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Cancer Research 68, 3133, May 1, 2008. doi: 10.1158/0008-5472.CAN-08-0064
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

Elise Meoli1, Ioannis Bossis1, Laure Cazabat3,4,5, Manos Mavrakis2, Anelia Horvath1, Sotiris Stergiopoulos1, Miriam L. Shiferaw1, Glawdys Fumey3,4,5, Karine Perlemoine3,4,5, Michael Muchow1, Audrey Robinson-White1, Frank Weinberg1, Maria Nesterova1, Yianna Patronas1, Lionel Groussin3,4,5, Jérôme Bertherat3,4,5 and Constantine A. Stratakis1

1 Section on Endocrinology and Genetics, Program in Developmental Endocrinology and Genetics, and 2 Section on Organelle Biology, Program in Cell Biology and Metabolism, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland; 3 Institut National de la Santé et de la Recherche Médicale U567, Département d'Endocrinologie, Métabolisme and Cancer, Institut Cochin; 4 Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104; and 5 Centre de Référence des Maladies Rares de la Surrénale, Service d'Endocrinologie, Hôpital Cochin, Université Paris 5, Paris, France

Requests for reprints: Constantine A. Stratakis, Section on Endocrinology and Genetics, Program in Developmental Endocrinology and Genetics, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892. Phone: 301-496-4686; Fax: 301-402-0574; E-mail: stratakc{at}mail.nih.gov.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1{alpha}{Delta}184-236 (R1{alpha}{Delta}6)]. We compared in vitro R1{alpha}{Delta}6 with wild-type (wt) R1{alpha}. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1{alpha} and mutant (mt) R1{alpha}; we observed by confocal microscopy R1{alpha} tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C{alpha}). Introduction of the R1{alpha}{Delta}6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1{alpha} protein in wt-R1{alpha}– and R1{alpha}{Delta}6-transfected cells but the former also exhibited discrete aggregates of R1{alpha} that bound C{alpha}; these were absent in R1{alpha}{Delta}6-transfected cells and did not bind C{alpha} at baseline or in response to cyclic AMP. Other changes induced by R1{alpha}{Delta}6 included decreased nuclear C{alpha}. We conclude that R1{alpha}{Delta}6 leads to increased PKA activity through the mt-R1{alpha} decreased binding to C{alpha} and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis. [Cancer Res 2008;68(9):3133–41]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inactivating mutations of the PRKAR1A gene coding for the regulatory subunit type 1A (RI{alpha}) of cyclic AMP (cAMP)–dependent protein kinase A (PKA) have been found in sporadic tumors and in the germ line of the majority of patients with the "complex of myxomas, spotty skin pigmentation, and endocrine overactivity" or "Carney complex" (1, 2).

PRKAR1A germ-line or somatic mutations that lead to tumors are associated with increased PKA activity (1–4). RI{alpha} is the main PKA subunit mediating PKA type I (PKA-I) activity in endocrine and other tissues (3); most PRKAR1A mutations that have been identified led to nonsense mRNA, which was not made into protein through the process known as nonsense mRNA–mediated decay (2). It has been assumed that reduction of RI{alpha} levels, a consequent decrease in PKA-I, and an increase in type II PKA (PKA-II) are responsible for increased cAMP-responsive kinase activity in tissues and primary and transformed cell lines bearing PRKAR1A mutations (5–7). An overall increase in response to cAMP and higher PKA-II activity were also seen in mouse cells with ≤50% of the wild-type (wt) RI{alpha} protein level (8–10).

The first PRKAR1A mutation that led to an expressed RI{alpha} variant that was associated with inherited tumors was described in 2002 (11). The expression of the mutant (mt) protein lacking exon 6 [R1{alpha} {Delta}184-236 (R1{alpha}{Delta}6)] was associated with increased kinase activity because it led to increased phosphorylation of cAMP-responsive element binding protein (CREB) in transfected cells. Recently, we reported more PRKAR1A mutations leading to expressed mt-RI{alpha} variants; they, too, were associated with increased PKA activity in vitro (12, 13).

In the absence of decreased R1{alpha} protein levels, how do expressed PRKAR1A mutations lead to increased PKA activity? PKA, when not stimulated by cAMP, exists as a tetrameric holoenzyme that consists of a homodimer of regulatory subunits that bind two inactive catalytic subunits, one catalytic molecule to each regulatory subunit (14). The accepted model of PKA activation suggests that cooperative binding of two cAMP molecules to each regulatory subunit results in dissociation and the consequent release of the two catalytic subunits, which, in turn, are free to phosphorylate serine-threonine residues of target proteins (14, 15). There are four genes encoding the different regulatory subunits (RI{alpha}, RIβ, RII{alpha}, and RIIβ) and three encoding the catalytic subunits (C{alpha}, Cβ, and C{gamma}); of these, only RI{alpha}, RII{alpha}, and C{alpha} are widely expressed whereas the remaining have mostly tissue-specific expression (14). PKA-I activity is mediated mainly through the expression of RI{alpha}, whereas PKA-II depends on both RII{alpha} and RIIβ expression in endocrine and most other, nonneural, tissues; the balance between PKA-I and PKA-II has been proposed to be critical for the control of cellular growth, proliferation, and differentiation (14, 16). The best known function of the regulatory subunits is inhibition of the catalytic subunits, but an increasing body of evidence supports additional functions, including some that may be PKA-independent (15). We recently showed direct binding of mammalian target of rapamycin by RI{alpha} in vitro (17) and interactions of RI{alpha} with an outer mitochondrial membrane protein (18). The formation of heterodimers between PKA-I and PKA-II subunits (RI{alpha} and RIβ, and likely between RII{alpha} and RIIβ) has also been reported (19) and nuclear localization of regulatory subunits may point to additional, possibly PKA-independent, roles (16, 17, 20).

Thus, for the expressed variants of RI{alpha}, the possibilities for mechanisms associated with tumorigenesis can vary considerably: Dysregulated kinase activity may be caused by altered binding of the catalytic subunit, an increase in PKA-II, heterodimer formation, or altered sensitivity to cAMP (11–13); in addition, both direct and indirect interactions of RI{alpha} and/or PKA with other molecules and signaling pathways may be affected. Expressed PRKAR1A mutations seem to be associated with a more aggressive clinical phenotype (11–13), an observation that makes understanding their effect on PKA function of paramount importance to the development of therapies directed toward cAMP/PKA signaling and related tumorigenicity.

We report here our investigation of the first reported, naturally occurring and pathogenic PRKAR1A mutation that led to an expressed variant, R1{alpha} {Delta}184-236; we refer to it from here on as R1{alpha}{Delta}6 because it leads to deletion of the sequence coding for exon 6 of the PRKAR1A cDNA (11). We confirmed the association of this protein with increased kinase activity and showed that this was due to decreased binding to C{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. HeLa, H295R, HEK293, IMRO-90, and COS cells were obtained from the American Type Culture Collection. A primary fibroblastoid cell line (CAR20.15) was established, following standard methods, from a skin biopsy from a patient with Carney complex and a germ-line PRKAR1A-inactivating mutation (c.491_492delTG/p.Val164fsX4) that we have published elsewhere (1). Only HeLa and HEK293 cells were used for immunoblotting, immunoprecipitation, and confocal microscopy experiments.

mRNA quantification. Total RNA from cells was extracted with TRIzol reagent (Invitrogen) and was purified with the RNeasy Mini Kit (Qiagen). The quantitative real-time reaction was carried out and analyzed with ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The primers and probes for PRKAR1A, PRKAR1B, PRKAR2A, PRKAR2B, and PRKACA (BioServe Biotechnologies) have been published elsewhere (7). All results were normalized against the expression of a housekeeping gene (GAPDH). All points for the standard curves and samples were done in quadruplicates.

Antibodies and expression constructs. All antibodies for this study have previously been described (17); commercially available antibodies for the PKA subunits RI{alpha}, RII{alpha}, RIβ, RIIβ, and C{alpha} and for CREB and extracellular signal–regulated kinase (ERK)-1/2 were also those that we have previously used (7, 11). HA-RI{alpha} was prepared by subcloning a HindIII/XhoI fragment of the hemagglutinin (HA)-tagged human PRKAR1A cDNA from pREP4-HA-RIA (11), as we described elsewhere (17). The green fluorescent protein (GFP) and C{alpha}-Cerulean constructs were made as we have reported elsewhere (17); the HA-tagged PRKAR1A was also previously reported (11).

PKA and cAMP responses. PKA activity was measured as previously described (1, 3, 4, 7), using [{gamma}-32P]deoxy-ATP, in cell extracts that had been snap-frozen in liquid nitrogen. All determinations of PKA activity were done twice for each sample, which were corrected for protein concentration (per milligram of total protein), and then an average value was calculated for each experiment.

For cAMP responsiveness, transfected cell lines were treated with forskolin (10–5 mol/L), 8-bromo-cAMP (8-Br-cAMP; 2 mmol/L), or vehicle and observed continuously during 20 min after stimulation. Transfection of the empty GFP vector was used as a control for the specificity of the signal.

Immunoblotting, immunofluorescence, immunoprecipitation, and nuclear extracts. For immunoblotting (Western), equivalent amounts of protein were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. Density for each band was analyzed with a densitometer. Protein loading was normalized by probing the same membrane with anti–β-actin.

For immunofluorescence, cells were grown on coverslips and fixed in 4% formalin (15 min), followed by blocking in 0.1% saponin, 1% bovine serum albumin-PBS (10 min) and sequential incubations with the primary and secondary antibodies as in Western blotting. Cells were washed and mounted on slides with Fluoromount-G (Southern Biotechnology Associates) in preparation for microscopy as we described elsewhere (17).

For GFP-RI{alpha} and C{alpha} coimmunoprecipitations, captured immunoprecipitates were washed thrice with lysis buffer and subjected to Western blot analysis for RI{alpha} and C{alpha}, as we have reported elsewhere (17). Nuclear extracts and cytosolic preparations were prepared using standard methods (17).

Confocal microscopy and photobleaching. Cells were seeded overnight in Lab-Tek chambers (Nalge Nunc) and cotransfected with the plasmids of interest, using Lipofectamine 2000 (Invitrogen). Confocal microscopic images of cells 15 to 24 h posttransfection were captured on a Zeiss 510 or Zeiss ConfoCor-2 inverted microscope using the 413-nm line of a Kr laser with a 430- to 470-nm emission filter for Cerulean and the 488-nm line of an Ar laser with a 505- to 530-nm emission filter for enhanced GFP (17). Images were captured with a Plan-Apochromat 63x oil immersion objective (numerical aperture, 1.4). Cells expressing both proteins were selected for z-sectioning. Z stacks were taken using a pinhole of 1 airy unit for both channels. Images were analyzed with ImageJ and Zeiss Image Examiner software and prepared by Adobe Photoshop 7.0. Fluorescence loss in photobleaching experiments were done by repeatedly photobleaching a small region of interest and monitoring fluorescence depletion in distant regions over time, as previously described (17, 21).

R1{alpha} protein modeling. Modeling of RI{alpha} sequence was done after the ENSP00000351410 RI{alpha} 1–381 amino acid sequence.6 Crystal structures for R-C binding exist7 for bovine RI{alpha} and mouse C{alpha} (22, 23). Bovine RI{alpha} and mouse C{alpha} are highly homologous to their human counterparts: Bovine R1a (not including exon 6) has only three changes from the human molecule, and the mouse C{alpha} is 98% homologous with human C{alpha} with only two nonconservative changes.

Amino acids 184 to 236 were highlighted on the existing structure protein database (PDB) #1RL3 (24). Molecular graphics images were produced using the UCSF Chimera software from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, CA (25), as suggested elsewhere (22, 23).

Statistics. PKA activity, mRNA expression, and optical densitometry data were obtained in at least duplicate measurements and an average was calculated for each value. Comparisons were made by ANOVA using the PDIFF procedure of the Statistical Analysis System (SAS; SAS Institute) to compare differences between treatment means. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell morphology, PKA activity, and PKA subunit mRNA and protein levels. After introduction of mt-R1{alpha}{Delta}6, IMR-90 and the primary cell line CAR20.15 (Fig. 1A and B ) both assumed a more compact, randomly oriented phenotype with an overall decrease in cytoplasm and a prominence of the nucleus (Fig. 1C and D). These changes were especially obvious in IMR-90 cells (Fig. 1A and C). Introduction of mt-RI{alpha} in H295R, HeLa, and HEK293 cells also caused changes in morphology (see below) but did not affect their viability. For the rest of the experiments, only HeLa and HEK293 cells were used.


Figure 1
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  		Figure 1. IMRO (A and C) and CAR20.15 (B and D) fibroblasts before (A and B) and after the introduction of the R1{alpha}{Delta}6 construct. Transfected cells (C and D) showed long extrusions, a more prominent nucleus, and decreased cytoplasm. Gradually the transfected IMRO and CAR20.15 cell lines became apoptotic and did not sustain large numbers of transfected cells. Although similar morphologic changes were also seen after the introduction of R1{alpha}{Delta}6 in HeLa and HEK293 cells (see Fig. 4), these cell lines were successfully propagated and used for the experiments described in this report.

 

Figure 4
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  		Figure 4. Morphology of HeLa and HEK293 cells after introduction of the wt-RI{alpha} and RI{alpha}{Delta}6-GFP constructs: Puncta are seen in both cell types only after transfection with the wt-R1{alpha} construct, whereas both wt-R1{alpha} and R1{alpha}{Delta}6 have diffuse cytoplasmic localization and a small nuclear pool; introduction of the mt-RI{alpha} caused morphologic changes (projections, irregular cytoplasmic shape) similar to what were seen in IMRO and CAR20.15 cells (Fig. 1A–D) and reported elsewhere after the introduction of a catalytic subunit GFP construct (35).

 
In both cell lines, adequate expression of the RI{alpha} constructs was obtained as shown by both mRNA and protein studies (Fig. 2A and B ). PKA assays showed that what was suggested by transfections in COS cells (11) was true in both these and human cell lines: Groussin et al. had shown that introduction of the R1{alpha}{Delta}6 construct in COS cells increased forskolin-induced cAMP-responsive element activity significantly more than did the introduction of the wt-R1{alpha}. Figure 2C shows that introduction of the R1{alpha}{Delta}6 construct in HEK293 cells had the same effect as the wt-R1{alpha} on total kinase activity at baseline, but led to significant increases in cAMP-stimulated kinase acticity. The data in COS, H295R, and HeLa cells were similar but we also found significant differences between PKA assays done 24 or 48 hours posttransfection. Figure 2D shows that the ratio of free PKA versus total PKA was significantly greater in HEK293 cells (and had the tendency to be significantly higher in HeLa cells) 24 hours after transfection with the R1{alpha}{Delta}6 construct versus wt-R1{alpha}. In addition to CREB phosphorylation, we also saw greater phosphorylation of ERK1 and ERK2 in COS, H295R, and HeLa cells (data not shown), consistent with previous findings that had shown increased pERK1/2 and pCREB as a marker of abnormal PKA activity in the lymphocytes and adrenal cells and tumors of patients with germ-line PRKAR1A-inactivating mutations (3, 6, 11).


Figure 2
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  		Figure 2. A and B, successful introduction of the GFP constructs containing wt-R1{alpha} and R1{alpha}{Delta}6 in HeLa and HEK293 cells; *, P < 0.05; NS, nonsignificant. Y axis represents arbitrary optical densitometry units. C, in both cell types, PKA activity in response to cAMP and after addition of protein kinase inhibitor was greater in R1{alpha}{Delta}6-transfected cells (Y axis represents counts per minute per milligram of protein; *, P < 0.05); D, free PKA activity was increased in HEK293 and had a tendency to be higher in HeLa cells (*, P < 0.05; Y axis represents a unitless ratio).

 
The introduction of the wt-R1{alpha} and R1{alpha}{Delta}6 constructs in both HeLa and HEK293 cells had significant effects on the mRNA of three other PKA subunits (PRKACA, PRKA2B, and PRKAR1B) but no effects on PRKAR2A mRNA (Fig. 3A ). The wt and mt constructs differed only in their effects on PRKAR1B (P < 0.001; which, however, was barely expressed) and tended to be different in their effects on PRKACA (P = 0.08). There were no significant corresponding effects on the protein content of whole-cell extracts for the same subunits both at 24 and 48 hours after transfection (Fig. 3B), with the exception of endogenous R1{alpha}: The cells transfected with wt-RI{alpha} had modestly higher endogenous RI{alpha} levels at 48 hours after transfection (Fig. 3B, top left; P = 0.04); the ratio between RI and RII subunits, however, was not different at 24 or 48 hours.


Figure 3
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  		Figure 3. A, mRNA levels of PKA subunits in HEK293 cells after introduction of the wt-R1{alpha} and R1{alpha}{Delta}6-GFP constructs. *, P < 0.05. B, protein levels of PKA subunits in HEK293 cells 24 and 48 h after introduction of the wt-RI{alpha} and R1{alpha}{Delta}6-GFP constructs. *, P < 0.05; Y axis represents arbitrary optical densitometry units.

 
Cellular distribution of R1{alpha} and R1{alpha}{Delta}6; binding to C{alpha} and other PKA subunits. The increase of kinase activity caused by R1{alpha}{Delta}6 without a dramatic effect on the whole-cell content of the other PKA subunits suggested an effect on the holoenzyme and/or its catalytic activity that did not involve a major imbalance between PKA-I and PKA-II isozymes. We then determined the cellular compartmentalization of wt-R1{alpha} and mt-R1{alpha} at baseline and after activation of the cAMP signaling pathway; a construct of the human C{alpha} with Cerulean was also used so that we could observe any interactions between wt-RI{alpha}, mt-RI{alpha}, and C{alpha} in doubly transfected cells.

First, diffuse cytoplasmic localization was observed with both wt-RI{alpha} and mt-RI{alpha} constructs, but obvious puncta, dense aggregates, were observed in the cytoplasm only with the wt-RI{alpha} protein (Fig. 4 ). Confocal microscopy showed, in addition, some nuclear localization of R1{alpha} for both constructs; however, perinuclear puncta that were obvious at baseline disappeared after forskolin treatment, and nuclear translocation occurred only with the wt-R1{alpha} construct; there were no distinct perinuclear puncta in cells transfected with mt-RI{alpha}, and the mobility of the R1{alpha}{Delta}6 construct was not enhanced by forskolin or 8-Br-cAMP (Fig. 5A, top ). Cell morphology was different in cells transfected with the wt-RI{alpha} construct (Fig. 4), as we noted above with other cells (Fig. 1). These data have been replicated with a different GFP construct and in a number of other cell types (data not shown).


Figure 5
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  		Figure 5. A, different distribution and mobility of the wt-R1{alpha} and R1{alpha}{Delta}6-GFP constructs in HeLa cells in response to cAMP (see text for details). Immunohistochemical fluorescence shows that wt-R1{alpha}-GFP colocalizes (arrows) with C{alpha} (1), whereas there is less R1{alpha}{Delta}6-GFP colocalizing with C{alpha} (2); both wt-R1{alpha} and R1{alpha}{Delta}6-GFP constructs colocalized with RII{alpha}—only the R1{alpha}{Delta}6-GFP–transfected cells immunostained with RII{alpha} (arrows) are shown (3); finally, there was no obvious colocalization with RIIβ of neither construct—only the R1{alpha}{Delta}6-GFP–transfected cells immunostained with RIIβ are shown (4). B, immunoprecipitation experiment where three different preparations of HEK293 cells (1, wt-R1{alpha} GFP-transfected cells; 2, R1{alpha}{Delta}6-GFP–transfected cells) were immunoprecipated with a C{alpha}-specific antibody: In all preparations, C{alpha} recognizes what was immunoprecipitated (positive control); the RI{alpha}-specific antibody detects the endogenous R1{alpha} in all lanes, but only the wt-R1{alpha} GFP is present in R1{alpha}-C{alpha} complexes; RII{alpha} is shown in all lanes of C{alpha} immunoprecipitates as expected. C and D, differential distribution and colocalization (arrows) with a Ca-Cerulean construct of the wt-R1{alpha} (C) and R1{alpha}{Delta}6-GFP (D) constructs in HeLa cells: The puncta of R1{alpha} colocalize with C{alpha}, whereas there is no colocalization with the diffusely present R1{alpha}{Delta}6 molecules.

 
We next examined the mt-R1{alpha} interactions with C{alpha} and other subunits: Immunofluorescence studies suggested that R1{alpha}{Delta}6 was likely to not bind C{alpha}, at least not as effectively as wt-R1{alpha} (Fig. 5A, bottom). Immunoprecipitation experiments confirmed this observation: When cells transfected with wt-R1{alpha} or mt-R1{alpha} were immunoprecipitated with C{alpha}, only the wt-RI{alpha}–transfected cells showed any immunostaining with the RI{alpha}-specific antibody (Fig. 5B, top). When immunoprecipitated with RI{alpha}, cells transfected with mt-RI{alpha} showed significantly less immunostaining for C{alpha} than cells transfected with wt-RI{alpha} (Fig. 5B, bottom). Interestingly, immunofluorescence studies suggested that RII{alpha}, but not RIIβ (Fig. 5A, bottom) or RIβ (data not shown), may form heterodimers with mt-RI{alpha}. Indeed, in immunoprecipitation experiments, wt-R1{alpha} and mt-RI{alpha} showed that they both formed heterodimers with RII{alpha} when immunoprecipitated with C{alpha} (Fig. 5A, bottom) or with RI{alpha} (data not shown), but there were no quantitative differences in repeated experiments between the two constructs.

We then used confocal microscopy to study the interactions of wt-R1{alpha} and mt-RI{alpha} with C{alpha}-Cerulean in cotransfections: Again there was little, if any, interaction between mt-RI{alpha} and C{alpha} whereas several of the puncta containing wt-RI{alpha} showed that they contained C{alpha} (Fig. 5C and D). We also conducted fluorescence loss in photobleaching experiments, in which we repeatedly photobleached (i.e., noninvasively abolished) the fluorescence in a region in the cytoplasm of cells containing wt-RI{alpha} or mt-RI{alpha} cotransfected with C{alpha}-Cerulean both at baseline and after exposure to 8-Br-cAMP. There were no significant differences between wt-RI{alpha} and mt-RI{alpha} in these experiments (data not shown).

Projection of the mt-RI{alpha} deletion on the existing wt-RI{alpha}-C{alpha} interaction models (22, 23) showed that deletion of sequences corresponding to exon 6 would affect not only interactions with cAMP (which was predictable from elimination of the cAMP-binding domain A of RI{alpha}) but also one of the major grooves forming around C{alpha} in the RI{alpha}-C{alpha} dimer (Fig. 6A ).


Figure 6
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  		Figure 6. The deletion in R1{alpha}{Delta}6 highlighted within an available model of R1{alpha} when bound to cAMP (A) and in a complex with C{alpha}, structures were adapted from refs. 22–24. R1{alpha}{Delta}6 lacks the most conserved region of R1{alpha} (26), one that is essential for both cAMP binding and interaction with C{alpha}. Differences in cytoplasmic and nuclear RI{alpha} (B) and C{alpha} (C) after introduction of the wt-R1{alpha} and R1{alpha}{Delta}6-GFP constructs in HEK293 cells. D, representative blots from one experiment for all PKA subunits: There is R1{alpha} present in the nuclear extracts of all lanes, whereas there is no R1β in the nuclear extracts and little in cytosolic extracts (lanes 1 and 4, from wt-R1{alpha}-transfected cells; lanes 2 and 5, from R1{alpha}{Delta}6-transfected cells; lanes 3 and 6, from mock transfections); *, P < 0.05; NS, nonsignificant. Y axis represents random optical densitometry units.

 
Nuclear and cytoplasmic R1{alpha} and C{alpha}. Transfections with the wt-R1{alpha} and mt-R1{alpha} constructs showed a small but detectable pool of R1{alpha} in the nucleus in both HEK293 and HeLa cells. cAMP induced wt-R1{alpha} mobility and entry into nucleus both before and after introduction of the C{alpha} construct, whereas it had no effect on mt-R1{alpha}; introduction of C{alpha} did not change the lack of response to cAMP (Supplementary data). Western blotting of cytosolic and nuclear extracts of HEK293 cells showed that all PKA subunits could be found in the nucleus; C{alpha} was mostly nuclear and regulatory subunits were mostly cytosolic, as expected. There were differences in the cytosolic and nuclear localization of endogenous RI{alpha} and C{alpha} when preparations from wt-R1{alpha}– and mt-RI{alpha}–transfected cells were compared: The nuclear content of C{alpha} in mt-RI{alpha}–transfected cells was significantly lower (P < 0.001); mt-R1{alpha} also did not affect the cytosolic endogenous RI{alpha}, which was induced by the wt-RI{alpha} construct, whereas both wt-R1{alpha} and mt-RI{alpha} increased cytosolic endogenous C{alpha} (Fig. 6B–D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation is the first attempt to study the PKA effects of a naturally occurring PRKAR1A mutation that has been associated with an aggressive clinical phenotype (11). Although increased cAMP-responsive element activity had been shown in COS cells transfected with R1{alpha}{Delta}6, it was not known how this was mediated. Our studies are significant for both what they showed and what they did not show: We first showed that the mechanism responsible for the observation of increased PKA activity due to the R1{alpha}{Delta}6 mutation involves a C{alpha} that is not bound by mt-R1{alpha} and, thus, is most likely inappropriately regulated. Absence of amino acids 184 to 236 of the RI{alpha} protein abolished part of cAMP-binding domain A (which extends between amino acids 143 and 260) but retained cAMP-binding domain B (amino acids 261–374) and the entire amino (NH2) part (22–24) including the dimerization/docking domain. Recognition of RI{alpha} by C{alpha} is mediated through specific amino acid interactions that include the critical Tyr205 (26, 27) that is absent in R1{alpha}{Delta}6 (Fig. 6A); Glu200, Leu201, and Ile204 are also absent in R1{alpha}{Delta}6 and play an important role in RI{alpha}-C{alpha} binding (27). Therefore, deficient binding of C{alpha} by R1{alpha}{Delta}6 could have been predicted from existing models, with the caveat that the latter are derived from interactions of the murine C{alpha} and the bovine R1{alpha} (which are highly homologous molecules).

Second, we also showed that the introduction of R1{alpha}{Delta}6 in cells did not alter their content of other PKA subunits, and more specifically, it did not lead to an increase in PKA-II subunits (Fig. 6A). The balance between PKA-I and PKA-II isozymes has been considered critical for PKA control of growth, proliferation, and differentiation (14, 16). Although both human and mouse studies confirmed that R1{alpha} reduction was associated with increases in expression of RII{alpha} or RIIβ (8–10, 28), the question remained whether these were simply compensatory changes (29). The R1{alpha}{Delta}6 studies showed that, in at least the case of one mutation associated with aggressive human tumors, an in vitro increase of PKA-II could not be shown.

Several other findings are worthy of discussion, however, and require further investigation: Wt-R1{alpha} formed cytosolic aggregates, puncta, that have been seen by us (17, 30) and by other investigators and in various (although not all) types of cells, tagged with GFP (17, 30, 31) and untagged (32, 33). Zaccolo et al. (34) showed similar puncta with the RII{alpha}-GFP, but not C{alpha}-GFP, constructs. GFP tagged to the NH2 or the COOH terminus of R1{alpha} resulted in similar formations (17, 30), and R1{alpha}{Delta}6-GFP completely lacked them (Fig. 6A). Fluorescence loss in photobleaching experiments showed the dynamic relationship between the puncta and the R1{alpha} and C{alpha} cytosolic pools (17). We found that these formations in HeLa, MNTI, and HEK293 cells associated with late endosomes and autophagosomes in cultured cells (17, 30).

Thus, R1{alpha}{Delta}6 not only did not bind C{alpha} but also lacked the ability to associate with these cellular formations. This was not due to a defect in dimerization: R1{alpha}{Delta}6 bound both wt-R1{alpha} and with itself (Fig. 6B–D); this could also have been predicted by the retention of the dimerization/docking domain (26) by the mt protein. Binding to the C{alpha} was not necessary for these aggregates to form: We have shown elsewhere (17), and it was evident from our studies here, too (Fig. 5), that these puncta formed by wt-R1{alpha} did not always associate with endogenous (or transfected) C{alpha}.

Another significant observation was that cells transfected with R1{alpha}{Delta}6 changed morphology consistent with earlier published data (34, 35) and in other settings (36, 37), whereas cells transfected with wt-R1{alpha} retained their original shape and contour (Figs. 1 and 5). These morphologic changes have been linked to excess C{alpha} (34, 35) or increases in PKA activity (36–38) and are consistent with the observation that R1{alpha}{Delta}6 did not efficiently bind C{alpha}.

Nuclear localization of mammalian R1{alpha} has been seen before but has not been adequately studied, despite the knowledge that the yeast PKA regulatory subunit Bcy1p is primarily nuclear (39). Our studies showed that both wt-R1{alpha} and mt-R1{alpha}{Delta}6, as well as endogenous R1{alpha}, are present in nuclei of HEK293 and HeLa cells, consistent with observations in mouse oocytes (33), cardiac myocytes from various species and mouse embryonic cells (40, 41), cancer cell lines (20), and in other settings (31, 35), and despite some evidence to the contrary (42). The role of nuclear PKA-I remains unclear, but regulation of cell division and coordination of this event with the cell cycle are likely (40). Interestingly, because R1{alpha}{Delta}6 did not bind C{alpha} and our studies on the C{alpha} content of total cell lysates did not show C{alpha} changes (Fig. 6A), we looked at cytosolic and nuclear C{alpha} and found it to be decreased in R1{alpha}{Delta}6-transfected cells (Fig. 6B–D). Our hypothesis is that increased PKA activity in R1{alpha}{Delta}6-expressing cells is due to the unregulated, mostly PKA-II–bound, cytosolic C{alpha}; C{alpha} that is not bound by R1{alpha} would be unstable and unavailable to enter the nucleus, an event that is brought about by simple diffusion (43) and would not be prevented by cytosolic RII subunits because, interestingly, C{alpha} diffuses away from PKA-II but not from PKA-I (31). In addition, R1{alpha}{Delta}6-transfected cells had less endogenous cytosolic R1{alpha} levels (Fig. 6B–D). Thus, the net effect of R1{alpha}{Delta}6 introduction was to further decrease any R1{alpha}-C{alpha} binding that would normally take place in these cells in both the cytoplasm and the nucleus. It remains unclear how decreased nuclear C{alpha} presence affects other functions of PKA in the nucleus of R1{alpha}{Delta}6-transfected cells.

cAMP, either directly (8-Br-cAMP) or generated by forskolin, did not affect R1{alpha}{Delta}6 binding to C{alpha}. Recent data indicate that cAMP-bound PKA-I holoenzyme is stable yet catalytically active and that maximum cAMP levels do not lead to immediate or complete dissociation of the tetramer (31, 35, 44), consistent with our data. R1{alpha}{Delta}6 lacks cAMP-binding domain A (Fig. 6A) but one could hypothesize that it would respond to cAMP: cAMP binding to R1{alpha} is an orchestrated event that is assisted by the tertiary structure of the PKA holomer; one cAMP molecule binds first to cAMP-binding domain B of the inactive PKA tetramer–bound R1{alpha}. However, complete separation of the A and B domains (so that cAMP-binding domain B is "available" for binding) is not possible without formation of the R1{alpha}-C{alpha} complex (27, 31). Because R1{alpha}{Delta}6 is not capable of efficiently forming a dimer with C{alpha}, chances are that it would not efficiently bind cAMP, which explains why it did not respond to forskolin and 8-Br-cAMP in our experiments; apparently, increasing the amount of available C{alpha} also did not change that, as suggested by our cotransfection experiments.

We conclude that R1{alpha}{Delta}6, a variant of R1a that lacks cAMP-binding domain A, an "ancient signaling domain conserved in every genome" (26), does not bind C{alpha} and has other unique properties when introduced in human cell lines. R1{alpha}{Delta}6 also causes Carney complex and aggressive human tumors, and from that, one can speculate that a "free" C{alpha}, and not a switch to PKA-II, is the cause of tumorigenicity in states of PRKAR1A inactivation. Further studies are needed to confirm this suggestion that changes at least some of the dogmas related to the role of PKA in cancer.


   Acknowledgments
 
Grant support: U.S. National Institutes of Health, National Institute of Child Health and Human Development intramural project Z01-HD-000642-04 (C.A. Stratakis), research funds to J. Bertherat (Hopital Cochin, Paris, France), and Groupement d'Intérêt Scientifique-Institut National de la Santé et de la Recherche Médicale Institut des Maladies Rares and the Plan Hospitalier de Recherche Clinique to the Comete Network grant AOM 02068 (J. Bertherat and C.A. Stratakis).


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

E. Meoli, I. Bossis, and L. Cazabat contributed equally to this work.

Current address for I. Bossis: School of Veterinary Medicine, University of Maryland, College Park, MD 20742.

6 See http://www.ensembl.org. Back

7 See http://www.rcsb.org/pdb/cgi/pdbId=2QCS. Back

Received 1/ 7/08. Revised 1/30/08. Accepted 1/31/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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