Protein Kinase A Phosphorylation of Human Phosphodiesterase 3B Promotes 14-3-3 Protein Binding and Inhibits Phosphatase-catalyzed Inactivation*

Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B.

Cyclic AMP regulates a diverse array of cellular processes, including intermediary metabolism, vascular and visceral smooth muscle relaxation, hormonal secretion, cytoskeletal organization, as well as transcription, migration, proliferation, and apoptosis (reviewed in Refs. [1][2][3]. Although the elements regulating cAMP synthesis have been extensively studied (4,5), the regulation of cyclic nucleotide phosphodiesterase (PDE) 2mediated hydrolysis of cAMP and its impact on cellular functions have only recently received considerable attention (6 -12). Based on their sequence homologies, substrate specificities, and sensitivities to pharmacological inhibitors, mammalian PDEs have been divided into 11 distinct enzyme families (6 -12).
Two genes, phosphodiesterase 3A (PDE3A) and PDE3B, encode PDE3 family enzymes (6,12). PDE3A mRNA is enriched in cells of the cardiovascular system and in oocytes, whereas PDE3B mRNA is abundant in adipocytes, hepatocytes, and cells of hematopoietic lineage (13). Full-length PDE3A and PDE3B contain two N-terminal hydrophobic regions (NHR1 and NHR2) that target these enzymes to the endoplasmic reticulum and perhaps the plasma membrane (13)(14)(15)(16). Both PDE3A and PDE3B are substrates of protein kinase A (PKA) or protein kinase B (PKB), and activation of these kinases can result in phosphorylation-mediated activation of these enzymes in some cells (13)(14)(15)(16).
A consensus has emerged that protein-protein interactions play a central role in regulating cAMP-mediated signaling. Indeed, it is generally accepted that selective subcellular anchorage of PKA, through interaction with A-kinase anchoring proteins, allows selective coordination of PKA-dependent cellular events (17,18). Subcellular targeting of certain PDEs also has emerged as a mechanism whereby these enzymes can coordinate various cellular effects of cAMP (17,18). In this context, several individual variants of the phosphodiesterase 4 (PDE4) family of enzymes interact with proteins including A-kinase anchoring proteins, ␤-arrestins, and receptor for activated protein kinase C, and these interactions regulate PDE4 subcellular targeting and enzyme activity (17,18).
Although PDE3 activity can represent a significant fraction of total cAMP hydrolytic capacity in certain cell types (6,11), little is known concerning how protein-protein interactions coordinate the activity and subcellular targeting of PDE3 enzymes. An HSPDE3B interaction with the insulin receptor in human adipocytes has been reported (19). Recently, rat adipocyte PDE3B was reported to interact with caveolin-1 (20) placing this enzyme in lipid rafts in these cells (20,21). Disruption of an interaction between the murine PDE3B and phosphoinositide 3-kinase ␥ (PI3K␥) (22), likely coordinated by one of its regulatory subunits p87 PIKAP (PI3K␥ adapter protein of 87 kDa) (23), reduced cardiomyocyte contractility (22,23). Of more immediate relevance to the studies reported here, insulin was reported previously to stimulate a PI3K-dependent interaction between murine PDE3B and 14-3-3␤ in 3T3-L1 adipocytes (24). Binding of 14-3-3 proteins to numerous proteins, usually following their phosphorylation, allows 14-3-3 proteins to act as regulators, adaptors, or scaffolds for these proteins (25). A PKC-dependent phosphorylation of HSPDE3A also stimulates association of HSPDE3A with 14-3-3 proteins (26).
In this study, we report that PKA-mediated phosphorylation of HSPDE3B at Ser-318 activates this enzyme and promotes its interaction with 14-3-3 proteins. Although PKA is also shown to phosphorylate HSPDE3B at two other sites, Ser-73 and Ser-296, these events neither activated nor promoted HSPDE3B interactions with 14-3-3 proteins. Although PKB activated HSPDE3B, this kinase did not promote 14-3-3 protein binding of HSPDE3B or influence the effects of PKA. Taken together, our data are consistent with the novel hypothesis that PKAactivated, but not PKB-activated, HSPDE3B interacts with 14-3-3 proteins and that this selective protein-protein interaction protects the PKA-activated HSPDE3B from phosphatasemediated deactivation in cells.
Heterologous Expression of HSPDE3B Constructs-A cDNA encoding HSPDE3B (Dr. V. C. Manganiello, National Institutes of Health) was cloned into the mammalian expression vector pCMV-Tag2C (Stratagene) using BamHI. This construct was used to express HSPDE3B. In experiments in which HSPDE3B was expressed heterologously, transfections were carried out with amounts of plasmid to limit overexpression of HSPDE3B to levels not exceeding 4-fold those of endogenous HSPDE3B. With full-length HSPDE3B, levels of expression were determined by PDE activity assays. Point mutations that allowed substitution of alanine (Ala) for serine (Ser) at positions 73, 295, 296, or 318 within HSPDE3B were generated using the Quick-Change site-directed mutagenesis kit (Clontech) according to the manufacturer's protocol. Plasmids encoding wild type (WT), membrane-associated (MA), or dominant negative (DN) PKB were provided by Dr. D. Alessi (University of Dundee, Dundee, Scotland, UK). HSPDE3B constructs were transiently expressed in 293T or NIH 3T3 cells following transfection using FuGENE 6 (Roche Applied Science).
In Vitro Phosphorylation of HSPDE3B-GST-or FLAGtagged proteins were purified by conventional approaches using GSH-Sepharose or M2-agarose. HSPDE3B proteins were incubated with recombinant PKA catalytic subunit or activated recombinant PKB␤ (Upstate Biotechnology, Inc., Lake Placid, NY) in a buffer without or with 200 M ATP or 200 M ATP supplemented with [␥-32 P]ATP (10 Ci per reaction; 3000 Ci/mmol stock) for 1 h at 30°C. For reference, reactions were carried out with neither kinase nor ATP, with kinase or ATP alone, or with both ATP and kinase. At the completion of these reactions, proteins were subjected to SDS-PAGE (10 -12% gels).
Statistical Analysis-Some of the data describing proteinprotein interactions are shown as representative immunoblots. In all cases, data consistent with that shown in the representative immunoblot were obtained in at least three additional separate experiments. Numerical data are presented as means Ϯ S.E. and are from at least four independent experiments. Statistical differences were assessed using unpaired analysis of variance, with a Tukey post hoc test, or unpaired Student's t test, as appropriate, with a value of p Ͻ 0.05 considered statistically significant.
In Vitro and in Vivo Association of Purified HSPDE3B and GST-14-3-3 Proteins Is Stimulated following PKA Phosphorylation of HSPDE3B-Using a GSH-based adsorption assay (herein the "GST-14-3-3 pulldown"), purified HSPDE3B interacted with either immobilized GST-14-3-3 or GST-14-3-3␤ but not with immobilized native GST. Consistent with an important regulatory role for HSPDE3B phosphorylation in regulating this interaction, prior in vitro incubation of HSPDE3B with the C-subunit of PKA and ATP, but not the C-subunit alone, increased markedly these interactions (Fig. 2,  A and B). In contrast, although in vitro incubation of HSPDE3B with purified recombinant-activated PKB and ATP increased HSPDE3B activity by ϳ90 Ϯ 15% (n ϭ 3), it did not promote HSPDE3B binding to GST-14-3-3␤ proteins (Fig. 2B). Overall these data are consistent with the idea that HSPDE3B and 14-3-3 proteins interact directly in vitro and that phosphorylation of HSPDE3B by PKA, but not PKB, promoted this direct interaction. The modest HSPDE3B binding caused by ATP alone (Fig. 2A) was likely because of the presence of trace amounts of kinase activity in the purified HSPDE3B.
To investigate if PKA-mediated phosphorylation of HSPDE3B also promoted 14-3-3 binding in cells, we used a cell line that expressed HSPDE3B endogenously, namely HEK293T, "293T." Incubation of 293T cells with a combination of forskolin and IBMX (F/I) markedly increased HSPDE3B binding to GST-14-3-3␤ and, when tested by immunoprecipitation of HSPDE3B, the amount of 14-3-3␤ that associated with HSPDE3B (Fig. 2, C and D). Consistent with a role for PKA in coordinating the effects of F/I, prior incubation of cells with a PKA inhibitor (H89, 10 M) ablated this effect (Fig. 2D). Addition of a PKC inhibitor (5 M Bis-1; Fig. 2D) or a PI3K inhibitor (10 M LY294002; data not shown) did not alter F/I-induced HSPDE3B binding to GST-14-3-3␤. Addition of either the exchange proteins activated by cAMP-selective activator (10 M; 8-(4-chlorophenylthio)-2Ј-O-methyl-cAMP), insulin (100 nM; data not shown), or of an activator of conventional PKCs (100 nM PMA; Fig. 2D) did not impact HSPDE3B binding to GST-14-3-3␤. Taken together, these data are consistent with the idea that endogenous 293T HSPDE3B interacts with 14-3-3 proteins in a PKA-dependent manner and that this interaction is independent of activation, or inhibition, of exchange proteins activated by cAMP, insulin-dependent signaling, PKC, PI3K, or PKB.
All of our data were consistent with the idea that PKA-mediated phosphorylation of HSPDE3B at Ser-318 coordinated 14-3-3 binding. Thus, although F/I treatment of cells expressing an S318A mutant form of FLAG-HSPDE3B(AT) did not promote 14-3-3␤ binding of this protein, F/I treatment of 293T cells expressing HSPDE3B(AT) forms encoding S73A, S295A, or S296A mutants did promote their interactions with 14-3-3␤ (Fig. 4A). Obviating effects because of differences in the responses of cells expressing these constructs to F/I treatments, endogenous HSPDE3B expressed in these cells interacted with GST-14-3-3␤ identically, irrespective of the HSPDE3B(AT) fragments expressed (Fig. 4A). Similar results were obtained when HSPDE3B(AT) constructs were expressed in NIH 3T3 cells even thought these cells do not express MMPDE3B endogenously (not shown).
Interestingly, trace amounts (ϳ5%) of the HSPDE3B(AT) S318A, S73A/S318A, or S296A/S318A mutants were recovered in GST-14-3-3␤ pulldowns when 293T cells were incubated with F/I (Fig. 4A). These findings were consistent with data from unrelated studies in which we show that HSPDE3B(AT) can dimerize with endogenous full-length HSPDE3B in 293T cells. 3 Indeed, when S318A mutants of HSPDE3B(AT) were expressed in NIH 3T3 cells, a cell type that does not express MMPDE3B, these constructs were not detected in GST-14-3-3␤ pulldowns (not shown).

Full-length HSPDE3B Variants Encoding an S318A Mutation Do Not Bind GST-14-3-3␤-Incubation of 293T cells express-
ing full-length S73A (not shown) or S296A HSPDE3B mutants with F/I promoted their interactions with GST-14-3-3␤ (Fig.  4B). In contrast, F/I treatment of cells expressing an HSPDE3B S318A mutant did not (Fig. 4B). Again, these data are consistent with the idea that Ser-318 is the sole relevant phospho-acceptor site that promotes PKA-dependent interactions between HSPDE3B and GST-14-3-3␤. In addition to representing the HSPDE3B phosphorylation site responsible for coordinating HSPDE3B/14-3-3␤ binding (Fig. 4) and enzyme activation (Table 1), by using an antiserum directed against phosphorylated PKA substrates we found that Ser-318 may also represent a   In vitro (A and B) and in vivo (C and D) PKA, but not PKB, phosphorylation of HSPDE3B promotes 14-3-3 protein binding. A, purified HSPDE3B was incubated with PKA (10 units/ml), ATP (200 M), or PKA and ATP at 30°C for 1 h ("Experimental Procedures"). A, following these incubations, products were incubated with immobilized GST (lane 1), immobilized GST-14-3-3␤ (lane 2), or immobilized GST-14-3-3 (lane 3) and processed as described under "Experimental Procedures." B, FLAG-tagged HSPDE3B expressed in 293T cells was isolated by M2-agarose precipitation and eluted from this immune complex with FLAG peptide (100 M). Following incubation of the peptide-eluted HSPDE3B with ATP (200 M) or with ATP and activated PKB, or the PKA C-subunit for 20 min, the mixture was subjected to a 14-3-3␤ pulldown ("Experimental Procedures"). HSPDE3B was resolved by SDS-PAGE, transferred to nitrocellulose, and detected with the FLAG antisera (M5). C, 293T cells were incubated with Me 2 SO (C, 0.2%, v/v final) or F/I (100 M each) for 20 min. Treated cells were lysed, and a cleared cell lysate was prepared as described under "Experimental Procedures." Cleared cell lysates were incubated with 1 g of anti-HSPDE3B antisera and 20 l of protein A/G-Sepharose for 16 h at 4°C. Immune complexes were recovered by centrifugation (1,000 ϫ g), washed, and resuspended in SDS-PAGE loading buffer and processed for immunoblot (ib) analysis, and HSPDE3B and 14-3-3␤ in the immune complexes were detected as described under "Experimental Procedures." ip, immunoprecipitated. D, 293T cells were incubated with Me 2 SO (C, 0.2%, v/v final), F/I, or PMA, in the absence or presence of H89, or Bis-1, for 20 min. Following these incubations, cells were lysed, and the interaction between HSPDE3B and 14-3-3␤ was measured by a 14-3-3-pulldown assay. A representative immunoblot of the impact of these incubations on HSPDE3B interactions with 14-3-3␤ is shown, as is a blot showing that these incubations had no effect on HSPDE3B levels in 293T cells.  Binding of HSPDE3B(AT), but not HSPDE3B(CT), to 14-3-3␤. Constructs encoding either a FLAG-tagged amino-terminal fragment of HSPDE3B(AT) (aa 1-518; 45 kDa), a FLAG-tagged carboxyl-terminal fragment of HSPDE3B(CT) (aa 519 -1112; 55 kDa), or a control vector (Mock) were transiently transfected in 293T cells. After 24 h, transfected cells were incubated with Me 2 SO (0.2%v/v, Ϫ) or F/I (100 M each, ϩ) for 20 min. Treated cells were lysed, and lysates were subjected to 14-3-3 pulldown assays (see "Experimental Procedures"). A representative immunoblot depicting the selective interaction between HSPDE3B (AT) and 14-3-3␤ and the promotion of this interaction by F/I is shown. MARCH 30, 2007 • VOLUME 282 • NUMBER 13 major site of PKA phosphorylation of HSPDE3B in cells incubated with F/I (Fig. 5). Because it is currently unknown if this antiserum reacts with similar affinity to all phosphorylation consensus sequences, a more detailed analysis will be required to quantify the absolute levels of phosphate at each of these sites. Although in silico analysis (30) identified other potential PKA consensus sites within HSPDE3B, our data are consistent with the idea that only Ser-318 was required for 14-3-3␤ binding in 293T cells.

14-3-3 Proteins and HSPDE3B Activation
PKB Does Not Promote HSPDE3B/14-3-3 Interactions-A previous report proposed that an insulin-promoted, PKB-dependent phosphorylation of MMPDE3B at Ser-279 and Ser-302 (residues equivalent to Ser-295 and Ser-318 in HSPDE3B) activated this enzyme and promoted its interaction with 14-3-3␤ in murine adipocytes (24). In marked contrast to this earlier report, although our data unequivocally confirm that PKB can activate HSPDE3B (see above), they are completely inconsistent with the idea that phosphorylation of HSPDE3B by PKB at Ser-295, or any other site, promotes binding of this enzyme with 14-3-3␤. Similarly, our data are inconsistent with the idea that PKB-mediated actions on HSPDE3B alter the ability of PKA phosphorylation at Ser-318 to promote 14-3-3␤ binding (Fig. 6). Indeed, expression of wild type PKB (WT PKB), a membrane-targeted and constitutively activated PKB (MA PKB), or a dominant negative and "kinase-dead" PKB (DN PKB) in 293T cells did not alter either the basal levels of endogenous HSPDE3B binding to GST-14-3-3␤ in these cells nor the ability of F/I treatment to promote this binding (Fig. 6). Consistent with their state of activation, immunoblot analysis with a phospho-PKB antiserum indicated that a large fraction of the heterologously expressed wild type or activated PKBs were phosphorylated and that the DN PKB, which is kinase-dead was not (not shown). Similarly, because F/I treatment promoted S295A-HSPDE3B binding to 14-3-3␤ (Fig. 4A), it is also unlikely that Ser-295 was involved in coordinating the PKA-dependent binding of HSPDE3B to 14-3-3␤ in these cells.
Binding of the PKA Phosphorylated and Activated HSPDE3B to 14-3-3␤ Alters Its Susceptibility to Phosphate-catalyzed Dephosphorylation and Inactivation-Because our data showed that PKA phosphorylation of Ser-318 in HSPDE3B activated this enzyme and promoted its binding to 14-3-3␤, we hypothesized that this interaction might alter the susceptibility of this fraction of HSPDE3B to be dephosphorylated and inactivated by phosphatases. Our data are completely consistent with this novel idea. Indeed, although a 20-min incubation of M2-agarose-precipitated HSPDE3B from F/I-treated cells with calf intestinal alkaline phosphatase (CIAP) resulted in substantial dephosphorylation (Fig. 7) and enzyme inactivation (Table  2), this identical CIAP treatment of the PKA-activated, GST-14-3-3␤-bound HSPDE3B did not result in substantial dephosphorylation ( Fig. 7) or enzyme inactivation (Table 2). Because PKB phosphorylation of HSPDE3B did not promote GST-14-3-3␤ binding (Fig. 2), nor influence the extent of 14-3-3␤ binding of this enzyme caused by F/I (Fig. 6), it is highly unlikely that PKB-activated HSPDE3B would be similarly protected from phosphatase-mediated dephosphorylation and inactivation. A scheme depicting these concepts is presented in Fig. 8.

DISCUSSION
Many proteins involved in cellular signaling are promiscuous, regulating several distinct events simultaneously (33,34).

14-3-3 Proteins and HSPDE3B Activation
Although the events that allow coordination of the multiple actions of signaling proteins are as yet poorly understood, dynamic regulation of protein phosphorylation has emerged as an important point of control (33,34). In this study, we have confirmed that phosphorylation of HSPDE3B regulates this enzyme activity in cells, and we have uncovered a mechanism that may allow cells to discriminate between the PKA-and the PKB-activated populations of this enzyme. As described in the Introduction, cells express only one HSPDE3B variant, and this enzyme can be targeted to both the endoplasmic reticulum and, perhaps to a lesser extent, to the plasma membrane of cells (21,(35)(36). However, there is currently a paucity of information regarding the proteins with which these potentially separate populations of HSPDE3B might interact and on the impact such protein-protein interactions might have on HSPDE3B activity. Indeed, whereas PDE3B in rodent or human adipocytes were reported to interact with the insulin receptor and/or with caveolin-1, and the rodent enzyme was shown to interact with p87 PIKAP in heart, the impact of these events on PDE3 activity and on cellular events regulated by the hydrolysis of cAMP by this enzyme are as yet poorly understood.
Previously, the rat adipocyte PDE3B was reported to interact with 14-3-3␤ in an insulin-and PI3K activation-dependent manner. Indeed, in this earlier study, it was suggested that phosphorylation of Ser-279 or Ser-302, sites equivalent to Ser-295 and Ser-318 in HSPDE3B, coordinated this interaction (24). Herein we presented data demonstrating that members of the 14-3-3 protein family of scaffolding proteins (14-3-3␤ or 14-3-3) interact with HSPDE3B in human cells, and these interactions are markedly potentiated by incubation of these cells with cAMP-elevating agents. Moreover, using a strategy of selective PKA inhibition, we identified PKA as the cAMP effector coordinating this cAMP-mediated effect. In addition to establishing that 14-3-3 proteins interacted with HSPDE3B in cells expressing these proteins endogenously, and demonstrating that these events were coordinated through cAMP-mediated activation of PKA, we also made use of a combination of genetic screens and in situ site-directed mutagenesis studies to identify the HSPDE3B residue(s) coordinating this interaction. Our data identify two potential 14-3-3 protein-binding sites within HSPDE3B. First, based on our yeast two-hybrid analysis, we identified a 14-3-3-binding domain within the first 90 amino acids of HSPDE3B. Although this HSPDE3B domain contained potential PKA phosphorylation consensus sites and was phosphorylated by PKA in vitro, our mutagenesis studies indicated that site(s) within this domain were unlikely to be involved in coordinating the increase in 14-3-3 protein binding in mammalian cells in response to cAMP. More likely, based on the fact that this interaction was initially identified in yeast in which PKA had not been experimentally stimulated, this 14-3-3 protein interacting domain of HSPDE3B may be involved in coordinating basal levels of 14-3-3 protein-HSPDE3B binding observed in our studies. This proposal is consistent with a basal level of 14-3-3 protein bind- FIGURE 5. PKA phosphorylates HSPDE3B at several distinct sites. Plasmids encoding a series of full-length HSPDE3B in which individual or combinations of potential PKA sites had been mutated were transiently expressed in 293T cells. After 24 h, transfected cells were incubated with Me 2 SO (0.2% v/v, Ϫ) or F/I (100 M each, ϩ) for 20 min. Treated cells were lysed, and lysates were subjected to immunoprecipitation with M2-agarose. Immune complexes were boiled, subjected to SDS-PAGE, and transferred to nitrocellulose for immunoblot analysis using an antibody raised against phosphorylated PKA substrates (see "Experimental Procedures"). A, representative immunoblot demonstrates that Ser 3 Ala mutations of individual or combinations of each Ser-73, Ser-296, and Ser-318 decreased phosphorylation of the FLAG-tagged HSPDE3B. B, immunoblot of HSPDE3B expressed in 293T cells transfected with the various plasmids. Similar results were obtained in three separate experiments. FIGURE 6. Increased, or antagonized, PKB activity has no impact on basal or PKA-mediated interactions between HSPDE3B and GST-14-3-3␤. 293T cells were transiently transfected with plasmids encoding either wild type PKB (Wt PKB), a membrane-targeted and constitutively activated PKB (MA PKB), or a dominant negative and kinase-dead PKB (DN PKB). After 24 h, transfected cells were incubated with Me 2 SO (0.2% v/v, Ϫ) or F/I (100 M each, ϩ) for 20 min. Following this incubation, cells were processed identically to those described in the legend to Fig. 4. Upper panel depicts levels of association between endogenous 293T cell HSPDE3B and GST-14-3-3␤, whereas the middle and lower panels show the levels of expression of PKB, or HSPDE3B, in the cell lysates used in this experiment, respectively. Similar results were obtained in three separate experiments. FIGURE 7. 14-3-3␤-associated and PKA-phosphorylated HSPDE3B is protected from CIAP-dependent dephosphorylation. 293T cells were transiently transfected with plasmids encoding FLAG-tagged HSPDE3B. After 24 h, transfected cells were incubated with Me 2 SO (0.2% v/v, Control) or F/I (100 M each) for 20 min. Following these incubations, cells were processed for either M2-agarose or GST-14-3-3␤ pulldowns. In these experiments, 0.2 mg of 293T cell lysate from each incubation was added either to M2-agarose (20-l bed volume) or to 14-3-3-GST-coupled GSH-Sepharose (50-l bed volume) and processed as described (see "Experimental Procedures"). Following these pulldowns, resuspended pellets were equally divided such that half was incubated with CIAP buffer and the other half incubated with this buffer supplemented with CIAP (1 unit) for 20 min at 37°C. Reaction products were processed for immunoblot analysis. Samples were first probed with an antibody for phosphorylated PKA substrates (A) and subsequently with the M5 anti-FLAG antiserum (B). Amounts of immunoreactive proteins were quantified by densitometric analysis. Similar data were obtained in three separate experiments. levels and the signaling events associated with HSPDE3B activation in cells.