF1, and presumably
Ser927 of human Ras-GRF1, does not have a central
role in ERK1⁄ 2 activation The small differences in
Ras activation observed between wild-type Ras-GRF1
and the Ser916Ala mutant, both in vitro [12] and in an
assay of Ras-dependent neurite outgrowth from PC12
cells [14], may not be detectable at the level of
ERK1⁄ 2 phosphorylation owing to amplification of
the signal through the kinase cascade These results are
also in agreement with our previous report that
phos-phorylation at this site was insufficient to activate
Ras-GRF1 in the absence of other signals [12] It is
probable that phosphorylation at this site is only one
of several regulated phosphorylation events that occur
on Ras-GRF1 to regulate its activity in coordination
with increases in Ca2+, and so an effect from the
mutation of a single site may not be apparent The
importance of phosphorylation of Ras-GRF1 at this
residue is underlined by the demonstration that it is a
physiologically relevant phosphorylation event which
occurs at the equivalent site (Ser898) in the dendritic
tree of rat prefrontal cortical neurones [14] In addition
to regulation of the Ras GEF activity of Ras-GRF1,
other phosphorylation events, particularly on tyrosine
residues, may regulate its activity as a GEF for
another small G-protein, Rac [31]
Expression of recombinant, murine, HA-tagged
Ras-GRF1 (HA-Ras-Ras-GRF1) in HEK293 cells increased the
basal ERK1⁄ 2 phosphorylation compared to that of
nontransfected cells Serotonin caused additional
phos-phorylation of ERK1⁄ 2 in HEK293 cells cotransfected
with the 5-HT7(a)receptor and HA-Ras-GRF1, but the
combined effect of 5-HT7(a) activation and
HA-Ras-GRF1 expression was not much higher than the sum
of the separate effects on ERK1⁄ 2 phosphorylation If
endogenous Ras-GRF1 was the limiting factor in the
cascade from the 5-HT7(a) receptor to ERK1⁄ 2
phos-phorylation, one might expect that the overexpression
of HA-Ras-GRF1 would elicit greater effects than
observed on ERK1⁄ 2 phosphorylation On the other
hand, if endogenous Ras-GRF1 was not the limiting
factor in the cascade, one could hypothesize that the
effect of Ras-GRF1 overexpression on ERK1⁄ 2
phosphorylation would be similar to the sum of the
receptor-induced effect and increased basal
phosphory-lation, mediated from overexpressed Ras-GRF1,
poss-ibly localized in different cellular compartments from
the receptor
The increased ERK1⁄ 2 phosphorylation in the
pres-ence of HA-Ras-GRF1 was essentially eliminated in
the presence of dominant-negative Ras, RasN17, but
only slightly reduced by the Ca2+ influx inhibitor, CAI Both interventions prevented the serotonin-induced phosphorylation of ERK1⁄ 2 A truncated ver-sion of Ras-GRF1 (Ras-GRF1-D1-225) lacking the PH1-, coiled-coil and IQ domain and thus not expec-ted to bind Ca2+⁄ CaM, did not increase the basal
or serotonin-induced ERK1⁄ 2 phosphorylation The reduced ability of Ras-GRF1-D1-225 to induce ERK1⁄ 2 activation may be a result of the lost
Ca2+⁄ CaM-binding site of the IQ domain, but the missing PH1- and coiled-coil domains may also change the subcellular localization of this version of Ras-GRF1 These domains have been shown to contribute
to the regulation of Ras GEF activity [32] The sero-tonin-induced phosphorylation of Ras-GRF1-D1-225
at Ser916 indicates that the protein is located in cellu-lar compartments within the reach of kinases activated upon serotonin treatment We have previously shown that while increased intracellular Ca2+ is required for the stimulation of Ras-GRF1 activation by a Gi -cou-pled pathway [12], Ca2+does not stimulate Ras-GRF1 phosphorylation at Ser916 [14] It is probable that Ras-GRF1 can serve to integrate signals from the cAMP and Ca2+ second messenger cascades to deter-mine activation of the ERK1⁄ 2 cascade In addition to the influence of second messengers and phosphoryla-tion events on its activities, Ras-GRF1 can also be regulated by interaction with another small GTPase, Cdc42 [33], and can serve a scaffolding function that directs signalling downstream of Ras activation [34,35]
In rat adrenal glomerulosa cells, 5-HT7 receptors were shown to increase [Ca2+]i through T-type Ca2+ channels in a cAMP⁄ PKA-dependent manner [22,23] Increase in [Ca2+]i following stimulation of over-expressed 5-HT7(a) receptors in HEK293 cells has pre-viously been shown [36] and no evidence of coupling to
Gqor Giwas found We showed that serotonin stimu-lation of HEK293 cells stably expressing 5-HT7(b) receptors resulted in increased [Ca2+]i Serotonin-induced ERK1⁄ 2 phosphorylation was severely reduced in the presence of CAI, but the PKA-depend-ent phosphorylation of HA-Ras-GRF1 was not influ-enced by the presence of CAI In nonexcitable cells, CAI can specifically inhibit store-operated calcium channels and may thereby reduce the serotonin-induced sustained increase in [Ca2+]i, as has been shown for endothelin-1-induced Ca2+ increase in Rat1 cells [29] Whether HEK293 cells express T-type Ca2+ channels, or whether the increase in [Ca2+]iis mediated through a different mechanism, has not been addressed further in this study, and the results obtained with the calcium influx inhibitor, CAI, do not provide conclu-sive data concerning the nature of the calcium increase
Trang 10Ras-GRF1 is implicated in signalling from various
neurotransmitter receptors [9,12,37] The downstream
target of Ras-GRF1, Ras, may help to regulate
expres-sion of specific genes involved in processes such as
memory In Aplysia, the activation of MAP kinases by
Gs-coupled serotonin receptors is implicated in
mem-ory formation [38,39] There is increasing evidence for
the biological importance of the Ras⁄ MAP kinase
cas-cade in human learning and memory [40] Gs-coupled
serotonin receptors are found in the hippocampus
[41,42], and 5-HT7 receptors activate ERK1⁄ 2 in
cul-tured neurones [43] Ras-GRF1 is highly expressed in
hippocampal and other neurones, and
Ras-GRF1-defi-cient mice have memory defects [44,45] Therefore, a
possible involvement of Ras-GRF1 in the Ca2+- and
Ras-dependent activation of ERK1⁄ 2 through 5-HT7
receptors may be of physiological relevance Since the
original manuscript was submitted for publication,
Johnson-Farley and colleagues have shown interplay
between Gs- and Gq-coupled serotonin receptors in the
activation of ERK1⁄ 2 and PKB (Akt) in PC12 cells
[46] They found that PKA activation through Gs
-cou-pled serotonin receptors was Ca2+dependent, whereas
ERK1⁄ 2 phosphorylation was Ca2+ independent
Considering all the different pathways reported for the
activation of Ras and ERK1⁄ 2 downstream of
GPCRs, Ras-GRF1 could be one of possibly several
GEFs involved in the activation of Ras and
subse-quently ERK1⁄ 2 downstream of Gs-coupled serotonin
receptors This remains a challenge for future research
Experimental procedures
Materials
HEK293 cells were from the American Type Culture
Collec-tion (Manassas, VA, USA) Mouse monoclonal
antiphos-pho-ERK1⁄ 2 and rabbit polyclonal
anti-(phosphoSer916-Ras-GRF1) Ig (#3321) were from Cell Signaling Technology
(Beverly, MA, USA), sheep polyclonal antimouse
immuno-globulin–horseradish peroxidase conjugate (Ig-HRP) and
sheep anti-(rabbit IgG)–HRP were from Amersham
Pharma-cia Biotech (Little Chalfont, Bucks, UK), rabbit polyclonal
anti-ERK1⁄ 2 Ig was from Upstate Biotechnology (Lake
Placid, NY, USA), and rabbit polyclonal anti-(Ras-GRF1)
Ig (human, rat) was from Santa Cruz Biotechnology (Santa
Cruz, CA, USA) 5-HT, EGF, H89 and Dulbecco’s modified
Eagle’s medium (DMEM) were from Sigma (St Louis, MO,
USA) Hybond-P [poly(vinylidene difluoride)] membrane
was from Amersham LipofectamineTM2000 was from
Invi-trogen (Carlsbad, CA, USA) Fetal bovine serum was from
EuroClone (Milano, Italy) UltraCULTURETMgeneral
pur-pose serum-free medium, penicillin⁄ streptomycin and
l-glu-tamine were from Cambrex (Vervierse, Belgium) Supersignal West Dura extended-duration chemiluminescent substrate was from Pierce Biotechnology (Rockford, IL, USA), and the BC assay protein quantification kit was from Uptima (Monticon, France) BAPTA-AM was from Calbiochem (La Jolla, CA, USA)
Plasmids The pcDNA3.1(–) vector (Invitrogen), encoding the human 5-HT7(a) receptor, was as described previously [27] The pKH3 mammalian expression plasmids encoding the full-length murine wild-type HA-Ras-GRF1 and the Ser916Ala mutant were as described previously [9,12,47] GRF1-Ser916Asp, GRF1-Ser916Glu and HA-Ras-GRF1-D1-225 were constructed by PCR using appropriate mutagenic primers and the protocol previously described [12] and then confirmed by DNA sequencing The pCMV vector encoding dominant-negative Ras, RasN17, was from Clontech (Palo Alto, CA, USA) The pCMV5 vector enco-ding the human phosphodiesterase 4D2, hPDE4D2, was provided by M Conti (Department of Obstetrics and Gynaecology, Stanford, CA, USA)
Cell culture and transfection HEK293 cells were cultured in DMEM containing 10% (v⁄ v) fetal bovine serum and supplements (2 mm l-gluta-mine, 100 UÆmL)1penicillin, 100 lgÆmL)1 streptomycin), at
37C in a humidified atmosphere of 5% CO2 in air, and transfected at 60–70% confluence with the indicated cDNA(s) using Lipofectamine 2000, according to the manu-facturer’s protocol When necessary, empty vector [pcDNA3.1(–)] was included in the transfection to ensure that each dish received the same amount of DNA (1.0 or 2.9 lg of plasmid DNA per 35 or 60 mm dish, respectively) Cells expressing 5-HT7receptors were cultured in UltraCUL-TURETMserum-free medium with supplements, as described above, prior to starvation in DMEM without serum for the last 16–20 h before serotonin treatment and lysis ( 48 h after transfection for transiently transfected cells) Non-transfected cells were similarly starved in DMEM without serum before treatment (with EGF or thapsigargin) and lysis Where indicated, cells were preincubated with 20 lm H89,
20 lm CAI or 40 lm BAPTA-AM for 25 min prior to treat-ment with agonist Cells were stimulated for 5 min if not indicated otherwise All experiments were carried out in duplicate at least three times, if not otherwise indicated
Western Blotting Equal amounts of cell lysate proteins were separated by SDS⁄ PAGE and electroblotted onto poly(vinylidene difluo-ride) membranes The membranes were incubated with