Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells

cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro, whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant “desensitization” of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a “sensitized intermediate” state that is in effect primed to trigger PKAc activity.

The catalytic activity of PKAc is in part controlled by four functionally non-redundant regulatory subunit proteins (RI␣, RI␤, RII␣, and RII␤) (5)(6)(7)(8), which bind PKAc in tetrameric "holoenzyme" complexes to maintain kinase inactivity until stimulation with cAMP (1,9). The structural assembly of PKA holoenzymes and allosteric activation mechanism triggered by cAMP binding to R-subunits is well-understood (9 -15). However, unique biochemical features of RI␣ may serve to activate PKAc via non-canonical, cAMP-independent mechanisms. The linker-hinge region of PKA regulatory subunit proteins contains the autoinhibitory motif that allows for selective inhibition of PKAc at the kinase active site; however, this inhibitor sequence (IS) differs significantly between RI and RII (11,16). Although RII subunits have a PKA consensus phosphorylation sequence (RRXS) that is phosphorylated by PKAc, RI subunits have a "pseudosubstrate" IS (RRX(A/G)) that is unable to be phosphorylated by PKAc; therefore, substrate competition is required to trigger full PKA holoenzyme dissociation (17). Additionally, RI␣ subunit has three serine residues in the linker region that are putative phosphorylation sites: Ser 77 , Ser 83 , and Ser 101 (Fig. 1A) (18 -20). Previously, the Ser 101 residue (P ϩ 2 to the pseudosubstrate IS) was shown to be an in vitro substrate of cGMP-dependent protein kinase (PKG) (21,22). Given the nature of the in vitro methodology used previously to assess phosphorylation of this site, as well as the lack of identity of a well-defined PKG consensus phosphorylation sequence, the physiological relevance of this putative phosphorylation site remains uncharacterized. To expand upon this work, experiments were aimed at validating in vitro studies with purified recombinant proteins and determining whether this unique mechanism of PKA activation happens in cell culture models.
Recent structural information acquired in the last 10 -15 years concerning the nature of binding between PKAc and RI␣ allows for some conjecture about how modification of Ser 101 might lead to changes in type I PKA activity. The protein structure of the RI␣-PKAc heterodimer was solved by X-ray crystallography techniques by Kim et al. (12), and this structure showed how the linker region of RI␣ binds the active site cleft of PKAc. The IS makes direct contacts with residues from both the N-lobe and C-lobe of PKAc, whereas the CNB-A domain binds distally to the C-lobe. Because of the pseudosubstrate nature of the IS in RI␣, the high affinity binding of this motif to PKAc (in complex with ATP and two magnesium ions, Mg 2 ATP) presents a kinetic barrier for activation, whereas this is not critical for RII subunits capable of phosphorylation by PKAc (16,23). The binding affinity of RI␣ and PKAc in the presence of Mg 2 ATP is 0.1 nM versus 200 nM in the absence of nucleotide (24). ATP also binds with an affinity of 60 nM in the RI␣ holoenzyme, whereas the K m /K d is 25 M for the free protein. This gives a rationale for why modification of Ser 101 could possibly perturb the binding interaction of the linker region of RI␣ to PKAc.
A close-up view of this interfacial region helps to illustrate the importance of this residue in maintaining proper binding interactions with PKAc (Fig. 1B). Upon analysis of polar interactions in this region, one can see that the hydroxyl moiety of Ser 101 makes hydrogen bonds with residues from the ␣C-helix of PKAc (particularly residues Gln 84 and His 87 ). It is inferred that introduction of a phosphate group at this position will introduce steric hindrance that will likely alter the interaction of these residues. Furthermore, the interaction of Ser 101 with Gln 84 and His 87 facilitates a hydrogen-bond network near the activation-loop phosphorylation site Thr 197 in PKAc. Thus, we can further postulate that phosphorylation of Ser 101 will also bring about a negative charge-charge repulsion effect caused by the juxtaposition of two phosphate groups within this binding interface. These effects in combination would lend to open- Figure 1. A, cartoon diagram of RI␣ domain layout, with emphasis on the linker region sequence and phosphorylation sites Ser 77 , Ser 83 , and Ser 101 . RI␣ domains from the N terminus to the C terminus were as follows: dimerization/docking (D/D) domain, the pseudosubstrate inhibitory site (IS; sequence is shown in red underlined text), and tandem CNB domains. The presumed kinases for each putative phosphorylation site are listed below. The PKG phosphorylation site Ser 101 is indicated by a red asterisk. B, PyMOL structural representation of the RI␣-PKAc heterodimer complex (Protein Data Bank code 2qcs), focusing on the binding interface between the active site of PKAc and the linker region IS of RI␣ (light-gray cartoon, PKAc N-lobe; olive-green surface, PKAc C-lobe; teal cartoon, RI␣ CNB domains; red cartoon with sticks, RI␣ linker region containing the IS to PKAc). The serine residue of interest in RI␣ (Ser 101 , red) is depicted along with other critical residues at this binding interface, including 1) the pseudosubstrate alanine (Ala 99 ) and p ϩ 1 residue (Ile 100 ) from the RI␣ linker region; 2) two residues from the ␣C-helix of PKAc (Gln 84 and His 87 ) that form direct hydrogen bonds with the hydroxyl group of serine 101; and 3) activation loop phosphorylation site (Thr(P) 197 ) and its neighboring residues in PKAc (Arg 165 , Arg 189 , and Ser 195 ). Hydrogen bonds are depicted as dashed lines.

RIa Ser 101 phosphorylation by PKG activates PKAc in cells
ing of the active site cleft of PKAc. Taking these observations under consideration, we hypothesized that phosphorylation of Ser 101 by PKG would promote enhanced PKAc activity by removing the additional requirement of substrate competition in RI␣ holoenzyme activation. Finding evidence of direct PKG-PKA cross-talk in the context of cellular signaling would aid our understanding of how these two related pathways could regulate physiologically relevant systems where both proteins are expressed (i.e. smooth muscle and cardiac tissues) (25,26).

PKGI␣ phosphorylates RI␣ at serine 101 both in vitro and in cells
To extend previous reports demonstrating that PKA RI␣ could be phosphorylated by PKGI␣, we performed in vitro phosphorylation reactions using purified recombinant bovine RI␣ (bRI␣)-PKAc holoenzyme and PKGI␣. The reactions contained [␥-32 P]ATP, and phosphate incorporation was determined by autoradiography. We observed robust phosphorylation of bRI␣ only in the presence of PKGI␣ ( Fig. 2A, compare lanes 5-8 with lanes 1-4; quantification of data in Fig. 2B). RI␣ was phosphorylated by PKGI␣ to a similar extent in the absence and presence of cyclic nucleotides (lanes [5][6][7][8]. These results are a bit unexpected, because cyclic nucleotide free and cAMPbound PKGI␣ should not be as active as the cGMP-bound kinase. However, under the conditions used, it is likely that PKGI␣ activity was driven by PKGI␣ autophosphorylation and was cyclic nucleotide-independent. We originally reasoned that cAMP would disassociate bRI␣ from PKAc and thus provide easier access for PKGI␣ to phosphorylate Ser 101 ; however, although there was a trend toward increased RI␣ phosphorylation in the presence of cAMP, the difference did not reach significance (compare lane 5 with 7 and 6 with 8). Thus, it appears that holoenzyme association/dissociation has no effect on PKGI␣'s access to the phosphorylation site.
Next, we examined whether PKGI␣ could phosphorylate RI␣ in intact cells. HEK293T cells were transfected with expression vectors for PKGI␣ and FLAG-tagged WT or S101A-mutant human R⌱␣. The cells were labeled with [ 32 P]orthophosphate, and the differences between WT and S101A-mutant RI␣ phosphorylation were compared in the presence or absence of PKGI␣, as well as with and without 2-h stimulus with 8-CPT-cGMP (a membrane-permeable analog of cGMP). In cells transfected with WT RI␣ alone, we observed a small amount of basal RI␣ phosphorylation, which was slightly increased by treatment with 8-CPT-cGMP (Fig. 2C, compare lanes 1 and 2; quantification of data in Fig. 2D). In cells co-transfected with PKGI␣, treatment with 8-CPT-cGMP induced a much higher level of RI␣ phosphorylation, which was dramatically reduced in cells transfected with PKGI␣ and S101A-mutant RI␣ ( In these cell-based experiments, the faint phosphorylation signal observed for RI␣ in cells expressing S101A-mutant RI␣ indicates the presence of other phosphorylation sites that are targeted in cells. To address this, two other putative phosphorylation sites within RI␣ protein, namely Ser 77 and Ser 83 , were investigated (Fig. 2E). These two sites have been shown to be phosphorylated in human heart tissues in the context of ischemic heart disease (20) and thus may contribute to the background signal in our phosphorylation assays. Phosphorylation reactions were performed in HEK293T cells overexpressing FLAG-tagged constructs of either WT or a mutant version of R⌱␣ protein with both Ser 77 and Ser 83 mutated to alanine. All reactions were conducted with overexpressed PKGI␣ either with or without 2 h of cGMP stimulus. In cells overexpressing WT protein, low levels of RI␣ phosphorylation in cells without cGMP stimulus were observed as seen in previous experiments. However, cells expressing the S77A/S83A double mutation of RI␣ did not yield phosphorylation signal without cGMP stimulus, indicating that all background signal observed in non-stimulated samples is due to extraneous in-cell phosphorylation of these two particular sites. As before, immunoblots using anti-FLAG antibodies were performed to show equal expression of RI␣. These initial phosphorylation experiments performed under both in vitro and in-cell conditions have shown that serine 101 in RI␣ is indeed a bona fide phosphorylation target of PKGI␣ in mammalian cells.

Mutation at serine 101 in RI␣ induces PKAc activity without cAMP stimulus in vitro and in cells
As mentioned in the introduction, we hypothesized that phosphorylation of Ser 101 would serve to increase PKAc activity in cells. Therefore, we developed mutations at this site in RI␣, via site-directed mutagenesis of the serine to either a glutamate residue (S101E) or an alanine residue (S101A), to test whether this modification will weaken IS binding to the active site of PKAc and thus allow the competition of substrates to bind and be phosphorylated. After generating S101E and S101A mutant constructs for recombinant protein purification (full-length bovine RI␣ (1-379) in pRSET vector) and S101E mutant for mammalian cell transfection (full-length human RI␣ (1-379) in pcDNA3.1 vector), components of the PepTag phosphorylation assay were used to assess differences in PKAc activation for wildtype versus mutant protein under both in vitro and in-cell conditions. Under in vitro conditions, WT RI␣ maintains PKAc in an inhibited state without cAMP stimulus; however, both S101E and S101A mutants display high PKAc activity under basal (cAMP-free) condi-tions (Fig. 3, A and B). Increasing S101E protein concentration from 2ϫ, 5ϫ, and 10ϫ the relative stoichiometry of WT protein had no effect on inhibiting the observed high basal PKAc activity (Fig. 3C).
For in-cell analysis of PKAc activity, WT and S101E-mutant human RI␣ were overexpressed in MEF RI␣ Ϫ/Ϫ cells (mouse embryonic fibroblasts with a genetic knockout of PRKAR1␣). The RI␣ Ϫ/Ϫ cell line allows for a null background to compare the addition of exogenous RI␣ overexpression. The relative degree of PKAc phosphorylation activity was compared between non-transfected cells (control RI␣ Ϫ/Ϫ ) and cells overexpressing either WT or S101E-mutant human RI␣ (Fig. 3, D and E). Control cells displayed high substrate phosphorylation under non-stimulated conditions, thus serving as a positive control for PKAc activity and also confirming earlier observations that in the absence of RI␣, PKA activity is not well-regulated even though other R-subunit isoforms are present. In close corroboration with in vitro data, overexpression of WT RI␣ in cells inhibited PKAc activity in the absence of Fsk/IBMX. In contrast to WT, cells expressing the S101E mutant showed high activity without cAMP stimulus, similarly as observed under in vitro conditions. We also conducted immunoblot analysis of RI␣ and PKAc expression in MEF RI␣ Ϫ/Ϫ lysates for the corresponding activity assay samples examined (Fig. 3, D and F). Two major trends were observed: 1) in WT cells, the level of RI␣ was slightly decreased upon Fsk/IBMX stimulation; and 2) RI␣ expression appeared to be slightly higher in S101E cells as compared with cells expressing WT RI␣ (but only significant when comparing WT vehicle to S101E Fsk/IBMX samples). The competitive nature of the phosphorylation reactions employed suggests that the IS of RI␣(S101E) must have a lowered affinity for the PKAc active site cleft; otherwise the PKA substrate would not be able to bind effectively to allow for phosphotransfer activity of PKAc.
Two explanations are possible for the aforementioned results. One possibility is that the RI␣(S101E) mutant is simply unable to form a complex with PKAc; alternatively, the mutant RI␣ protein may still distally bind PKAc (via the R-subunit CNBs domains), even though the modified sequence in the IS region is loosely anchored to the PKAc active site cleft. Therefore, several techniques were explored to evaluate the relative capacity of S101E and S101A mutants of RI␣ to form holoenzyme.

Ser 101 mutant variants of RI␣ pulldown PKAc from HEK293T cell lysates via co-immunoprecipitation (co-IP)
To demonstrate that the Ser 101 mutants of RI␣ can form holoenzyme complexes in cells, FLAG-tagged versions of human RI␣ were overexpressed in HEK29T cells, whereupon we assessed whether the S101A and S101E mutant variants of RI␣ were able to pull down overexpressed HA-tagged PKAc protein from cell lysates via co-immunoprecipitation. In this experiment, immunoblotting for the presence of HA-tagged protein in anti-FLAG IP samples can be used to qualitatively determine the state of holoenzyme complex formation in cells. We observed HA-tagged PKAc in the immunoprecipitate of all RI␣ proteins tested (WT, S101A, and S101E), sig-

RIa Ser 101 phosphorylation by PKG activates PKAc in cells
nifying that these mutations do not influence the formation of heterodimer complexes between PKAc to RI␣ (Fig. 4A). Furthermore, co-immunoprecipitation of PKAc was also possible in the presence of excess substrate (Kemptide), indicating that excess substrate does not promote holoenzyme dissociation (Fig. 4B).

Figure 3. Mutation of bovine RI␣ at serine 101 induces PKAc activity without cAMP stimulus in vitro.
A, analysis of in vitro PKAc kinase activity using the PepTagா activity assay kit, comparing the inhibitory capacity of either WT, phosphomimetic mutant (S101E), or alanine mutant (S101A) variants of purified bovine RI␣ protein (n ϭ 3). B, densiometric quantification of three experiments performed as described in A, expressed as means Ϯ S.D. Statistical significance is indicated as compared with RI␣(WT) without cAMP. **, p Ͻ 0.001; ***, p Ͻ 0.0001. C, qualitative analysis of in vitro PKAc kinase activity using the PepTagா activity assay kit, comparing the inhibitory capacity of either WT or phosphomimetic mutant (S101E) variants of purified bovine RI␣ protein. In this experiment, full-length RI␣ S101E dimer protein was added in doses of progressively higher molar stoichiometry as compared with PKAc protein. Lane 1, no protein control; lanes 2-12, 200 nM PKAc; lanes 3 and 4, 120 nM WT dimer; lanes 5 and 6, 120 nM S101E dimer; lanes 7 and 8, 240 nM S101E dimer; lanes 9 and 10, 480 nM S101E dimer; lanes 11 and 12, 1.2 M S101E dimer (n ϭ 1). D, analysis of PKAc kinase activity using the PepTagா activity assay kit, comparing WT or phosphomimetic mutant (S101E) variants of human RI␣ overexpressed in MEF RI␣ Ϫ/Ϫ cells (mouse embryonic fibroblasts with genetic knockout of PRKAR1␣). To stimulate cAMP production in these cells, Fsk/IBMX (10 and 100 M) were added for 10 min prior to cell lysis and subsequent activity assay (n ϭ 3). The lower panels are immunoblots of RI␣, PKAc, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control for the given treatment conditions. E, densiometric quantification of three activity assay experiments performed as described in D, expressed as means Ϯ S.D. Statistical significance is indicated as compared with WT without Fsk/IBMX. *, p Ͻ 0.05. F, densiometric quantification of three immunoblot experiments performed as described in D, expressed as means Ϯ S.D. Statistical significance is indicated as compared with WT without Fsk/IBMX. *, p Ͻ 0.05. Ctrl, control.

RIa Ser 101 phosphorylation by PKG activates PKAc in cells Protein-fragment complementation assay (PCA) shows Ser 101 mutants form cAMP-sensitive complexes in cells
To further confirm that mutant RI␣ forms stable holoenzyme complex in cells, a bioluminescent PCA (developed by Stefan and co-workers. (27); see "Experimental procedures") was implemented. This assay uses differential tagging of PKA regulatory and catalytic subunit proteins with fragments of Renilla luciferase (RLuc) to create a "split-reporter" system that can be used to monitor R-C complex formation as a function of luciferase-fragment complementation. Upon cellular overexpression of RLuc-tagged versions of PKAc and either wildtype, S101A, or S101E variants of RI␣, the relative degree of RI␣-PKAc protein-protein interaction can be measured in intact cells. Moreover, PKA holoenzyme dissociation can be measured by luminescence signal decrease upon cellular stimulation with isoproterenol (a ␤-adrenergic receptor agonist known to stimulate cAMP production in cells). We observed that Ser 101 mutants of RI␣ still displayed a similar degree of complex formation as compared with wildtype in non-stimulated cells (Fig. 5). Furthermore, bioluminescence signal was diminished upon addition of isoproterenol in all protein constructs tested, thus showing that the Ser 101 mutant variants of RI␣-PKAc complexes are still sensitive cAMP and thus allow for holoenzyme dissociation under stimulatory conditions. cAMP response of bRI␣ proteins shows high inhibitor binding for S101E, but not S101A mutant For a more quantitative understanding of PKAc binding affinities for wildtype and mutant RI␣ subunits in vitro, we utilized the ligand-regulated competition (LiReC) fluorescence polarization assay (see "Experimental procedures") (28). This competitive inhibitor-binding assay is used to detect the relative fluorescence polarization of FAM-IP20, a fluorescein-conjugated PKA inhibitor peptide that is derived from the heat stable protein kinase inhibitor. In the LiReC assay, the relative increase of the fluorescence polarization signal measured as the IP20 probe binds PKAc is indicative of the decrease in anisotropic tumbling of the immobilized inhibitor. First, a cAMP response assay was performed to compare FAM-IP20 binding to PKAc in the presence of either the WT, S101A, and S101E versions of RI␣ (Fig. 6). Whereas the S101A mutant behaved similarly as WT protein, the S101E mutant displayed significantly higher levels of FAM-IP20 binding in the absence of cAMP. In this instance, the S101E and S101A mutants showed lower EC 50 values as compared with WT, yet Hill slope values for both mutants were not significantly changed.

R-subunit inhibition response assay shows S101E binds PKAc with lower relative affinity
To more conclusively determine whether RI␣(S101E) with its modified linker region can functionally bind PKAc, the assay was modified to assess the inhibitory capacity for the RI␣ subunit in the presence of a constant concentration of PKAc (10 nM) and in the absence of cAMP (Fig. 7). WT, S101A, and S101E proteins were compared to discern any differences in formation of holoenzyme complexes for these serine 101 mutants. We observed that significantly higher levels of S101E mutant were required to out-compete FAM-IP20 for binding to PKAc (IC 50 S101E ϭ 212.5 nM Ϯ 1.55). In contrast, S101A behaved very similar to WT RI␣ (IC 50 WT ϭ 4.5 nM Ϯ 1.05; IC 50 S101A ϭ 5.05 nM Ϯ 1.08), indicating that both proteins have an affinity for PKAc of less than 10 nM, which is the concentration of PKAc in the assay. These data suggest that the presence of the glutamic acid residue specifically and significantly affects the affinity of the RI␣ subunit to the PKAc active site, whereas the more conservative alanine substitution can function similar to unmodified wildtype protein at 10 nM concentrations. Thus, these experiments were able to illustrate that RI␣(S101E) can functionally bind PKAc to out-compete FAM-IP20, but  PCA (see "Experimental procedures") was performed to measure RI␣-PKAc protein association in isoproterenol-stimulated HEK293T cells, comparing WT to mutant RI␣ (S101A and S101E). In this assay, increased bioluminescence signal indicates complementation of the luciferase "split reporter" caused by protein-protein interaction mediated by holoenzyme association. Moreover, PKA holoenzyme dissociation is measured by luminescence signal (relative light units) decrease upon cellular stimulation with isoproterenol (a ␤-adrenergic receptor agonist known to stimulate cAMP production in cells). Shown is a representative of n ϭ 3 independent experiments; Ϯ standard deviation from triplicates.

RIa Ser 101 phosphorylation by PKG activates PKAc in cells
the relative inhibitory equilibrium constant was approximately 2 orders of magnitude higher compared with the wildtype RI␣ control.

Discussion
To summarize the work presented here, we have revisited and additionally built upon a former concept within the field of protein kinase signaling, which highlights a potential cross-talk mechanism between the key contributors of cyclic nucleotide signaling in eukaryotic cells (PKA and PKG). Our evidence has validated that Ser 101 in RI␣ is a target of PKG both in vitro and in cells, and furthermore we showed that introduction of a phosphorylation modification (or phosphomimetic mutation) at the serine 101 site in RI␣ leads to a state of heightened PKAc activity while still maintaining a RI␣-PKAc complex. These data significantly shift the paradigm regarding our understanding of Type I PKA signaling, in that modification of the RI␣ linker region can serve to circumvent previously described kinetic restrictions of holoenzyme dissociation and thus trigger what we denote as "desensitized" PKAc activity. We have thus created a revised model of RI␣ holoenzyme activation to better illustrate how the effect of PKG phosphorylation could lead to significant changes in the equilibrium of RI␣-PKAc association and dissociation (Fig. 8). In this model, modification of RI␣ leads a "sensitized intermediate" state of the RI␣ holoenzyme that is capable of phosphotransfer activity because of the lack of linker region accessibility at the PKAc active site. The addition of physiological levels of cAMP thus can trigger rapid dissociation of the holoenzyme complex, therefore creating a "desensitized" state of activity because of the reduced affinity of the modified Figure 7. R-subunit inhibition response assay shows that S101E binds PKAc with lower relative affinity. LiReC fluorescence polarization assay (see "Experimental procedures") compared inhibitory capacity of regulatory subunit protein between WT (black), S101E mutant (red), and S101A mutant  . cAMP response of bRI␣ proteins shows high inhibitor binding for S101E, but not S101A mutant. A LiReC fluorescence polarization assay (see "Experimental procedures") compared cAMP response between WT (black), S101E mutant (red), and S101A mutants (blue) forms of bRI␣. A, graph of fluorescence polarization data reported in millipolarization units (mP). B, table of EC 50 values and Hill slope. (Each condition was performed in quadruplicate, n ϭ 4; error bars and table data are representative of Ϯ S.E.).

Figure 8. PKG phosphorylation model of type I PKA activation.
A cartoon diagram of a modified, three-state model of type I PKA holoenzyme activation based on RI␣ phosphorylation by PKG is shown. Our model illustrates that type I holoenzyme may not transition directly from an inactive holoenzymecomplex state to a completely dissociated and active state. Instead, our data suggest that phosphorylation of serine 101 in RI␣ lowers the affinity between the IS of RI␣ and the active site of PKAc, thus leading to a "sensitized intermediate" state that displays PKAc catalytic activity while maintaining a holoenzyme complex configuration. This intermediate would then respond to physiological levels of cAMP toward a "desensitized" active state of the dissociated holoenzyme complex. Our model also denotes the potential for reversibility of linker region phosphorylation by protein phosphatases (PP).

RIa Ser 101 phosphorylation by PKG activates PKAc in cells
RI␣ to reassociate with PKAc. Although this study focused upon PKG phosphorylation of Ser 101 in RI␣, our supplementary investigation of alternative phosphorylation sites in the linker region portion of the protein (i.e. Ser 77 and Ser 83 ) has further highlighted the potential significance of linker region modification as a regulatory mechanism for type I PKA in cells. Because these sites are differentially phosphorylated in heart tissues with and without onset of heart failure (20), it is possible that these linker region phosphorylation sites in RI␣ are specifically involved in regulating response to stress in the context of cardiac disease. Further investigation is also required to address the presumed role of protein phosphatases in allowing reversibility of phosphorylation at these linker region sites.
In terms of chemical equilibrium, we propose that modified RI␣ (i.e. the sensitized intermediate) has a significant (nearly 2 orders of magnitude) decrease in binding affinity to PKAc as compared with the 0.1 nM K d of the inactive holoenzyme. This assertion could very well explain discrepancies observed in related scientific literature, where it has been shown that elevation of cAMP under physiological conditions may not be sufficient to explain how PKA is activated upon stimulatory signaling in the cell (29). However, further examination is required to determine the exact nature of binding in the "sensitized intermediate" protein complex, with particular regard to 1) pseudosubstrate binding/accessibility to the active site cleft of PKAc, and 2) effects upon allosteric interactions in the modified holoenzyme. In future studies, surface plasmon resonance methods will be used to quantitatively determine the binding affinity of Ser 101 mutants for PKAc. Utilization of alternative biophysical methodologies such as hydrogen/deuterium exchange mass spectrometry could also help to better explore the relative degree of conformational dynamics for WT and S101E proteins binding to PKAc by quantifying the degree of solvent exposure upon differing timescales of protein-protein interactions.
Although this study provides additional biochemical characterization of PKG phosphorylation of Ser 101 in RI␣ in a cellular overexpression system, a final goal for this work is to assess the significance of this mechanism within in vivo systems. Because both PKG and PKA signaling play critical roles within cardiac and smooth muscle cell physiology (26,30,31), our ongoing research is aimed at detecting endogenous phosphorylation of RI␣ in differentiated muscle cell types, either treated with or without stimulus to drive PKG activity. Along this line of investigation, determining the role of oxidative stress signaling in the activation of this cross-talk mechanism will be of particular importance, because reactive nitrogen/oxygen signaling has been shown to result in alterations of heart physiology as manifested through both PKA and PKG (32)(33)(34). Given that both PKG and PKA have been implicated as targets in disease conditions such as dilated cardiomyopathy (35)(36)(37), heart failure with preserved ejection fractions (38, 39), and ischemia reperfusion injury (40,41), characterization of RI␣ phosphorylation within endogenous tissues may further bolster the significance of this mechanism within the context of clinically relevant cardiovascular diseases.

Materials
Antibodies for PKAc and RIa were from BD Biosciences (San Jose, CA); antibodies for glyceraldehyde-3-phosphate dehydrogenase were from Santa Cruz Biotechnology (Dallas, TX). Anti-HA and anti-species secondary antibodies were obtained from Santa Cruz Biotechnology. Other antibodies, chemicals, and reagents were obtained from Sigma unless otherwise stated.

Recombinant DNA and site-directed mutagenesis
Constructs of full-length human RIa gene (hRIa 1-379) incorporated into the pCDNA3.3 mammalian expression vector were used for cellular transfection experiments in MEFs. Point mutations (hRIa[S101A] and hRIa[S101E]) were introduced by site-directed mutagenesis via QuikChange PCR as per the manufacturer's instructions (Stratagene, La Jolla, CA). The same genetic material was also incorporated into pRSET-Xa3 vector for bacterial recombinant protein expression. Point mutations (bRIa[S101A] and bRIa[S101E]) were introduced into both constructs by site-directed mutagenesis via QuikChange PCR as per the manufacturer's instructions (Stratagene). FLAG-tagged RI␣ was constructed by PCR using an untagged RI␣ vector as a template. The PCR product was digested and inserted into the pFLAG-D expression vector. The S101A and S101E mutant RI␣ constructs were generated using overlapping extension PCR. All constructs that went through a PCR step were sequenced to ensure that the expected coding sequence was present.

Expression and purification of recombinant proteins from Escherichia coli
The C-subunit was expressed in E. coli and purified as described (42). Full-length (1-379) constructs of either wildtype, S101E, and S101A mutants of bRIa (in pRSET-Xa3 vector) were expressed in BL21(DE3) cells and purified by cAMP affinity chromatography in 20 mM MES (pH 6.5), 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, and 5 mM DTT. RI␣ proteins were further purified on a gel filtration column using Superdex 200 and concentrated for purposes of biochemical assays using Amicon centrifugal filters (EMD Millipore, Billerica, MA).

Cell culture: Growth, transfection, and treatments
HEK293T and mouse embryonic fibroblast (MEF) cells were used throughout this study. Propagating cultures were RIa Ser 101 phosphorylation by PKG activates PKAc in cells grown in 10-cm dishes using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. MEF cultures contained 1% penicillin/streptomycin, unless otherwise stated.

Phosphorylation of RI␣ in HEK293T cells
HEK293T cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a 37°C incubator with a 5% CO 2 atmosphere. The day before transfection, the cells were spit into a 6-well cluster dish such that the cells were 90 -95% confluent at the time of transfection. The cells were transfected with the indicated expression constructs with Lipofectamine 2000 using conditions recommended by the manufacturer (Thermo Fisher). The next day, the medium was removed, and 1 ml of phosphate-free media and 100 Ci of 32 PO 4 was added to each well. The cells were incubated at 37°C for 2 h, and then 250 M 8-CPT-cGMP was added to the appropriate wells. The cells were incubated for an additional 2 h at 37°C, at which point the medium was removed, the cells were washed once with ice-cold PBS, and the cells were lysed on the plate in PBS, 0.1% Nonidet P-40, 1ϫ protease inhibitor mixture (Calbiochem, San Diego, CA), and 1ϫ HALT phosphatase inhibitor (Cell Signaling Technology, Danvers, MA). Cleared lysates were added to 20 l of anti-FLAG affinity gel and incubated for 1 h at 4°C with constant mixing. The beads were washed and boiled in 30 l of 1ϫ SDS sample buffer, and phosphorylation was analyzed by SDS-PAGE/autoradiography. The relative amount of immunoprecipitated FLAG-tagged RI␣ was determined by immunoblotting.

PepTagா non-radioactive PKA phosphorylation assay
Assays using both cell lysates and recombinant proteins were performed in a modified procedure from the manufacturer's directions. For cell-based experiments, MEF cell samples were plated on 6-well dishes at passage 3. Transfections were conducted using Lipofectamine 2000 (Thermo Fisher) as per the manufacturer's directions. For activity experiments, MEF cells were serum-starved for 2 h before conducting 10 min of Fsk/ IBMX stress treatment (i.e. 20 M forskolin and 100 M IBMX). 10 l of cell lysate (of normalized protein concentration) was added to 5 l each of "5ϫ peptide," "5ϫ buffer," and distilled water for all reactions. The samples were mixed and incubated at room temperature for 30 min and then heat-inactivated at 95°C for 10 min. For recombinant protein experiments: RI␣ and PKAc proteins were incubated in situ at a 1.2:1 molar stoichiometry within a 10-l volume (for RI␣(1-379) dimer protein, 5 l of 1.2 M R-subunit was added to 5 l of 2 M PKAc). Then 5 l each of 5ϫ peptide, 5ϫ buffer, and either 5 mM cAMP or distilled water was added to start the reactions. Samples were mixed and incubated at room temperature for 30 min and then heat-inactivated at 95°C for 10 min. For all experiments, phosphopeptides were separated by 0.8% agarose gel as per the manufacturer's directions. Densiometric analysis of gel images was performed using ImageJ. Activity is expressed as the percentage of phosphorylated peptide, calculated by the ratio of phosphorylated peptide signal divided by total signal.

Co-immunoprecipitation
Expression vectors encoding HA-tagged PKAc and WT or Ser 101 mutant FLAG-tagged RI␣, or empty vector were transfected into 293T cells using Lipofectamine 2000. After 20 h, the cells were lysed in PBS, 0.1% Nonidet P-40 with 1ϫ protease inhibitor mixture. The lysates were cleared by centrifugation at 16,000 ϫ g for 10 min, and supernatants were incubated with anti-FLAG M2 affinity gel for 1 h at 4°C with constant mixing. The beads were washed three times with PBS, 0.1% Nonidet P-40, proteins were eluted by boiling in 2ϫ Laemmli buffer, and bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. For some experiments, 500 M Kemptide was added during the immunoprecipitations.

PCA using fragmented RLuc
Plasmid constructs of PKAc tagged with F1 fragment (PKAc-F1) and RI␣ tagged with F2 fragment (RI␣-F2) were generated previously (27). S101E and S101A mutations of RI␣ were introduced into RI␣-F2 by site-directed mutagenesis via QuikChange PCR as per the manufacturer's instructions (Stratagene). PKAc-F1 and either WT, S101E, or S101A versions of RI␣-F2 plasmids were co-transfected into HEK293T cells, and bioluminescence signal was compared under basal conditions, as well as with 15 min of 10 nM isoproterenol stimulus.

Statistics
All activity, immunoblot, and PCA data are presented as means Ϯ S.D., and all fluorescence polarization assay data are presented as means Ϯ S.E. GraphPad Prism 4 software (GraphPad Software, Inc., San Diego, CA) was used for all statistical analysis. Statistical analyses were performed by unpaired Student's t test of planned comparisons.