(Received for publication, November 21, 1995; and in revised form, January 2, 1996)
From the
The cAMP/cAMP-dependent protein kinase (A-kinase) and
Ca/calmodulin-dependent protein kinase (Cam-kinase)
signal transduction pathways are well known to regulate gene
transcription, but this has not been demonstrated directly for the
cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction
pathway. Here we report that transfection of G-kinase into
G-kinase-deficient cells causes activation of the human c-fos promoter in a strictly cGMP-dependent manner. The effect of
G-kinase appeared to be mediated by several sequence elements, most
notably the serum response element (SRE), the AP-1 binding site (FAP),
and the cAMP response element (CRE). The magnitude of G-kinase
transactivation of the fos promoter was similar to that of
A-kinase, but there were significant differences between G-kinase and
A-kinase activation of single enhancer elements and of a chimeric
Gal4-CREB transcription factor. Our results indicate that G-kinase
transduces signals to the nucleus independently of A-kinase or
Ca
, although it may target some of the same
transcription factors as A-kinase and Cam-kinase.
The c-fos gene product is part of the AP-1
transcription factor complex which plays a central role in regulating
cell growth and differentiation(1) . c-fos transcription is rapidly induced by a variety of stimuli including
activation of A-kinase ()by cAMP, protein kinase C by
phorbol esters, or Cam-kinase IV by
Ca
(2, 3) . We (4) and others (5, 6) have shown that treating cells with cGMP
analogs or increasing the intracellular cGMP concentration induces
c-fos mRNA expression; in some instances co-induction with
Ca
ionophores or antioxidants was
required(7, 8) . Unlike cAMP which acts predominantly
through A-kinase activation, cGMP has several intracelluar target
proteins: G-kinase, cGMP-gated ion channels, and cGMP-stimulated or
cGMP-inhibited phosphodiesterases(9) . To test whether
c-fos induction by cGMP analogs was mediated by G-kinase, we
used G-kinase-deficient baby hamster kidney (BHK) cells and a
chloramphenicol acetyltransferase (CAT) reporter construct under
control of the human fos promoter (pFOS-CAT,(10) ). We
found that reporter gene expression from this construct was stimulated
by cGMP analogs only in BHK cells co-transfected with a G-kinase
expression vector, but not in cells co-transfected with empty vector.
Transactivation of the fos promoter in the presence of
G-kinase was characterized further and found to be independent of
A-kinase or Ca
.
Plasmids-The G-kinase expression vector
encoding human G-kinase type I has been described(11) ;
the parent vector is pRC/CMV (Invitrogen). pFC400, pFC225, pFC122, and
pFC87 were derived from pFOS-CAT by deletion of the appropriate
restriction fragment(10) ; the remaining Fos promoter
constructs, pTRE-CAT, pCRE-CAT, pSRE-CAT, Gal4-CREB,
pRSV-PKI
, and pRSV-PKI
were described
previously(12, 13, 14, 15, 16) .
To determine the amount of G-kinase or A-kinase
that was activated in vivo in BHK cells treated with 8-Br-cGMP
or 8-Br-cAMP, respectively, cells were washed rapidly in situ in large volumes of ice-cold phosphate-buffered saline and
snap-frozen on dry ice. Cells were disrupted by several 10-s pulses of
sonication, centrifuged at 11,000 g for 30 s, and
assays were performed at 4 °C immediately thereafter to minimize
8-Br-cGMP dissociation from G-kinase(19) . To correct for the
activity of A-kinase and other protein kinases, G-kinase activity in
G-kinase-transfected cells was measured in the presence of PKI, and
Kemptide phosphorylation in mock-transfected cells was subtracted from
that in the G-kinase-transfected cells. A-kinase activity was
determined as the difference between Kemptide phosphorylation in the
absence and presence of 10 µM PKI(16) .
We have previously shown that NO-releasing agents and membrane-permeable cGMP analogs induce c-fos mRNA expression in rat embryonal fibroblasts; these cells contain NO-stimulatable guanylate cyclase activity and G-kinase activity(4) . To test whether c-fos mRNA induction by cGMP analogs was mediated by G-kinase, we co-transfected the reporter plasmid pFOS-CAT (10) with a G-kinase expression vector (11) into BHK cells which contain extremely low endogenous G-kinase activity. In G-kinase-transfected cells, the enzyme was easily detected by Western blotting (Fig. 1), and total cellular G-kinase activity was 0.83 ± 0.09 nmol/min/mg of protein compared to 0.02 ± 0.01 nmol/min/mg of protein in cells transfected with control expression vector (``empty'' pRC/CMV). From Western blots using bovine lung G-kinase as a standard and by comparing enzyme activity in transfected BHK cells to that of the purified bovine lung enzyme(18) , we estimate the intracellular concentration of G-kinase in the entire transfected BHK cell population to be 3-6 pmol/mg of protein; taking into consideration that the transfection efficiency was 67-80%, the concentration of G-kinase in successfully transfected cells is in the range of physiological G-kinase concentrations found in smooth muscle cells, neuronal cells, and platelets (1-9 pmol/mg of protein)(9) .
Figure 1:
G-kinase expression in transfected BHK
cells. BHK cells were transfected with G-kinase vector (lanes 3 and 5) or control vector (lanes 4 and 6) as described under Methods. Expression of G-kinase
protein was examined by Western blotting using a G-kinase-specific
antibody(11) . Either a homogenate of 10 cells (lanes 3 and 4) or 30 µg of protein of a 100,000
g supernatant (lanes 5 and 6) were
analyzed. Lanes 1 and 2, 50 and 100 ng of purified
bovine lung G-kinase, respectively.
CAT
expression from the pFOS-CAT reporter was induced by 8-Br-cGMP only in
G-kinase-transfected cells and was strictly dependent on the amount of
G-kinase vector transfected (Fig. 2A) and the 8-Br-cGMP
concentration in the culture medium (Fig. 2B). Thus,
activation of the fos promoter by 8-Br-cGMP required
transfected G-kinase and was not due to cross-activation of A-kinase.
Maximal induction of pFOS-CAT was 5-fold at 1 mM 8-Br-cGMP, a concentration consistent with that reported to be
required for various physiological responses in whole
cells(4, 20, 21) . When
8-chlorophenylthio-cGMP was used to replace 8-Br-cGMP, maximal pFOS-CAT
expression in G-kinase-transfected cells occurred at 0.25 mM (data not shown). In comparison, activation of endogenous A-kinase
by 1 mM 8-Br-cAMP maximally induced pFOS-CAT expression 7-fold (Fig. 2B). For both G-kinase and A-kinase there was an
excellent correlation between pFOS-CAT activity (Fig. 2B) and the amount of in vivo kinase
activity (inset, Fig. 2B). These data are
further evidence that 8-Br-cGMP induced pFOS-CAT expression by
activating G-kinase and show that the amount of G-kinase activity in
the G-kinase-transfected cells was comparable with the amount of
endogenous A-kinase activity (taking into consideration that the
transfection efficiency was 67-80%). CAT expression from the
promoterless parent plasmid of pFOS-CAT(10) , and from several
other promoter constructs containing the Rous sarcoma virus long
terminal repeat, the chicken
-actin promoter, and the SV40 early
promoter (4) was not influenced by G-kinase in the absence or
presence of 8-Br-cGMP, indicating that the effect of G-kinase required
sequence elements present in the fos promoter.
Figure 2:
Activation of pFOS-CAT by transfected
G-kinase or endogenous A-kinase. A, activity of pFOS-CAT was
measured in BHK cells co-transfected with 50 ng of pFOS-CAT, 50 ng of
pRSV-Luc, and increasing amounts of G-kinase expression vector; the
total amount of transfected DNA was kept constant by adding control
expression vector (pRC/CMV). Cells were either left untreated ()
or were treated with 0.5 mM 8-Br-cGMP (
) for 8 h prior
to determining CAT and luciferase activity. Luciferase activity from
pRSV-Luc served to control for transfection efficiency and varied by
<25% among samples. Relative CAT activity represents the
CAT/luciferase activity ratio of each sample normalized to that of
untreated cells transfected with control vector. Results are the mean
± S.D. of three independent experiments. B, activity of
pFOS-CAT was measured in BHK cells co-transfected with 50 ng of
pFOS-CAT, 50 ng of pRSV-Luc, and either 500 ng of control vector
(
,
) or 500 ng of G-kinase expression vector (
).
Cells were treated with the indicated concentration of 8-Br-cGMP
(
,
) or 8-Br-cAMP (
) as described above. The data
are the mean ± S.D. of three independent experiments. Inset, the amount of in vivo G-kinase and A-kinase
activity was measured in G-kinase-transfected BHK cells treated with
8-Br-cGMP (
) and in mock-transfected cells treated with 8-Br-cAMP
(
), respectively, as described under Methods. To
minimize 8-Br-cGMP dissociation from G-kinase, protein kinase
activities were measured at 4 °C immediately after cell
extraction(19) .
The pFOS-CAT reporter construct contains approximately 700 base pairs of the human fos gene 5`-flanking region including several known enhancer elements: a sis-inducible element, a serum-inducible element (SRE), an AP-1 binding site (FAP, which resembles the phorbol ester response element, TRE), and a cAMP-response element (CRE) (Fig. 3)(2, 10, 12) . fos promoter induction by A-kinase is mediated by the SRE, FAP, and CRE, with each element functioning independently without synergism(12, 22) . A series of promoter deletions and mutations generally gave a similar pattern for both G-kinase- and A-kinase-mediated transactivation (Fig. 3); deletions of the fos promoter up to -72 nucleotides from the transcription start were inducible, but deletion of the CRE (pFC53) abolished inducibility by both kinases. However, the constructs pFC87 and pTF5/53 showed much greater induction by A-kinase than G-kinase suggesting that the two kinases do not regulate the fos promoter via identical mechanisms. The high inducibility of pTF5/53 by cAMP (which has been noted before(12) ) may be mediated by the C/EBP-related transcription factor NFIL-6(23) .
Figure 3: Identification of fos promoter sequences which mediate the effect of G-kinase. A schematic diagram of pFOS-CAT is shown containing 700 base pairs of the human c-fos 5`-flanking region; regions corresponding to the sis-inducible element, SRE, FAP, CRE, direct repeats (D.R.), and TATA box are shown relative to the transcription start site (arrow)(12) . Deletion constructs extending from variable 5` sites are indicated. A linker scanning mutation in pFC60LS (in the CRE) is indicated by XXX. The dotted lines indicate internal deletions. Transfections were performed as described under Methods with 50 ng of pRSV-Luc, 50 ng of the indicated CAT reporter plasmid, and either 400 ng of G-kinase expression vector or the same amount of control vector. Stimulation of endogenous A-kinase by 8-Br-cAMP (cAMP) or of transfected G-kinase by 8-Br-cGMP (cGMP/GK) was calculated as the difference in CAT expression in the presence and absence of 1 mM cyclic nucleotide. Results are the mean of at least three independent experiments which differed by <15% of the mean.
To study G-kinase transactivation of specific enhancer elements, we used synthetic promoter constructs containing multimerized TRE, CRE, or SRE consensus sequences in the context of an enhancerless thymidine kinase promoter(13, 14) . Reporter gene expression from all three constructs was stimulated 4-5-fold by 8-Br-cGMP/G-kinase, similar to the 5-fold induction of pFOS-CAT (Fig. 4). In contrast, 8-Br-cAMP activation of endogenous A-kinase induced CAT expression from pTRE-CAT <2-fold and from pCRE-CAT >24-fold but from pFOS-CAT and pSRE-CAT about 5-7-fold. These results are further evidence that transcription factors binding to TRE and CRE sequences are regulated differently by the two kinases and suggest that the contribution of single enhancer elements to the transactivation of the fos promoter by G-kinase and A-kinase may differ. Moreover, responsiveness of TRE- and CRE-containing promoters to G-kinase and A-kinase may be cell type-specific and depend on the presence of certain transcription factors(4) .
Figure 4: Effect of transfected G-kinase and endogenous A-kinase on the activity of minimal promoter constructs containing multimerized TRE , CRE, and SRE enhancer elements. BHK cells were co-transfected with 50 ng of pRSV-Luc, 50 ng of the indicated CAT reporter plasmid, and either 400 ng of control vector or G-kinase expression vector; cells were left untreated(-) or treated with 1 mM 8-Br-cGMP (cGMP) or 8-Br-cAMP (cAMP) for 8 h as indicated. The parent plasmid pBL-CAT-5 (open bars) contains an enhancerless thymidine kinase promoter; CAT expression from this plasmid was very low and not influenced by G-kinase or A-kinase. pTRE-CAT (narrow diagonal bars) contains 10 copies of the SV40 enhancer AP-1 binding site (13) , pCRE-CAT (filled bars) contains four copies of the fos CRE(14) , and pSRE-CAT (wide diagonal bars) contains four copies of the fos SRE(14) . pFOS-CAT (stippled bars) is shown for comparison. For each construct, CAT/luciferase activity ratios were normalized as described in Fig. 2. Results are the mean ± S.D. of at least three independent experiments.
CREB binds constitutively to the fos promoter CRE(22) , and its transactivation potential is
modulated by phosphorylation, especially phosphorylation of Ser by A-kinase or Cam-kinase
IV(2, 3, 15) . To determine if G-kinase
modulates the transactivation potential of CREB, we used a Gal4-CREB
fusion construct containing the DNA binding domain of the yeast
transcription factor Gal4 and lacking the CREB COOH-terminal leucine
zipper dimerization motif
(Gal4-CREB
zip(15, 24) ). This construct was
co-transfected with either G-kinase or control vector and the reporter
plasmid pGAL4-Luc, which contains Gal4 DNA binding sites(3) .
Treatment of G-kinase-transfected cells with 8-Br-cGMP increased
transactivation of pGAL4-Luc by Gal4-CREB
zip 3.5-fold (Fig. 5A). 8-Br-cGMP had no effect on CREB
transactivation in cells transfected with control vector indicating no
significant cross-activation of A-kinase by 8-Br-cGMP. However,
A-kinase activation by 8-Br-cAMP caused a 16-fold increase in
CREB-mediated transactivation. A Ser
Ala mutation
in CREB (Gal4-CREB
zip-M1) (15) rendered CREB unresponsive
to either G-kinase or A-kinase activation (Fig. 5A).
These results suggest that G-kinase modulation of CREB activity
requires Ser
and are consistent with the finding that
G-kinase phosphorylates a synthetic peptide derived from the sequence
surrounding Ser
of CREB with a 4-fold lower V
than that of A-kinase(25) . At
present, we do not know whether G-kinase directly phosphorylates CREB in vivo; the enzyme could also modulate the activity of
proteins which cooperate with CREB, e.g. CBP(26) . We
did not observe any effect of G-kinase on the transactivation potential
of Gal4-Fos, Gal4-c-Jun, or Gal4-JunB fusion constructs (27) (data not shown).
Figure 5:
Effect of G-kinase on CREB-mediated
transactivation and effect of PKI on pFOS-CAT expression induced by
G-kinase and A-kinase. A, BHK cells were co-transfected with
100 ng of the reporter plasmid pGAL4-Luc(3) , 100 ng of
pRSV-Gal (
-galactosidase expression vector serving as
internal control), 3 ng of either pGal4-CREB
zip (a chimeric
transcription factor containing the CREB transactivation domain fused
to the Gal4 DNA binding domain, filled bars) or
pGal4-CREB
zip-M1 (pGal4-CREB
zip with a Ser
Ala mutation in CREB, diagonal bars), and 400 ng
of either G-kinase or control vector. Cells were treated with 1 mM 8-Br-cGMP (cGMP) or 8-Br-cAMP (cAMP) for 8 h as
indicated. CAT activity was normalized to
-galactosidase activity
expressed from pRSV-
Gal; the ratio of CAT/
-galactosidase
activity in the absence of Gal4-CREB constructs was assigned a value of
1. B, cells were co-transfected with pFOS-CAT and G-kinase or
control vector as described in Fig. 4except that 200 ng of
either pRSV-PKI
(encoding for wild type PKI(16) , cross-hatched bars) or pRSV-PKI
(encoding for a
PKI mutant which does not inhibit A-kinase(16) , open
bars) were added. Cells were treated with cyclic nucleotides as
described above. A and B, results are the mean
± S.D. of three independent
experiments.
To exclude that G-kinase activation
of CRE-containing promoters required endogenous A-kinase activity, we
co-transfected an expression vector encoding either the specific
A-kinase inhibitor peptide PKI (PKI), or a noninhibitory
mutant of PKI (PKI
(16) ), together with pFOS-CAT
and either G-kinase or control vector. Co-transfection of PKI
inhibited cAMP-induced reporter gene expression by >70%, but
had no significant effect on G-kinase-mediated transactivation (Fig. 5B).
Since 8-Br-cGMP has been reported to
amplify Ca induction of the fos promoter in
PC12 cells(7) , we examined the effect of the Ca
ionophores A23187 and ionomycin (7) on pFOS-CAT activity
in BHK cells transfected with G-kinase or control vector. There was no
significant effect of the ionophores (0.1-3 µM) on
pFOS-CAT expression in the presence or absence of 8-Br-cGMP (data not
shown), suggesting that BHK cells may be lacking the Cam-kinase isozyme
required for Ca
induction of the fos promoter(3) .
G-kinase is important in the regulation
of smooth muscle tone, platelet function, neuronal long-term
potentiation, and hematopoietic cell
differentiation(9, 28, 29) . Some of these
functions involve lowering of cytosolic Ca,
inhibition of phosphatidylinositol breakdown, and phosphorylation of
cytoskeletal proteins(9, 21) . This report
demonstrates that G-kinase can regulate gene expression and that the
transcription factor CREB and the c-fos gene are important
common nuclear targets for the cGMP-, cAMP- and
Ca
-activated signal transduction pathways. Additional
transcription factors which may mediate transactivation of the fos promoter by G-kinase include other members of the CREB/ATF or
Fos/Jun families; these proteins can form a large number of different
complexes, including heterodimers between proteins from both families
which bind to TRE- and CRE-like consensus
sequences(1, 30, 31) . Nuclear localization
of G-kinase has not been established, but nuclear staining by a
G-kinase-specific antibody has been observed in immunofluorescence
studies of human macrophages(32) , and G-kinase contains an
amino acid motif (residues 404-410 of G-kinase I
) which
closely resembles the nuclear localization signal of human interleukin
1
(33) . c-fos is the first gene whose expression
is regulated by G-kinase; however, other candidate genes include junB, tumor necrosis factor
, and elastin since their
mRNA expression is induced by cGMP
analogs(4, 20, 34) .