Protein Kinase A Type I and Type II Define Distinct
Intracellular Signaling Compartments
Giulietta Di Benedetto, Anna Zoccarato, Valentina Lissandron, Anna Terrin, Xiang Li,
Miles D. Houslay, George S. Baillie, Manuela Zaccolo
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Abstract—Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of
cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics,
resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform
distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we
provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that
PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales,
thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond
to distinct, spatially restricted cAMP signals generated in response to specific G protein– coupled receptor agonists and
regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA
isoforms thus leads to phosphorylation of unique subsets of downstream targets. (Circ Res. 2008;103:836-844.)
Key Words: cAMP 䡲 compartmentalization 䡲 compartmentation 䡲 adrenergic stimulation
䡲 prostaglandin 䡲 protein kinase A
P
coupled to specific GPCRs to degrade cAMP selectively in
response to a given stimulus.8
Cardiac myocytes express all four types of PKA isozymes,
PKA-RI␣, PKA-RII␣, PKA-RI␤, and PKA-RII␤.9 PKA isoforms show different subcellular localization, with PKA-RII
being mainly associated with the particulate fraction of cell
lysates whereas PKA-RI has been found preferentially in the
cytosol.10,11 PKA isoforms also show different biochemical
properties. PKA-RI is more readily dissociated by cAMP than
PKA-RII,12,13 and the recent structure solution of holoenzyme
complexes14,15 shows critical isoform-specific features that
specifically regulate inhibition and cAMP-induced activation
of PKA-RI and PKA-RII. Given the distinct biochemical
properties and the specific subcellular localization of PKA
isozymes, it is not surprising that the biological role of
PKA-RI and PKA-RII is nonredundant, as demonstrated by
genetic and biochemical studies (reviewed elsewhere16).
However, how individual PKA isoforms serve to deliver a
specific response remains unknown. In particular, it remains
to be established how spatial control of the cAMP signal and
activation of individual PKA isoforms are coordinated to
perform a specific biological function.
Here, we set out to answer the question of whether
confined pools of cAMP elicited in response of specific
extracellular stimuli selectively activate individual PKA iso-
rotein kinase A (PKA) is a key regulatory enzyme in the
heart that is involved in the catecholamine-mediated
control of excitation– contraction coupling, as well as in a
myriad of other functions including activation of transcription factors and control of metabolic enzymes. The second
messenger cAMP activates PKA by binding to the regulatory (R) subunits, causing release of the activated catalytic
(C) subunits.
The fact that, following cAMP-engagement, PKA mediates
a plethora of cellular responses has raised the question of how
specificity is maintained. In recent years, features of this
pathway that contribute to specificity have been uncovered.1
A key role is played by AKAPs (A kinase anchoring
proteins), a family of proteins that act as molecular scaffolds
to anchor PKA in the vicinity of specific substrate molecules,2 thus focusing PKA activity toward relevant substrates.
A second mechanism contributing to specificity revolves
around the spatial control of the cAMP signal itself. Restriction of intracellular diffusion of cAMP has been shown by
using a variety of approaches,3–5 including direct imaging of
gradients of cAMP in response to activation of various G
protein– coupled receptors (GPCRs).6 A key role in shaping
cAMP intracellular pools is played by phosphodiesterases
(PDEs), the enzymes that hydrolyze cAMP.7 Indeed, individual PDE isoforms have been shown to be functionally
Original received February 28, 2008; revision received August 14, 2008; accepted August 19, 2008.
From the Dulbecco Telethon Institute (G.D.B., A.Z., V.L., M.Z.), Venetian Institute of Molecular Medicine, Padova, Italy; and Neuroscience and
Molecular Pharmacology (A.T., X.L., M.D.H., G.S.B., M.Z.), Faculty of Biomedical & Life Sciences, University of Glasgow, Scotland, United
Kingdom.
Correspondence to Dr Manuela Zaccolo, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University Avenue,
G12 8QQ, Glasgow, UK. E-mail [email protected]
© 2008 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.108.174813
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forms. By using FRET- and FRAP-based imaging approaches
we show that, in cardiomyocytes, PKA-RI and PKA-RII, by
anchoring to endogenous AKAPs, define distinct compartments within which cAMP is specifically controlled by
different subsets of PDEs. In addition, we demonstrate that
cAMP levels rise selectively in the PKA-RI and PKA-RII
compartments in a stimulus-specific manner, leading to the
phosphorylation of unique subsets of downstream PKA targets. The generation of distinct pools of cAMP within cells
that allows for the selective activation of individual PKA
isoforms is instrumental for the cell to modulate specific
physiological functions and points to means for developing
strategies for selective pharmacological intervention.
Materials and Methods
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Primary cultures of neonatal cardiac ventriculocytes from 1- to 3-day
old rats were prepared as described.6 All the details concerning
generation of constructs, cells transfection, Western blotting, immunostaining and confocal imaging, FRAP experiments, FRET imaging
and RT-PCR are described in the expanded Materials and Methods
section in the online data supplement, available at http://circres.
ahajournals.org.
Results
Generation of cAMP Sensors Selectively Targeted
to the PKA-RI and PKA-RII Compartments
We set out to assess whether PKA-RI and PKA-RII are
selectively and independently activated by specific extracellular stimuli and generated 2 FRET-based probes that, by
selectively targeting to the same subcellular compartments as
the endogenous PKA isoforms, monitor the cAMP signals
generated at these sites. We took advantage of the unique
dimerization/docking domain sequences that have been
shown17 to mediate anchoring of PKA-RI and PKA-RII
subunits to AKAPs. Thus, by fusing the dimerization/docking
domain from either RI␣ (amino acids 1 to 64) or RII␤ (amino
acids 1 to 49) to the N terminus of the soluble Epac-1
sensor,18 we generated the sensors RI_epac and RII_epac
(Figure IA in the online data supplement). For both sensors,
it is believed that the binding of cAMP to the cAMP-binding
domain will result in a conformational change that causes an
increase in the distance of the cyan fluorescent protein and
yellow fluorescent protein moieties, with a consequent reduction of the FRET signal, as shown for the parent sensor.18 The
modified sensors have been tested with respect to maximal
FRET response and to dose-response behavior (supplementary materials and supplemental Figure I). We found that
RI_epac and RII_epac are equally sensitive to cAMP
changes.
RI_epac and RII_epac Show a Different and
AKAP-Mediated Localization in Cardiac Myocytes
To assess the ability of the modified sensors to effectively
bind to AKAPs, we coexpressed RI_epac with ezrin and
RII_epac with AKAP79. Ezrin is a dual-specificity AKAP
localized at the cortical cytoskeleton and at microvilli,19
whereas AKAP79 localizes at the plasma membrane (Figure
1).20 Coexpression in CHO cells of RI_epac with ezrin results
in the relocalization of the sensor to the sub–plasma membrane region and to microvilli, whereas coexpression of
Figure 1. Targeted FRET-based cAMP sensors. Confocal
images of CHO cells expressing either RI_epac or RII_epac
alone (left panels) or in combination with ezrin and AKAP 79,
respectively (right images). The middle images show the localization of green fluorescent protein (GFP)-tagged ezrin and
GFP-tagged AKAP79 in CHO cells. Scale bars⫽10 ␮m.
RII_epac with AKAP79 results in the relocalization of the
sensor at the plasma membrane, confirming that the modified
sensors localize within the cell where AKAPs are present.
To verify if RI_epac and RII_epac are targeted to different
subcellular compartments in neonatal cardiac myocytes, the
localization of the sensors was analyzed by confocal microscopy. As a localization marker, the Z-line protein Zasp fused
to the red fluorescent protein mRFP (zasp-RFP) was coexpressed in combination with either RI_epac or RII_epac. As
illustrated in Figure 2A and 2I, RI_epac shows a tight striated
pattern overlaying with both the Z and the M sarcomeric lines
(see line intensity profiles at the bottom of Figure 2). In
contrast, the distribution of RII_epac shows a very strong
localization that corresponds to the M line and a much weaker
localization overlaying the Z line (Figure 2B and 2L). Such
localization is identical to the localization of overexpressed
full-length RI and RII subunits and corresponds to the
localization of endogenous RI and RII subunits (supplemental
Figure II). To assess whether the differences in localization
were attributable to anchoring of the sensors to endogenous
AKAPs, we used the AKAP-competing peptides RIAD21 and
SuperAKAP-IS.22 These peptides have been shown to compete selectively with the binding of PKA-RI and PKA-RII to
endogenous AKAPs. In particular, the RIAD peptide displays
more than 1000-fold selectivity for RI over RII,21 whereas the
peptide SuperAKAP-IS is 10 000-fold more selective for the
RII isoform relative to RI.22 Challenge of cells expressing
RI_epac with RIAD and of cells expressing RII_epac with
SuperAKAP-IS completely abolished the striated pattern of
localization of the sensors (Figure 2C and 2D), whereas
RIAD and SuperAKAP-IS did not have any effect on the
localization pattern of, respectively, RII_epac and RI_epac
(not shown).
The accepted paradigm for the PKA signaling pathway
is that type II PKA is associated with particulate subcellular fractions via binding to AKAPs, whereas type I PKA
is primarily cytoplasmic.11 Our confocal imaging studies
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Figure 2. RI_epac and RII_epac localize in different
subcellular compartments via binding to AKAPs.
Confocal images of cardiomyocytes expressing
either RI_epac or RII_epac alone (A and B) or in
combination with the competing peptides RIAD (C)
or SuperAKAP-IS (D). Localization of the marker
zasp-RFP in the same cell is shown in E through
H. The overlay between probe localization and
zasp-RFP is shown in I through N. The bottom
images show, for each cell, the intensity profile of
the probe signal (in blue) and of the zasp-RFP signal (in red) in the region indicated by the black
line. Scale bars⫽10 ␮m.
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suggest, however, that RI is also anchored in neonatal
cardiac myocytes. To assess the extent of anchoring of the
RI_epac and RII_epac sensors, we conducted fluorescence
recovery after photobleaching (FRAP) experiments. As
shown in Figure 3A, following fluorescence bleaching in
cells expressing either the untagged, cytosolic cAMP
sensor Epac-1 or 1 of the targeted sensors, we found that
fluorescence recovery occurred with a t1/2 of 5.7⫾0.4
seconds (n⫽12) for Epac1, 14.3⫾1.9 seconds (n⫽10) for
RI_epac, and 21.6⫾1.7 seconds (n⫽10) for RII_epac.
Fluorescence recovery was almost complete for the parent
Epac-1 probe (fractional recovery of 94.2⫾1.2%). However, this was reduced to 73.1⫾3.4% for RI_epac and to
45.4⫾5.0% for RII_epac. These results confirm that although the Epac-1 parent probe is free to diffuse in the
cytosol, the diffusion of both RI_epac and RII_epac is
substantially constrained. However, despite the mobility of
both the RI_epac and RII_epac being restricted, we noted
that RI_epac was 1.6 times as mobile as RII_epac.
To assess the extent to which the reduced mobility of RI_epac
and RII_epac was attributable to anchoring to endogenous
AKAPs, we repeated the FRAP experiments in cardiomyocytes
expressing each of the targeted sensor in combination with
specific competing peptides. Figure 3B shows a significant
increase in the fractional recovery both for cells expressing
RI_epac in combination with RIAD (85.3⫾2.3% [n⫽10]) and
for cells expressing RII_epac in combination with
SuperAKAP-IS (80.1⫾2.5% [n⫽11]). Accordingly, we found a
decrease in the recovery times with a t1/2 of 8.4⫾0.8 seconds
(n⫽10) in cells expressing RI_epac⫹RIAD and of 8.9⫾0.9
seconds (n⫽11) in cells expressing RII_epac⫹SuperAKAP-IS.
These results demonstrate that RI_epac and RII_epac specifically localize to different intracellular compartments primarily
by binding to specific endogenous AKAPs.
Figure 3. AKAP binding
limits the intracellular
mobility of RI_epac and
RII_epac. Mean FRAP
curves recorded in cardiomyocytes expressing
Epac-1 (white), RI_epac
(red), or RII_epac (green) in
the absence (A) and in the
presence (B) of the competing peptides RIAD and
SuperAKAP-IS, as indicated. Error bars indicate
SEM.
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Figure 4. Cyclase and PDE activity in the PKA-RI and PKA-RII compartments. Representative kinetics of FRET changes recorded in
cardiomyocytes expressing either RI_epac (gray circles) or RII_epac (black circles) in response to the application of 5 ␮mol/L forskolin
(A), 100 ␮mol/L IBMX (B), 10 ␮mol/L cilostamide (C), 10 ␮mol/L EHNA (D), and 10 ␮mol/L rolipram (E). F, Summary of all the experiments performed in the above conditions. Error bars indicate SEM. *0.01 ⬍ P⬍0.05. ns indicates not significant.
cAMP Levels Are Specifically Regulated in the
PKA-RI and PKA-RII Compartments
In a first set of experiments, we wanted to assess whether the
PKA-RI and PKA-RII compartments have equal access to
cAMP. Cardiac myocytes expressing either RI_epac or RII_epac
were challenged with 5 ␮mol/L forskolin. As shown in Figure
4A and 4F, the 2 sensors detected a comparable rise in [cAMP]
with ⌬R/R0 ⫽2.1⫾0.4% (n⫽7) for RI_epac and ⌬R/
R0⫽2.9⫾0.4% (n⫽14) for RII_epac (P⫽0.24), indicating that
the compartments hosting PKA-RI and PKA-RII are equally
associated to adenylyl cyclases and have potentially access to
comparable cAMP levels.
Next, we wanted to assess the role of PDEs in the
control of cAMP levels in both PKA compartments. When
myocytes were challenged with isobutylmethylxanthine
(IBMX) (100 ␮mol/L), no significant difference was
detected in the level of cAMP in the 2 compartments
(⌬R/R 0 ⫽4.5⫾0.3%; n⫽20 for RI_epac and ⌬R/
R0⫽4.9⫾0.4%; n⫽15 for RII_epac; P⫽0.45) (Figure 4B
and 4F), suggesting that the basal level of cAMP is under
comparable levels of PDE control in both PKA-RI and
PKA-RII compartments in these cells.
To determine whether specific subsets of PDEs control the
cAMP signal in the 2 PKA compartments, cardiac myocytes
expressing either RI_epac or RII_epac were treated with
selective PDE inhibitors. As shown in Figure 4C and 4F,
PDE3 inhibition assessed using the selective inhibitor cilostamide (10 ␮mol/L) generated a small and similar cAMP rise
in both PKA compartments (⌬R/R0⫽0.8⫾0.2%; n⫽9 for
RI_epac and ⌬R/R0⫽0.6⫾0.2%; n⫽11 for RII_epac;
P⫽0.58). PDE2 inhibition assessed using the selective inhib-
itor, EHNA (10 ␮mol/L) resulted in a ⌬R/R0⫽2.2⫾0.8%
(n⫽15) when detected by RI_epac and in a ⌬R/
R0⫽0.3⫾0.2% (n⫽12) when detected by RII_epac
(P⫽0.04), indicating that, in basal conditions, PDE2 activity is prominent in the PKA-RI compartment but very
low in the RII_epac compartment (Figure 4D and 4F).
PDE4 inhibition, assessed using the selective inhibitor
rolipram (10 ␮mol/L), generated a ⌬R/R0⫽0.7⫾0.2%
(n⫽13) when detected by RI_epac and a ⌬R/
R0⫽2.6⫾0.7% (n⫽16) when detected with RII_epac
(P⫽0.02) (Figure 4E and 4F), indicating that, contrary to
PDE2, PDE4 exerts its activity mainly in the PKA-RII
domain and to a much lower extent in the PKA-RI
compartment. These results show that cAMP levels are
differently regulated in the PKA-RI and PKA-RII compartments and point to a specific association of individual PKA
isoforms with selected subsets of PDEs in these cells.
Individual GPCRs Generate a cAMP Signal
Selectively in the PKA-RI or
PKA-RII Compartments
We next asked whether the PKA-RI and PKA-RII compartments may be coupled to specific GPCRs such that individual
PKA isoforms respond selectively to cAMP signals elicited
by different agonists. As shown in Figure 5A, the application
of isoproterenol (10 nmol/L), a specific activator of ␤-adrenoreceptors, generated a rise in [cAMP] more pronounced in
the PKA II domain as compared to the PKA I domain
(⌬R/R 0 ⫽2.5⫾0.5% [n⫽13] for RI_epac and ⌬R/
R0⫽4.5⫾0.7% [n⫽9] for RII_epac; P⫽0.007). Addition of
IBMX (100 ␮mol/L) abolishes such a difference. This result
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Figure 5. Effect of GPCR stimulation on the cAMP signal in the
PKA-RI and PKA-RII compartments. Representative kinetics of
FRET change on application of 10
nmol/L isoproterenol (A), 100
nmol/L GLP-1 (B), 300 nmol/L
glucagon (C), and 1 ␮mol/L PGE1
(D) recorded in cardiomyocytes
expressing RI_epac (gray circles)
of RII_epac (black circles). E,
Summary of the experiments performed in the above conditions. F
and G, FRET change detected in
cardiomyocytes coexpressing
either RI_epac in combination with
the competing peptide RIAD (gray
bars) or RII_epac in combination
with SuperAKAP-IS (black bars)
and stimulated with 10 nmol/L
isoproterenol (F) or with 1 ␮mol/L
PGE1 (G). Error bars indicate
SEM. *0.01⬍P⬍0.05;
**0.001⬍P⬍0.01; ***P⬍0.001.
is in agreement with our previous studies showing that
␤-adrenergic stimulation generates a restricted pool of cAMP
that activates selectively AKAP-anchored PKA-RII.6 By
contrast, challenging the myocytes with GLP-1 (100 nmol/L)
resulted in a larger [cAMP] increase in the PKA-RI compartment as compared to the PKA-RII compartment (⌬R/
R0⫽3.7⫾0.8%, n⫽15 for RI_epac and ⌬R/R0⫽1.0⫾0.4%,
n⫽12, for RII_epac; P⫽0.008) (Figure 5B). Similarly, 300
nmol/L glucagon (Figure 5C) and 1 ␮mol/L prostaglandin E1
(PGE1) (Figure 5D) generated a higher [cAMP] increase in
the PKA-RI associated compartment than in the PKA-RII
compartment, showing ⌬R/R0⫽1.3⫾0.4% (n⫽7) for RI_epac
and ⌬R/R0⫽0.2⫾0.1% (n⫽6) for RII_epac (P⫽0.017) in the
case of glucagon and ⌬R/R0⫽1.4⫾0.2% (n⫽35) for RI_epac
and ⌬R/R0⫽0.6⫾0.2% (n⫽30) for RII_epac (P⫽0.01) in the
case of PGE1. In the presence of the specific competing
peptide RIAD, the cAMP signal generated by the application
of isoproterenol (10 nmol/L) was now clearly detected by
RI_epac (⌬R/R0⫽5.0⫾1.0% [n⫽9]; P⫽0.008 versus RI_epac
alone; Figure 5F). Coexpression of SuperAKAP-IS and
RII_epac did not affect RII_epac ability to detect the
isoproterenol-induced cAMP signal (Figure 5F). Similarly,
when RII_epac was coexpressed with the selective competing
peptide SuperAKAP-IS, the amplitude of the cAMP signal
generated by application of 1 ␮mol/L PGE1 was now clearly
detected by RII_epac (⌬R/R0⫽2.8⫾0.7% [n⫽17]; P⫽0.0001
versus RII_epac alone; Figure 5G), whereas coexpression of
RIAD and RI_epac had no effect on the amplitude of the
signal detected on PGE1 application (Figure 5G). In addition,
we found that treatment with RIAD significantly reduced the
velocity of the FRET change of RI_epac to PGE1 (supplemental Figure III).
Taken together, these results demonstrate that individual
GPCR agonists generate spatially restricted pools of cAMP
that selectively activate individual AKAP-anchored PKA
isoforms. Removal of PKA isoforms from their anchoring
sites allows them to diffuse and to be activated by pools of
cAMP that would not normally affect them.
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Figure 6. Specific GPCR agonist stimulation results in a different pattern of downstream PKA targets phosphorylation. A,
Western blots of cardiac myocyte lysates
probed for PLB, TnI, and the ␤2AR with
corresponding phosphoblots after treatment with 10 nmol/L isoproterenol and
1 ␮mol/L PGE1. Quantifications are means
of at least 3 separate experiments. Error
bars indicate SEM. **0.001⬍P⬍0.01;
*0.01⬍P⬍0.05. B, Representative Western
blots of cardiac myocyte lysates probed
for PLB and TnI with corresponding phosphoblots after treatment with isoproterenol
10 nmol/L with and without pretreatment
with KT5720 (2 ␮mol/L).
Compartmentalized PKA Isozymes Phosphorylate
Selected Subsets of Downstream Targets
We next wanted to assess whether the selectivity of the cAMP
signals generated in the PKA-RI and PKA-RII compartments
on stimulation of individual GPCRs has a functional relevance and results in a specific pattern of PKA-mediated
phosphorylation of downstream targets. To this aim, we
studied the phosphorylation level of several PKA targets after
stimulation with either isoproterenol or PGE1. The phosphorylation level of phospholamban (PLB), troponin (Tn)I, and
the ␤ adrenergic receptor type 2 (␤2AR) was markedly
increased (Figure 6A) on stimulation with isoproterenol (1
nmol/L) but not on stimulation with PGE1 (10 ␮mol/L).
Stimulation of myocytes with isoproterenol in the presence of
the PKA inhibitor KT5720 (2 ␮mol/L) completely abolished
the increased phosphorylation level of both PLB and TnI
(Figure 6B), confirming the involvement of PKA. Unexpectedly, PGE1-stimulated cells showed a reduction in the phosphorylation level of PLB and TnI as compared to untreated
cells. Further analysis indicated that the reduced phosphorylation of these targets was dependent on the activity of
phosphatases (supplemental Figure IV).
Based on these results we can conclude that individual
agonists, via activation of specific PKA isoforms, affect
distinct subsets of downstream targets.
A Gi-Mediated Mechanism Contributes to Control
the cAMP Signal, Leading to the Phosphorylation
of PLB and TnI
Four different receptors for PGE have been described (EP1 to
EP4) with EP2 and EP4 being coupled to G␣s and EP3 being
coupled to G␣i.23 We asked whether the reduction in the
phosphorylation level of PBL and TnI observed on PGE1
stimulation may be attributable to activation of an EP3
receptor and subsequent G␣i-mediated inhibition of cyclase
activity. RT-PCR analysis of mRNA extracted from neonatal rat cardiomyocytes confirmed that these cells express
high levels of message for the G␣i-coupled EP3 receptor, as
well as message for the G␣s-coupled EP2 and EP4 receptors
(Figure 7A).
To evaluate the occurrence and the relevance of Gi activation on the local control of the cAMP signal generated by
PGE1, we performed imaging experiments applying PGE1 on
cardiomyocytes that had been pretreated with the Gi-inhibitor
pertussis toxin (PTX). As shown in Figure 7B, application of
1 ␮mol/L PGE1 following 2 to 4 hours pretreatment with 2
␮g/mL PTX generated a comparable cAMP increase in the
PKA-RI and PKA-RII compartments. In particular, PTX
pretreatment results in a much-increased cAMP signal in the
PKA-RII compartment (⌬R/R0⫽1.7⫾0.4% [n⫽11] versus
0.6⫾0.2% [n⫽30] in the absence of PTX; P⫽0.006). Indeed,
this equals the amplitude of the signal generated in the
PKA-RI compartment (⌬R/R0⫽1.4⫾0.2%; P⫽0.54; see Figure 7B). No significant effect of PTX pretreatment was
observed on the amplitude of the cAMP signal observed in
the PKA-RI compartment (⌬R/R0⫽2.1⫾0.5% [n⫽8] as compared to ⌬R/R0⫽1.4%⫾0.2% in control cells; P⫽0.13).
To assess the functional relevance of such a G␣i-mediated
control of the cAMP signal, we measured the level of
phosphorylation of PLB and TnI in cardiomyocytes pretreated with PTX for 1, 2, or 4 hours before the application of
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Figure 7. A Gi-mediated mechanism contributes to the control of the cAMP signal in the PKA II compartment. A, RT-PCR analysis of
the EP receptors expressed in neonatal rat cardiomyocytes. Hypoxanthine phosphoribosyltransferase (HPRT) is used as a control for
input mRNA concentration. B, FRET changes recorded in cardiomyocytes expressing either RI_epac (gray bars) or RII_epac (black bars)
with or without pretreatment with PTX (2 ␮g/mL for 2 to 4 hours) and challenged with 1 ␮mol/L PGE1. C, Western blots of cardiac myocyte lysates probed for PLB and TnI with corresponding phosphoblots after treatment with 1 or 10 nmol/L isoprenaline and 1 ␮mol/L
PGE1 with and without pretreatment with PTX (2 ␮g/mL for 1, 2, and 4 hours) Quantifications are means of at least 3 separate experiments. Error bars indicate SEM. *0.01⬍P⬍0.05.
PGE1. Figure 7C shows that, on inhibition of G␣i, PGE1
stimulation induce a significant increase in the phosphorylation level of both these PKA-RII–specific targets (compare
Figures 6 and 7C). Thus, a G␣i-mediated mechanism appears
to contribute significantly to the control of the cAMP signal
generated by PGE1 stimulation in the PKA-RII domain.
Discussion
The organization of the signaling machinery in discrete
compartments is increasingly recognized as a critical feature
for the specificity of the cAMP/PKA system in cardiomyocytes.24 Here, we used real-time imaging of cAMP to study
how the diversity of PKA isoforms may contribute to the
transduction of specific downstream signals. We did this by
targeting FRET-based reporters of cAMP concentration at
intracellular sites where endogenous PKA-RI and PKA-RII
isoforms localize. We demonstrate that PKA-RI and PKA-RII
reside in physically segregated and distinct compartments
within which the level of cAMP appears to be selectively
regulated by different subsets of PDEs, although the exact
contribution of individual PDEs would require simultaneous
inhibition of combinations of different isoforms. We show
that the cAMP signal is specifically generated either in the
PKA-RI or in the PKA-RII compartment depending on the
GPCR agonist applied and that cAMP does not diffuse from
one compartment to the other so as to cross-activate PKA
isozymes, allowing fidelity of the response (Figure 8). One
functional consequence of such compartmentalization is that
isoproterenol stimulation leads to the specific phosphorylation of PLB, TnI, and ␤2AR, whereas PGE1 stimulation does
not affect these substrates, demonstrating that individual PKA
isoforms are coupled with defined subsets of targets and that
PKA isoforms activity is not promiscuous.
Our findings are consistent with previous work25–27 showing that, in cardiac myocytes, there is a clear dichotomy
between the effects of PGE1 and isoproterenol on a variety of
cAMP-dependent events. These studies demonstrated that
isoproterenol generates an increase in contractile force25
Di Benedetto et al
Compartmentalized Signaling by PKA Isoforms
843
Figure 8. Model of PKA isoforms compartmentalization. A, Activation of the PGE receptor system leads to the generation of a restricted cAMP pool affecting PKAI. The signal
in the specific PKA-RII compartments may be switched off with the contribution of an
EP3-dependent, Gi-mediated mechanisms. B, Only when the ␤AR is activated via binding of the appropriate agonist a cAMP pool is generated in the PKA-RII compartment
and this isoform is activated to phosphorylate a specific subset of downstream targets.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
enhances ventricular pressure development and induces phosphorylation of target proteins such as phosphorylase kinase
and TnI,26 whereas PGE1, although elevating cAMP to
comparable levels and inducing activation of PKA similar to
isoproterenol, does not show any effect on contractility or on
the phosphorylation of these target proteins.
The general notion concerning subcellular localization of
PKA isoforms in cardiac myocytes is that PKA-RII holoenzyme is localized whereas PKA-RI is mainly cytosolic.
Biochemical studies on cell homogenates showed that PKARII subunits are bound to particulate material in heart cells,
whereas RI subunits are largely soluble.10,11 Interestingly, the
selective effects of ␤-adrenergic stimulation on heart function
has been shown to correlate with activation of a membranebound fraction of PKA, whereas PGE1 stimulation increases
the activity of a soluble pool of PKA.26 Our data now provide
a mechanism for these observation by showing that catecholamines generate restricted pools of cAMP that selectively
engage PKA-RII isoforms. However, the notion of a PKA-RI
migrating freely in the cytosol, as suggested on the basis of
previous biochemical studies, is difficult to reconcile with
any selectivity in the activation of PKA isozymes, particularly considering that PKA-RI is more readily activated by
cAMP as compared to PKA-RII.13 In tissues other than the
heart, dual-specificity AKAPs capable of binding PKA-RI
and -RII have been described,28 and AKAP-mediated localization of PKA-RI to the neuromuscular junction29 or to
interphase microtubules and specific regions of the mitotic
spindle has been shown.30 The discovery in Caenorhabditis
elegans of a specific RI-binding AKAP that does not interact
with RII subunits31 suggests the potential for nonredundant
PKA-RI localization and function and a specific role for
localized PKA-RI in modulating T-cell receptor signaling has
been demonstrated.32 Thus, by performing FRAP experiments on intact cells, here we find that neonatal cardiac
myocytes express a considerable amount of PKA-RI anchor
sites, as shown by the slow fluorescence recovery time and
the large immobile fraction detected in cells expressing the
RI_epac sensor. This suggests that a large fraction of the
PKA-RI isozyme may in fact be anchored in the heart in vivo.
The fact that PKA-RI is found mainly in the supernatant of
heart cell homogenates may reflect relatively low affinity
interactions between PKA-RI and the protein binding partners that are lost during the homogenization and washing
steps. Notwithstanding this, the nature of the PKA-RI anchoring proteins in cardiac myocytes remains to be established.
Catecholamine-mediated sympathetic control of cardiac stimulation is a major compensatory mechanism that maintains or
augments systolic and diastolic ventricular function during
physiological stress or pathological conditions. In particular,
catecholamines selectively improve diastolic function by reducing myofilament calcium sensitivity through phosphorylation of
proteins such as TnI and accelerate sequestration of calcium into
the sarcoplasmic reticulum through phosphorylation of PLB and
release of its inhibitory effect on the SERCA pump.33 Enhanced
and sustained cardiac adrenergic drive, however, is known to be
deleterious and to contribute, in part, to development and
progression of pathological states such as heart failure.34 Our
results indicate that key proteins involved in the catecholaminemediated regulation of cardiac contractility are under the control
of a restricted pool of cAMP that selectively activates a subset of
PKA-RII isozymes, thus leading to a coordinated and specific
response. In addition, we show that a supplementary element
contributing to specificity is provided by a Gi-mediated mechanism that contributes to keep the PKA-RII compartment clear of
cAMP unless the appropriate catecholaminergic stimulus impinges on the cell. Such a mechanism may indeed serve to
protect the heart from excessive adrenergic stimulation in pathological conditions. Interestingly, PGE1 stimulation is known to
protect myocardial tissue from injury following ischemia and
reperfusion,35 an effect that has been suggested to depend on
PGE-mediated stimulation of EP3 receptors in cardiac myocytes36 and the consequent Gi-mediated inhibition of cAMP
synthesis.37
In summary, the present work provides original insight into
the mechanisms that underpin cAMP/PKA specificity of response by demonstrating that, in cardiac myocytes, both
PKA-RI and PKA-RII isoforms subsets anchor to subcellular
844
Circulation Research
October 10, 2008
sites via binding to endogenous AKAPs. This defines exclusive
signaling domains within which the cAMP signal is uniquely
generated via activation of specific GPCR and their associated G
proteins and is uniquely modulated by the activity of different
subsets of PDEs, resulting in stimulus-specific phosphorylation
of downstream protein targets. These results provide a mechanism for the different function of PKA-RI and PKA-RII subsets
and provide a functional rationale for the design of isoformspecific activators and inhibitors of PKA.
Acknowledgments
We thank Martin Lohse for providing Epac-1, John Scott and Kjetil
Tasken for the constructs for RIAD and for SuperAKAP-IS, Kjetil
Tasken for ezrin and ezrin-GFP, Matteo Vatta for wt-Cypher/ZASPGFP, and Marc Dell’Acqua for AKAP-79 and AKAP-79-GFP.
Sources of Funding
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
This work was supported by Telethon Italy grant GGP05113; Human
Frontier Science Program Organization grant RGP0001/2005-C (to
M.Z.); Fondation Leducq grant O6 CVD 02 (to M.Z.); and the
European Commission grant LSHB-CT-2006-037189 (to M.Z.,
G.S.B., and M.D.H.); and Medical Research Council grant
G0600765 (to M.D.H. and G.S.B.).
Disclosures
None.
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Protein Kinase A Type I and Type II Define Distinct Intracellular Signaling
Compartments
Giulietta Di Benedetto, Anna Zoccarato, Valentina Lissandron, Anna Terrin, Xiang Li, Miles D.
Houslay, George S. Baillie and Manuela Zaccolo
Circ Res. 2008;103:836-844; originally published online August 28, 2008;
doi: 10.1161/CIRCRESAHA.108.174813
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2008 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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World Wide Web at:
http://circres.ahajournals.org/content/103/8/836
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2008/08/28/CIRCRESAHA.108.174813.DC1
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Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Supplemental Materials and Methods
Reagents
DMEM-Hepes, Medium 199, PBS, FBS, L-glutamine, penicillin and TRIzol
reagent
were
purchased
from
Invitrogen.
Prostaglandin
E1
(PGE1),
isoproterenol, glucagon-like peptide 1 (GLP1), glucagon, forskolin, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), cilostamide, rolipram, isobutyl-methylxanthine (IBMX), pertussis toxin (PTX), okadaic acid and KT 5720 were
obtained from Sigma-Aldrich. All chemicals were analytical grade. Restriction
enzymes, T4 ligase, and shrimp alkaline phosphatase (SAP) were purchased
from New England Biolabs, Taq polymerase from Euroclone. Transfectin Lipid
Reagent and Superscript IIITM were obtained from BioRAD.
Constructs generation
The Epac-1 genetically encoded sensor for cAMP1 was kindly provided by M.
Lohse (Institute of Pharmacology and Toxicology, University of Würzburg,
Germany). RI_epac and RII_epac were generated by fusion to the N-terminus
of Epac-1 of the sequence encoding for the dimerization-docking (DD) domain
of RIα (64 aa) or RIIβ (49 aa), respectively. Between the DD domain and
Epac-1, the 27 aa linker A (EAAAK)5A was inserted. Such a linker was
chosen because it folds as a monomeric hydrophilic α-helix and is effective in
separating heterofunctional domains in a fusion protein, avoiding inadequate
interactions between the domains and allowing them to work independently 2.
The mRFP-ZASP and CFP-ZASP constructs were generated by substituting
the mRFP3 and ECFP fluorophores to the GFP in the wt-Cypher/ZASP-GFP4,
kindly provided by Dr M. Vatta. The RI-ECFP construct was generated by
substituting the RIα to the RIIβ in the RII-ECFP5. Finally, RI-RFP and RII-RFP
were constructed by substituting The mRFP fluorophore to the ECFP in the
RI-ECFP and RII-ECFP.
The constructs containing the cDNA for RIAD and SuperAKAP-IS were
generated by external PCR starting from the RIAD-GFP and SuperAKAP-ISGFP constructs that were kindly provided from Dr. J.D. Scott (Oregon Health
and Science University, U.S.A.)
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Cell culture and transfection
Primary cultures of cardiac ventricular myocytes from 1- to 3-day old Sprague
Dawley rats (Charles River Laboratories, Wilmington, MA) were prepared as
described5 and transfected with Transfectin Lipid Reagent (BioRAD), following
the supplier instruction. Imaging experiments were performed 24–48 h after
transfection.
Western blotting
Treated cardiac myocytes were washed twice with ice cold PBS (phosphate
buffered saline) before cellular lysates were prepared. Cellular lysates were
prepared in lysis buffer containing 25mM Hepes, pH 7.5, 2.5 mM EDTA, 50
mM NaCl, 30 mM sodium pyrophosphate, 10% (v/v) glycerol and 1% (v/v)
Triton X-100 and Complete TM EDTA-free protease inhibitor cocktail tablets
(Roche).Protein concentration of lysates were quantified using the Bradford
assay and all samples were equalised for protein concentration. Proteins were
separated by gel electrophoresis and transferred to nitrocellulose. The
following antibodies were used to detect phospho-proteins and native proteins
in lysates from control and treated cells: Phospho-Troponin I (cardiac)
Ser23/24 (Cell Signaling, product code 4004), Troponin I
(Cell Signaling,
product code 4002), Phospho-phospholamban Ser 16 (Upstate, product code
07-052),
Phospholamban
(Upstate,
product
code
05-205),
Phospho-
βadrenergic receptor2 Ser345/346 (Santa Cruz, product code sc-16718), βadrenergic receptor2 (Santa Cruz, product code sc-569). Quantification of
phospho-proteins was done using densitometry software Quantity One.
Arbitrary phosphorylation units were calculated and results plotted against
controls. Plotted results represent the mean of at least three independent
experiments with standard errors.
Immunostaining and Confocal Imaging
Cardiac ventricular myocytes were co-transfected with different combination
of constructs or decorated with anti-PKA RI or anti-PKA RIIβ antibodies (BD
Transduction Laboratories). Alexa fluor 568-conjugated antimouse antibody
(Molecular Probes) were used as secondary antibodies. CHO cells were
transfected with either RI_epac or RII_epac, alone or in combination with,
respectively, ezrin and AKAP79. Confocal images were acquired 24-48 hours
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
after transfection by using the broadband confocal Leica TCS SP5 system
(Leica Microsystems) and a HCX PL APO 63x1.4NA oil-immersion objective.
Cells were maintained in Hepes-buffered, calcium-free, Ringer-modified saline
(125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgS04, 5.5 mM glucose and
20 mM Hepes, pH 7.5), at room temperature (20-22°C), and excited using the
458-nm line of an argon laser for imaging CFP and the 543-nm line of a
helium-neon laser for imaging mRFP.
FRAP experiments
FRAP experiments were performed 24-48 hours after transfection on a Leica
TCS SP5 confocal system. Experiments and data analysis were performed by
using the Leica FRAP application wizard. Cardiomyocytes trasfected with
either RI_epac or RII_epac were maintained in the Ringer-modified saline
described for confocal imaging, with the addition of 1 mM CaCl2, at room
temperature, and excited using the 514-nm line of an argon laser for imaging
YFP. Image acquisition was performed by using a HCX PL APO 63x1.4NA oilimmersion objective. Each experiment was conducted at 100% laser power,
acquiring 5 images with AOTF set to 5%, one bleaching image with AOTF set
to 100%, and 70 images of fluorescence recovery with AOTF set to 5%. The
time between frames was minimized.
The fluorescence recovery half-time, t1/2, was calculated as the time
necessary for the fluorescence signal to recover to 50% of the asymptote
intensity. Values are expressed as the mean ± the standard error of the mean
(SEM). To determine the mobile fraction we compared the fluorescence in the
bleached region after full recovery (F∞ROI) with the fluorescence before
bleaching (Fi) and immediately after bleaching (F0). The mobile fraction Mf
was calculated as:
100 ∗
FCELL-PRE (F∞ROI - F0 )
∗
(Fi - F0 )
FCELL( t )
where FCELL-PRE is the mean fluorescence intensity in the whole cell before
bleaching and FCELL( t ) is the fluorescence outside the bleached area at each
time point. The first term in the above equation contains a correction for
photobleaching during the acquisition of the post-bleach images.
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Fluorescence Resonance Energy Transfer Imaging
FRET imaging experiments were performed 24-48 h after cardiomyocytes
transfection. Cells were maintained at room temperature in the Ringermodified saline (see above) additioned of CaCl2 (1 mM), and imaged on an
inverted microscope (Olympus IX50) with a FLUAR 100xNA1.3 oil-immersion
objective (Zeiss). The microscope was equipped with a CCD camera
(Sensicam
QI,
PCO,
U.S.A.),
a
software-controlled
monochromator
(Polychrome IV, TILL Photonics, Germany), and a beam-splitter optical device
(Multispec Microimager; Optical Insights, U.S.A.). Images were acquired using
custom-made
software
and
processed
using
ImageJ
(http://rsb.info.nih.gov/ij/). FRET changes were measured as changes in the
background-subtracted 480/545-nm fluorescence emission intensity on
excitation at 430 nm and expressed as either R/R0, where R is the ratio at
time t and R0 is the ratio at time = 0 sec, or ΔR/R0, where ΔR = R – R0. Values
are expressed as the mean ± SEM.
RT-PCR
Total RNA was isolated from cultured rat neonatal cardiac myocytes and from
freshly isolated rat adipocytes from epididymal fat pads by using the TRIzol
reagent (Invitrogen). An aliquot of total RNA was retrotranscribed by
Superscript IIITM (Invitrogen) to generate cDNA. mRNAs for the four EP
receptors genes and for the hypoxanthine-guanine phosphoribosyltransferase
(HPRT) gene were amplified from the cDNA. For the oligonucleotide
sequences used for the detection of cDNA specific for the EP1, EP2, EP3, EP4
and HPRT see the supplementary materials. Each amplification reaction,
containing all reagents except the primers, was split in two. Primers to amplify
one of the four EP receptors were added to one half of the reaction, whereas
in the primers to amplify the HPRT gene were added to the other half. Cycling
conditions were: 94°C for 30 s, Tann (68°C for EP1 and EP4, 60°C for EP3 and
56°C for EP2 and HPRT) for 30 s, 72°C for 30 s, 35 cycles. The predicted
sizes of the amplified fragments were of 135 bp for EP1, 126 bp for EP2, 122
bp for EP3, 130 bp for EP4 and 160 bp for HPRT.
Oligonucleotides used for the detection of cDNA specific for the EP1, EP2,
EP3, EP4 and HPRT genes are shown below, together with the annealing
temperature used for each amplification.
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
EP1
Fwd 5’-GAGCCCCCTGCTGGTATTGG-3’
Rev 5’-GCGCAGCAGGATGTACACCC-3’
Tann: 68°C
EP2
Fwd 5’-CTGCCTTTCACAATCTTTGC-3’
Rev 5’- TCTAAGGATGACAAAAACCC-3’
Tann: 56°C
EP3
Fwd 5’-GCCGCTATTGATAATGATGC-3’
Rev 5’-GCGAAGCCAGGCGAACGGCG-3’
Tann: 60°C
EP4
Fwd 5’-TCCATTCCGCTCGTGGTGCG-3’
Rev 5’-TCCAAGGGTCCAGGATGGGG-3’
Tann: 68°C
HPRT Fwd 5’-AGTCCCAGCGTCGTGATTAG-3’
Rev 5’-CCATCTCCTTCATGACATCTCG-3’
Tann: 56°C
Supplemental Information
RI_epac and RII_epac are equally sensitive to cAMP.
In order to test the performance of the modified sensors, CHO cells
expressing either the original Epac-1 or one of the novel targeted sensors
were challenged with the direct activator of adenylyl cyclase, forskolin (5 μM)
together with the non-selective PDE inhibitor, IBMX (100 μM) so as to elicit a
saturating cAMP response8. The average maximal FRET change for the three
sensors is shown in Online Fig IB. When compared to Epac-1 (ΔR/R0 = 1.00
± 0.08, n = 11), both targeted probes show a 50% reduction in the maximal
FRET response, with a ΔR/R0 = 0.51 ± 0.03 (n = 35) for RI_epac and a
ΔR/R0 = 0.49 ± 0.04 (n = 20) for RII_epac, respectively. This indicates that
fusion of the DD domain to Epac-1 roughly halves the maximal FRET change.
We found that such a reduction in FRET response is likely due to the
presence of the DD domain (see below).
A
dose-response
curve
generated
by challenging
CHO
cells
expressing either RI_epac or RII_epac with increasing concentrations of
forskolin shows a complete overlap for the two sensors with an EC50 of about
1 μM (Online Fig IC). These results show that although fusion of the DD
domain to the cAMP sensor affects its performance, the resulting RI_epac
and RII_epac probes maintain a reasonable maximal FRET response (about
10%) and are equally sensitive to cAMP changes.
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Fusion of the DD domain to the Epac-1 sensor is responsible for the
observed reduction of its maximal FRET change.
Fusion of the DD domain to Epac-1 resulted in a 50% reduction of the
maximal FRET change for both targeted sensors. We wondered if this
reduction was due to anchoring of the sensors to endogenous AKAPs and the
consequent steric hindrance which would limit the conformational change
needed for maximal FRET. To test this hypothesis, we coexpressed RI_epac
and RII_epac with the AKAP competing peptides RIAD6 and SuperAKAP-IS7,
respectively. These peptides have been shown to compete selectively with
the binding of PKA-RI and PKA-RII to endogenous AKAPs. Challenge of CHO
cells expressing either RI_epac in combination with RIAD or RII_epac in
combination with SuperAKAP-IS resulted in a maximal FRET change not
significantly different from the maximal FRET change recorded in cells
expressing the sensor alone (Online Fig ID). These results indicate that the
N-terminal fusion of the DD domains per se is responsible for the reduction in
the maximal FRET change of the targeted sensors.
Mobilization of the RI_epac sensor reduces the speed of FRET change in
response to PGE1.
Our results demonstrate that application of selected GPCR agonists
generates distinct pools of cAMP that are sensed selectively by either
RI_epac or RII_Epac. It is expected that mobilization of the targeted sensor
from their anchoring sites results in a slower FRET change in response to the
specific stimulus as a consequence of the increased distance of the sensor
from the selected pool of cAMP. Indeed, we found that the response to PGE1
is much slower if detected by RI_epac in the presence of RIAD than the FRET
change detected by RI_epac alone (P = 0.0008) (see Online Fig III). The
same effect was not found in the case RII_epac. In this case the response to
isoproterenol resulted to be equally fast when detected by RII_epac alone or
by RII_epac in the presence of SuperAKAP-IS (P = 0.98). The t/2 of the
response (i.e. the time to reach half-maximal FRET change) was of about 40
seconds in both cases. It should be noted, however, that the response to
isoproterenol is one order of magnitude faster than the response to PGE1.
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Nikolaev et al.1, in their description of the parent cAMP sensor Epac-1,
reported that, in HEK-β1AR cells challenged with isoprenaline 1 μM, the t/2 of
the sensor response is about 50 seconds. In addition, even when cells were
treated with saturating stimuli (e.g. 25μM forskolin in the presence of 100mM
IBMX) we were not able to detect a t/2 faster than 40 s. Thus, a t/2 of about
40 seconds for the response to isoproterenol, either detected by RII_epac or
by RII_epac in the presence of SuperAKAP-IS, seems to be the fastest
possible detectable by the sensor. Therefore, a faster cAMP increase
generated by isoproterenol in the PKA type II microdomain can not be
appreciated due to limiting probe activation time.
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Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Online Figure Legends
Online Fig I. (A) Schematic representation of RI_epac and RII_epac (B)
Average maximal FRET change for Epac-1 (white), RI_epac (gray) and
RII_epac (black), elicited by forskolin 5 μM and IBMX 100 μM in CHO cells.
Error bars indicate SEM. ***, P < 0.001. (C) Dose-response curves for
RI_epac (gray circles) and RII_epac (black circles), expressed in CHO cells
treated with forskolin concentration ranging from 2.5 nM to 100 μM. Error bars
indicate
SEM.
(D)
Average
maximal
FRET
changes
detected
in
cardiomyocytes co-expressing either RI_epac in combination with the
competing peptide RIAD (gray bars) or RII_epac in combination with
SuperAKAP-IS (black bars) and stimulated with forskolin 5 μM and IBMX 100
μM. Error bars indicate SEM.
Online Fig II. A) Localization of endogenous R subunits. Confocal images of
neonatal cardiac myopcytes decorated with antibodies specific for for RI (RI
end) and RII (RII end). B) Confocal images of cardiomyocytes co-expressing
either the full-length, CFP-tagged, RI or RII (in blue) and zasp-RFP (in red).
The overlay is shown in green. Panels on the right show, for each cell, the
intensity profile of the RI/RII-CFP signal (in blue) and of the zasp-RFP signal
(in red) in the region indicated by the black line. C) Confocal images of
cardiomyocytes co-expressing either RI_epac or RII_epac (in blue) and the
full-length, RFP-tagged, PKA RI or RII (in red). The overlay is shown in green
Right panels show, for each cell, the intensity profile of the probe signal (in
blue) and of the RI/RII-RFP signal (in red) in the region indicated by the black
line.
Online Fig III. t/2 of FRET changes detected in cardiomyocytes coexpressing either RI_epac in combination with the competing peptide RIAD
and stimulated with 1 μM PGE1 (gray bars), or RII_epac in combination with
SuperAKAP-IS and stimulated with 10 nM isoproterenol (black bars). Error
bars indicate SEM. ***, P < 0.001.
Online Supplement
Di Benedetto et al., Protein kinase A type I and type II…
Online Fig IV. Phosphatase inhibitors rescue Gi-mediated reduction in basal
levels of phospho-Tn I and phospho-PLB. Representative western blots of
cardiac myocyte lysates probed for phospholamban and Troponin I with
corresponding phospho-blots after treatment with 1 μM PGE1 with and without
pre-treatment of okadaic acid (100 nM).
Online Figure IV