- Email: [email protected]
A-kinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus
Brief Communication
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A-kinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus Antonio Feliciello*†, Ying Li*, Enrico V. Avvedimento‡, Max E. Gottesman† and Charles S. Rubin* A-kinase anchor protein 75 (AKAP75) binds regulatory subunits (RIIa and RIIb) of type II protein kinase A (PKAII) isoforms and targets the resulting complexes to sites in the cytoskeleton that abut the plasma membrane [1–7]. Co-localization of AKAP75–PKAII with adenylate cyclase and PKA substrate/effector proteins in cytoskeleton and plasma membrane effects a physical and functional integration of up-stream and downstream signaling proteins, thereby ensuring efficient propagation of signals carried by locally generated cyclic AMP (cAMP) [4–9]. An important, but previously untested, prediction of the AKAP model is that efficient, cyclic nucleotide-dependent liberation of diffusible PKA catalytic subunits from cytoskeletonbound AKAP75–PKAII complexes will also enhance signaling to distal organelles, such as the nucleus. We tested this idea by using HEK-A75 cells, in which PKAII isoforms are immobilized in cortical cytoskeleton by AKAP75. Abilities of HEK-A75 and control cells (with cytoplasmically dispersed PKAII isoforms) to respond to increases in cAMP content were compared. Cells with anchored PKAII exhibited a threefold higher level of nuclear catalytic subunit content and 4–10-fold greater increments in phosphorylation of a regulatory serine residue in cAMP response element binding protein (CREB) and in phosphoCREB-stimulated transcription of the c-fos gene. Each effect occurred more rapidly in cells containing targeted AKAP75–PKAII complexes. Thus, anchoring of PKAII in actin cortical cytoskeleton increases the rate, magnitude and sensitivity of cAMP signaling to the nucleus. Addresses: *Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York, New York 10461, USA. †Institute of Cancer Research, Columbia University, New York, New York 10032, USA. ‡Dipartimento di Biologia e Patologia Molecolare e Cellular, Centro di Endocrinologia ed Oncologia Sperimentale CNR, Facolta’ di Medicina, Universita’ Federico II, 80131 Napoli and Dipartimento Medicina Sperimentale e Clinica, Facolta’ di Medicina, 88100 Catanzaro, Italy. Correspondence: Charles S. Rubin E-mail: [email protected] Received: 11 August 1997 Revised: 29 September 1997 Accepted: 14 October 1997
Results and discussion The coupling of adenylate cyclase activation at the cell surface to mRNA induction involves dissociation of PKA holoenzyme, translocation of the catalytic (C) subunits to the nucleus, phosphorylation of the transcription factor CREB at Ser 133 and phosphoCREB/co-activator mediated activation of gene transcription (reviewed in [10]). To study effects of AKAP75 on nuclear cAMP signaling, the AKAP-deficient human HEK293 cell line [11] was used as a recipient for transgenes encoding anchor proteins. Stably transfected cells that express AKAP75 or AKAP45 were created [6,7,11]. AKAP45, which is derived from AKAP75 by deletion of residues 1–180, binds PKAII isoforms, but lacks domains that mediate anchoring [6,11,12]. AKAP75 targeted and anchored >90% of RIIα and RIIβ in the cortical actin cytoskeleton that lies adjacent to the plasma membrane [6,7]. In contrast, RII isoforms were dispersed in the cytoplasm of control cells and in cells expressing AKAP45 [6,7,11]. Transcription of c-fos is activated through an upstream cAMP-responsive element (CRE) [10]. Thus, effects of anchored PKA on transcription were assessed by monitoring synthesis of c-fos mRNA. Cells were incubated in medium containing 0.1% fetal calf serum for 12 hours and were then treated with forskolin (FSK) to stimulate adenylate cyclase and 3-isobutyl-1-methylxanthine (IBMX) to inhibit cAMP phosphodiesterases. In control cells, a low level of c-fos mRNA was detected 30 minutes after activation of adenylate cyclase. The c-fos mRNA content increased over the next 60 minutes and reached a peak value 90 minutes after addition of FSK (Figure 1a,b). Induction of c-fos mRNA by cAMP in HEK-A75 cells was more rapid and robust. A substantial amount of c-fos mRNA was detected 30 minutes after exposure to FSK (Figure 1a). Content of c-fos mRNA peaked at 45 minutes and then declined slowly during the succeeding 45 minutes (Figure 1a,b). Levels of c-fos mRNA were 4–10-fold higher in HEK-A75 cells than in control cells during the initial phase (0–45 minutes) of adenylate cyclase activation (Figure 1b). By 90 minutes, c-fos transcripts were equally abundant in these cell lines. The kinetics of c-fos mRNA accumulation in HEK-A45 cells was indistinguishable from that observed in control cells. Subsequent experiments were focused on the physiologically relevant, early phase of cAMP-mediated signal transduction.
Current Biology 1997, 7:1011–1014 http://biomednet.com/elecref/0960982200701011 © Current Biology Ltd ISSN 0960-9822
HEK-A75 cells generated 4–10-fold more c-fos mRNA than did control cells over a range of activator concentrations (Figure 1c). To exclude the possibility that AKAP75
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Figure 1 HEK293 HEK-A75 HEK-A45 Time (min) 0 15 30 45 0 15 30 45 0 15 30 45
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Northern blot analyses of c-fos mRNA content in HEK293, HEK-A75 and HEK-A45 cells. (a) Accumulation of c-fos mRNA in cells incubated with 40 µM FSK and 0.1 mM IBMX. Representative autoradiograms are shown. (b) The c-fos mRNA content was quantified by scanning densitometry [6] at the indicated time points. Cells were incubated with 40 µM FSK. Levels of c-fos mRNA are presented as arbitrary densitometric units (ADU) and represent the mean of three independent experiments that yielded similar results. (c) Accumulation of c-fos mRNA in control and HEK-A75 cells exposed to the indicated FSK concentrations for 30 min. Representative autoradiograms are shown. Mean densitometric values (in ascending order of FSK concentration) for c-fos mRNA content (above the basal level) were
determined from three independent experiments (expressed as ADU). HEK293: 3, 5, 4; HEK-A75 13, 65, 55. (d) Cells were treated for 30 min with the cAMP analog CPT-cAMP, at the indicated concentrations. Representative autoradiograms are shown. Mean values for c-fos mRNA (above the basal value) from two independent experiments (expressed as ADU) were HEK293: 0, 4, 6; HEK-A75: 23, 44, 35. Data are presented in ascending order of CPT-cAMP concentration. (e) Cells grown in 10% fetal calf serum were incubated with the indicated amounts of CPT-cAMP for 30 min. Autoradiograms are shown. Mean values for CPT-cAMP stimulated c-fos mRNA accumulation (from three independent experiments, expressed as ADU) were HEK293: 8, 17; HEK-A75: 39, 45; HEK-A45: 4, 5.
increased the sensitivity of adenylate cyclase to FSK, we monitored c-fos mRNA accumulation after incubating cells with 8-chlorophenylthio-cAMP (CPT-cAMP), a stable cAMP analog. CPT-cAMP selectively elicited a large increase in c-fos mRNA content in cells containing anchored PKAII (Figure 1d). A similar induction of c-fos transcripts by CPT-cAMP was observed in HEK-A75 cells incubated with 10% fetal calf serum (Figure 1e). Thus, serum hormones had little effect on the pattern of
cAMP-mediated c-fos mRNA accumulation in the three cell lines. Phosphotransferase activity of the C subunits increased threefold in nuclei of FSK-treated HEK-A75 cells (Figure 2a,b). Catalytic activity of C subunits in the nucleus increased only slightly in similarly treated control and HEK-A45 cells. The physiological significance of enhanced nuclear translocation of C subunits was
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Figure 2
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Stimulation of nuclear translocation of C subunits by FSK. C subunit phosphotransferase activity was assayed in nuclear extracts isolated from cells incubated in the presence or absence of FSK. C activity is expressed as the fold increase over basal activity in response to (a) time of exposure to 40 µM FSK and 0.1 mM IBMX, or (b) various FSK concentrations (cells harvested 20 min after addition of drug). Values represent phosphotransferase activity inhibitable by 50 µM PKI, which selectively binds and inactivates C subunits. Nuclear C-specific activity is expressed as the mean ± s.e. from three independent experiments. C activity in untreated cells was: 0.8 ± 0.12, 0.2 ± 0.03 and 0.8 ± 0.14 pmol per µg protein per min for HEK293, HEK-A75 and HEK-A45 cells, respectively.
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evaluated by assaying CREB phosphorylation at Ser 133 by western immunoblot analysis (Figure 3). The level of phosphorylated CREB increased modestly in control HEK293 cells exposed to FSK. Peak phosphorylation was evident 20 minutes after addition of drug (Figure 3a). The kinetics and extent of phosphorylation of CREB in HEK-A45 cells were similar. In HEK-A75 cells, a substantial increase in CREB phosphorylation was observed 10 minutes after adenylate cyclase activation. The concentration of phosphoCREB rose sharply for the succeeding 30 minutes. At each time point, the amount of phosphoCREB was 5–10- fold higher in HEKA75 cells than in control cells. CREB phosphorylation was also markedly increased in HEK-A75 cells at low FSK concentrations, thereby documenting an enhanced sensitivity to cAMP in cells with anchored PKAII (Figure 3b). Total CREB protein in nuclear extracts did not vary significantly during these experiments (Figure 3a,b). Several stable, independently isolated lines of HEK293 and hamster AV-12 cells [7] that overexpress AKAP75, exhibit properties that are similar to those reported for HEK-A75 cells. HEK-A75, HEK-A45 and control cells contain similar levels of PKAII isoforms [6,7]. Synthesis and accumulation of AKAP75 (in HEK-A75 cells) elicits redistribution of holoenzyme from a large volume (cytoplasm) to a more limited ‘compartment’ occupied by portions of cortical actin cytoskeleton and plasma membrane [6,7]. In accord with the AKAP model [1,2] and laws of mass action, generation of a high concentration of PKAII in the vicinity of adenylate cyclase presumably optimizes binding of locally produced cAMP to PKAII holoenzyme, thereby facilitating dissociation of active C subunits. Initially, PKA-mediated signaling will be focused on co-localized substrates, but subsequent C subunit diffusion is also expected.
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Increases in nuclear translocation of C subunits, CREB phosphorylation and c-fos mRNA synthesis in FSKtreated HEK-A75 cells suggest that anchored PKAII serves as an easily activated pool of diffusible C subunits that mediate cAMP signaling to the nucleus. Enrichment of PKAII in cortical cytoskeleton can substantially increase target cell responses to modest levels of hormones (that is, partial activation of adenylate cyclase), just as increased receptor number classically increases target cell sensitivity to low ligand concentrations. Moreover, immobilized PKAII is exposed to a maximal level of cAMP before the cyclic nucleotide is diluted and degraded (via cAMP phosphodiesterases) in cytoplasm. HEK-A45 cells, which contain unanchored AKAP–PKAII complexes, do not exhibit enhanced signaling to the nucleus. Thus, targeting and immobilization of PKAII is the key determinant of signal amplification; formation of an AKAP–PKAII complex per se is necessary, but not sufficient, for upregulation of cAMP-controlled gene transcription. In vivo fluxes in hormone concentration often occur on a timescale of seconds to several minutes. In this context, the ability of cells containing anchored PKAII to respond rapidly and with increased potency and sensitivity to modest increases in cAMP (that is, low physiological levels of hormones) confers a considerable biological advantage. Other recent observations have linked intracellular targeting of PKAII to gene regulation. Overexpression of AKAP45 in thyroid cells causes displacement of immobilized PKAII from perinuclear sites (Golgi) to cytoplasm and depresses cAMP-controlled thyroglobulin gene expression [13]. Overexpression of AKAP75 and RII in variant PC12 cells that are defective in cAMP signal transduction restored cAMP-stimulated activation of a chimeric reporter gene [14]. In these studies, however,
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Figure 3
HEK293 HEK-A75 HEK-A45 Time (min) 0 10 20 40 0 10 20 40 0 10 20 40 –43 kDa pCREB
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The authors dedicate this paper to G. Salvatore (Second Medical School, Naples, Italy) for his scientific and personal support. We thank C. Schindler for useful comments, R. Prywes for the c-fos cDNA clone and R.S. Watnick for help in preparing the graphics. A.M. Alba provided expert secretarial assistance. This work was supported by grants from the National Institute of Health (GM22792, C.S.R.), Associazione Italiana Ricerca sul Cancro, Progetti Finalizzati CNR ‘Ingegneria Genetica’ Italy (E.V.A.) and the Lucille P. Markey Charitable Trust (M.E.G.). A. F. was supported by a Fellowship of the Associazione Italiana Ricerca sul Cancro.
References HEK293 0.4 4 40
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Current Biology
cAMP/PKA-dependent phosphorylation of CREB at Ser 133. Proteins (30 µg) in nuclear extracts from untreated and FSK-stimulated cells were fractionated by denaturing electrophoresis. A western blot was probed with antiserum directed against an epitope in CREB that includes phosphorylated Ser 133 (upper panels in (a) and (b)) or with non-discriminating anti-CREB antibodies (lower panels). Arrows indicate phosphorylated CREB (pCREB) and total CREB proteins. (a) Cells were incubated with 40 µM FSK and 0.1 mM IBMX and harvested at the indicated times. Representative immunoblots are shown. Relative levels of the 42 kDa phosphoCREB doublet (above basal values) are expressed as the mean of three independent experiments. The results were HEK293: 3, 21, 19; HEK-A75: 42, 88, 150; HEK-A45: 8, 8, 10. Values are given in ascending order of incubation times. (b) Cells were harvested 20 min after exposure to the indicated concentrations of FSK. Typical immunoblots are shown. Relative levels of phosphoCREB (above basal values), expressed as the mean of three independent experiments, were HEK293: 1, 21, 26; HEK-A75: 7, 80, 265. Values are presented in ascending order of FSK concentration.
the exact location and composition of PKAII–anchor protein complexes were not determined; phosphorylation and activation of CREB was not addressed and data documenting positive regulation of an endogenous, cAMP- and PKA-regulated target gene (for example cfos) was lacking. Our present results show that assembly of AKAP75–PKAII complexes in cortical cytoskeleton strongly promotes CREB activation via phosphorylation at Ser 133 and a high level of expression of mRNA encoding a key transcription factor for immediate early genes, c-Fos. Regulation of AKAP75 content by hormones or developmental factors could provide a novel mechanism for regulating the sensitivity of cells to agents that activate adenylate cyclase.
Materials and methods Cell culture, transfections, assay for c-fos mRNA, protein kinase assay, and western analysis were carried out by standard methods [6,11–13,15,16] and are given in more detail in the Supplementary material published with this paper on the internet.
1. Rubin CS: A-kinase anchor proteins and the intracellular targeting of signals carried by cyclic AMP. Biochim Biophys Acta 1994, 1224:467-479. 2. Scott JD, McCartney S: Localization of A-kinase through anchoring proteins. Mol Endocrionol 1994, 8:5-11. 3. Faux MC, Scott JD. Molecular glue: kinase anchoring and scaffold proteins. Cell 1996, 85:9-12. 4. Glantz SB, Amat JA, Rubin CS: cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase IIb. Mol Biol Cell 1992, 3:12151228. 5. Carr DW, Stofko-Hahn RE, Fraser IDC, Cone RD, Scott JD: Localization of cAMP-dependent protein kinase to postsynaptic densities by a kinase anchoring proteins. J Biol Chem 1992, 267:16816-16823. 6. Ndubuka C, Li Y, Rubin CS: Expression of A-kinase anchor protein 75 depletes type II cAMP-dependent protein kinases from the cytoplasm and sequesters the kinases in a particulate pool. J Biol Chem 1993, 268:7621-7624. 7. Li Y, Ndubuka C, Rubin CS: A-kinase anchor protein 75 targets regulatory (RII) subunits of cAMP-dependent protein kinase II to the cortical actin cytoskeleton in non-neuronal cells. J Biol Chem 1996, 271:16862-16869. 8. Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD, Westbrook GL: Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 1994, 368:853-856. 9. Johnson BD, Scheurer T, Catterall WA: Voltage-dependent potentiation of L-type Ca2+ channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1994, 91:11492-11496. 10. Montminy M. Transcriptional regulation by cyclic AMP. Annu Rev Biochem 1997, 66:807-822. 11. Hirsch AH, Glantz SB, Li Y, You Y, Rubin CS: Cloning and expression of an intronless gene for AKAP75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase IIb. J Biol Chem 1992, 267:2131-2134. 12. Glantz SB, Li Y, Rubin CS: Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase IIb to the cytoskeleton. J Biol Chem 1993, 268:12796-12804. 13. Feliciello A, Giuliano P, Porcellini A, Garbi C, Obici S, Mele E, et al.: The v-Ki-Ras oncogene alters cAMP nuclear signaling by regulating the location and expression of cAMP-dependent protein kinase IIb. J Biol Chem 1996, 271:25350-25359. 14. Cassano S, Gallo A, Buccigrossi V, Porcellini A, Cerillo R, Gottesman ME, Avvedimento EV: Membrane localization of cAMP-dependent protein kinase amplifies cAMP signaling to the nucleus in PC12 cells. J Biol Chem 1996, 271:29870-29875. 15. Gius D, Cao XM, Rauscher FJ, Cohen DR, Curran T, Sukhatme VP: Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CArG elements. Mol Cell Biol 1990, 10:4243-4255. 16. Montminy MR, Bilezekjian LM: Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 1987, 328:175-178.
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A-kinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus Antonio Feliciello*†, Ying Li*, Enrico V. Avvedimento‡, Max E. Gottesman† and Charles S. Rubin* A-kinase anchor protein 75 (AKAP75) binds regulatory subunits (RIIa and RIIb) of type II protein kinase A (PKAII) isoforms and targets the resulting complexes to sites in the cytoskeleton that abut the plasma membrane [1–7]. Co-localization of AKAP75–PKAII with adenylate cyclase and PKA substrate/effector proteins in cytoskeleton and plasma membrane effects a physical and functional integration of up-stream and downstream signaling proteins, thereby ensuring efficient propagation of signals carried by locally generated cyclic AMP (cAMP) [4–9]. An important, but previously untested, prediction of the AKAP model is that efficient, cyclic nucleotide-dependent liberation of diffusible PKA catalytic subunits from cytoskeletonbound AKAP75–PKAII complexes will also enhance signaling to distal organelles, such as the nucleus. We tested this idea by using HEK-A75 cells, in which PKAII isoforms are immobilized in cortical cytoskeleton by AKAP75. Abilities of HEK-A75 and control cells (with cytoplasmically dispersed PKAII isoforms) to respond to increases in cAMP content were compared. Cells with anchored PKAII exhibited a threefold higher level of nuclear catalytic subunit content and 4–10-fold greater increments in phosphorylation of a regulatory serine residue in cAMP response element binding protein (CREB) and in phosphoCREB-stimulated transcription of the c-fos gene. Each effect occurred more rapidly in cells containing targeted AKAP75–PKAII complexes. Thus, anchoring of PKAII in actin cortical cytoskeleton increases the rate, magnitude and sensitivity of cAMP signaling to the nucleus. Addresses: *Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York, New York 10461, USA. †Institute of Cancer Research, Columbia University, New York, New York 10032, USA. ‡Dipartimento di Biologia e Patologia Molecolare e Cellular, Centro di Endocrinologia ed Oncologia Sperimentale CNR, Facolta’ di Medicina, Universita’ Federico II, 80131 Napoli and Dipartimento Medicina Sperimentale e Clinica, Facolta’ di Medicina, 88100 Catanzaro, Italy. Correspondence: Charles S. Rubin E-mail: [email protected] Received: 11 August 1997 Revised: 29 September 1997 Accepted: 14 October 1997
Results and discussion The coupling of adenylate cyclase activation at the cell surface to mRNA induction involves dissociation of PKA holoenzyme, translocation of the catalytic (C) subunits to the nucleus, phosphorylation of the transcription factor CREB at Ser 133 and phosphoCREB/co-activator mediated activation of gene transcription (reviewed in [10]). To study effects of AKAP75 on nuclear cAMP signaling, the AKAP-deficient human HEK293 cell line [11] was used as a recipient for transgenes encoding anchor proteins. Stably transfected cells that express AKAP75 or AKAP45 were created [6,7,11]. AKAP45, which is derived from AKAP75 by deletion of residues 1–180, binds PKAII isoforms, but lacks domains that mediate anchoring [6,11,12]. AKAP75 targeted and anchored >90% of RIIα and RIIβ in the cortical actin cytoskeleton that lies adjacent to the plasma membrane [6,7]. In contrast, RII isoforms were dispersed in the cytoplasm of control cells and in cells expressing AKAP45 [6,7,11]. Transcription of c-fos is activated through an upstream cAMP-responsive element (CRE) [10]. Thus, effects of anchored PKA on transcription were assessed by monitoring synthesis of c-fos mRNA. Cells were incubated in medium containing 0.1% fetal calf serum for 12 hours and were then treated with forskolin (FSK) to stimulate adenylate cyclase and 3-isobutyl-1-methylxanthine (IBMX) to inhibit cAMP phosphodiesterases. In control cells, a low level of c-fos mRNA was detected 30 minutes after activation of adenylate cyclase. The c-fos mRNA content increased over the next 60 minutes and reached a peak value 90 minutes after addition of FSK (Figure 1a,b). Induction of c-fos mRNA by cAMP in HEK-A75 cells was more rapid and robust. A substantial amount of c-fos mRNA was detected 30 minutes after exposure to FSK (Figure 1a). Content of c-fos mRNA peaked at 45 minutes and then declined slowly during the succeeding 45 minutes (Figure 1a,b). Levels of c-fos mRNA were 4–10-fold higher in HEK-A75 cells than in control cells during the initial phase (0–45 minutes) of adenylate cyclase activation (Figure 1b). By 90 minutes, c-fos transcripts were equally abundant in these cell lines. The kinetics of c-fos mRNA accumulation in HEK-A45 cells was indistinguishable from that observed in control cells. Subsequent experiments were focused on the physiologically relevant, early phase of cAMP-mediated signal transduction.
Current Biology 1997, 7:1011–1014 http://biomednet.com/elecref/0960982200701011 © Current Biology Ltd ISSN 0960-9822
HEK-A75 cells generated 4–10-fold more c-fos mRNA than did control cells over a range of activator concentrations (Figure 1c). To exclude the possibility that AKAP75
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Figure 1 HEK293 HEK-A75 HEK-A45 Time (min) 0 15 30 45 0 15 30 45 0 15 30 45
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Northern blot analyses of c-fos mRNA content in HEK293, HEK-A75 and HEK-A45 cells. (a) Accumulation of c-fos mRNA in cells incubated with 40 µM FSK and 0.1 mM IBMX. Representative autoradiograms are shown. (b) The c-fos mRNA content was quantified by scanning densitometry [6] at the indicated time points. Cells were incubated with 40 µM FSK. Levels of c-fos mRNA are presented as arbitrary densitometric units (ADU) and represent the mean of three independent experiments that yielded similar results. (c) Accumulation of c-fos mRNA in control and HEK-A75 cells exposed to the indicated FSK concentrations for 30 min. Representative autoradiograms are shown. Mean densitometric values (in ascending order of FSK concentration) for c-fos mRNA content (above the basal level) were
determined from three independent experiments (expressed as ADU). HEK293: 3, 5, 4; HEK-A75 13, 65, 55. (d) Cells were treated for 30 min with the cAMP analog CPT-cAMP, at the indicated concentrations. Representative autoradiograms are shown. Mean values for c-fos mRNA (above the basal value) from two independent experiments (expressed as ADU) were HEK293: 0, 4, 6; HEK-A75: 23, 44, 35. Data are presented in ascending order of CPT-cAMP concentration. (e) Cells grown in 10% fetal calf serum were incubated with the indicated amounts of CPT-cAMP for 30 min. Autoradiograms are shown. Mean values for CPT-cAMP stimulated c-fos mRNA accumulation (from three independent experiments, expressed as ADU) were HEK293: 8, 17; HEK-A75: 39, 45; HEK-A45: 4, 5.
increased the sensitivity of adenylate cyclase to FSK, we monitored c-fos mRNA accumulation after incubating cells with 8-chlorophenylthio-cAMP (CPT-cAMP), a stable cAMP analog. CPT-cAMP selectively elicited a large increase in c-fos mRNA content in cells containing anchored PKAII (Figure 1d). A similar induction of c-fos transcripts by CPT-cAMP was observed in HEK-A75 cells incubated with 10% fetal calf serum (Figure 1e). Thus, serum hormones had little effect on the pattern of
cAMP-mediated c-fos mRNA accumulation in the three cell lines. Phosphotransferase activity of the C subunits increased threefold in nuclei of FSK-treated HEK-A75 cells (Figure 2a,b). Catalytic activity of C subunits in the nucleus increased only slightly in similarly treated control and HEK-A45 cells. The physiological significance of enhanced nuclear translocation of C subunits was
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Figure 2
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Stimulation of nuclear translocation of C subunits by FSK. C subunit phosphotransferase activity was assayed in nuclear extracts isolated from cells incubated in the presence or absence of FSK. C activity is expressed as the fold increase over basal activity in response to (a) time of exposure to 40 µM FSK and 0.1 mM IBMX, or (b) various FSK concentrations (cells harvested 20 min after addition of drug). Values represent phosphotransferase activity inhibitable by 50 µM PKI, which selectively binds and inactivates C subunits. Nuclear C-specific activity is expressed as the mean ± s.e. from three independent experiments. C activity in untreated cells was: 0.8 ± 0.12, 0.2 ± 0.03 and 0.8 ± 0.14 pmol per µg protein per min for HEK293, HEK-A75 and HEK-A45 cells, respectively.
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evaluated by assaying CREB phosphorylation at Ser 133 by western immunoblot analysis (Figure 3). The level of phosphorylated CREB increased modestly in control HEK293 cells exposed to FSK. Peak phosphorylation was evident 20 minutes after addition of drug (Figure 3a). The kinetics and extent of phosphorylation of CREB in HEK-A45 cells were similar. In HEK-A75 cells, a substantial increase in CREB phosphorylation was observed 10 minutes after adenylate cyclase activation. The concentration of phosphoCREB rose sharply for the succeeding 30 minutes. At each time point, the amount of phosphoCREB was 5–10- fold higher in HEKA75 cells than in control cells. CREB phosphorylation was also markedly increased in HEK-A75 cells at low FSK concentrations, thereby documenting an enhanced sensitivity to cAMP in cells with anchored PKAII (Figure 3b). Total CREB protein in nuclear extracts did not vary significantly during these experiments (Figure 3a,b). Several stable, independently isolated lines of HEK293 and hamster AV-12 cells [7] that overexpress AKAP75, exhibit properties that are similar to those reported for HEK-A75 cells. HEK-A75, HEK-A45 and control cells contain similar levels of PKAII isoforms [6,7]. Synthesis and accumulation of AKAP75 (in HEK-A75 cells) elicits redistribution of holoenzyme from a large volume (cytoplasm) to a more limited ‘compartment’ occupied by portions of cortical actin cytoskeleton and plasma membrane [6,7]. In accord with the AKAP model [1,2] and laws of mass action, generation of a high concentration of PKAII in the vicinity of adenylate cyclase presumably optimizes binding of locally produced cAMP to PKAII holoenzyme, thereby facilitating dissociation of active C subunits. Initially, PKA-mediated signaling will be focused on co-localized substrates, but subsequent C subunit diffusion is also expected.
20 30 Time (min)
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Increases in nuclear translocation of C subunits, CREB phosphorylation and c-fos mRNA synthesis in FSKtreated HEK-A75 cells suggest that anchored PKAII serves as an easily activated pool of diffusible C subunits that mediate cAMP signaling to the nucleus. Enrichment of PKAII in cortical cytoskeleton can substantially increase target cell responses to modest levels of hormones (that is, partial activation of adenylate cyclase), just as increased receptor number classically increases target cell sensitivity to low ligand concentrations. Moreover, immobilized PKAII is exposed to a maximal level of cAMP before the cyclic nucleotide is diluted and degraded (via cAMP phosphodiesterases) in cytoplasm. HEK-A45 cells, which contain unanchored AKAP–PKAII complexes, do not exhibit enhanced signaling to the nucleus. Thus, targeting and immobilization of PKAII is the key determinant of signal amplification; formation of an AKAP–PKAII complex per se is necessary, but not sufficient, for upregulation of cAMP-controlled gene transcription. In vivo fluxes in hormone concentration often occur on a timescale of seconds to several minutes. In this context, the ability of cells containing anchored PKAII to respond rapidly and with increased potency and sensitivity to modest increases in cAMP (that is, low physiological levels of hormones) confers a considerable biological advantage. Other recent observations have linked intracellular targeting of PKAII to gene regulation. Overexpression of AKAP45 in thyroid cells causes displacement of immobilized PKAII from perinuclear sites (Golgi) to cytoplasm and depresses cAMP-controlled thyroglobulin gene expression [13]. Overexpression of AKAP75 and RII in variant PC12 cells that are defective in cAMP signal transduction restored cAMP-stimulated activation of a chimeric reporter gene [14]. In these studies, however,
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Acknowledgements
Figure 3
HEK293 HEK-A75 HEK-A45 Time (min) 0 10 20 40 0 10 20 40 0 10 20 40 –43 kDa pCREB
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–43 kDa
CREB (b) FSK (µM) – pCREB
The authors dedicate this paper to G. Salvatore (Second Medical School, Naples, Italy) for his scientific and personal support. We thank C. Schindler for useful comments, R. Prywes for the c-fos cDNA clone and R.S. Watnick for help in preparing the graphics. A.M. Alba provided expert secretarial assistance. This work was supported by grants from the National Institute of Health (GM22792, C.S.R.), Associazione Italiana Ricerca sul Cancro, Progetti Finalizzati CNR ‘Ingegneria Genetica’ Italy (E.V.A.) and the Lucille P. Markey Charitable Trust (M.E.G.). A. F. was supported by a Fellowship of the Associazione Italiana Ricerca sul Cancro.
References HEK293 0.4 4 40
CREB
–
HEK-A75 0.4 4 40 –43 kDa
–43 kDa
Current Biology
cAMP/PKA-dependent phosphorylation of CREB at Ser 133. Proteins (30 µg) in nuclear extracts from untreated and FSK-stimulated cells were fractionated by denaturing electrophoresis. A western blot was probed with antiserum directed against an epitope in CREB that includes phosphorylated Ser 133 (upper panels in (a) and (b)) or with non-discriminating anti-CREB antibodies (lower panels). Arrows indicate phosphorylated CREB (pCREB) and total CREB proteins. (a) Cells were incubated with 40 µM FSK and 0.1 mM IBMX and harvested at the indicated times. Representative immunoblots are shown. Relative levels of the 42 kDa phosphoCREB doublet (above basal values) are expressed as the mean of three independent experiments. The results were HEK293: 3, 21, 19; HEK-A75: 42, 88, 150; HEK-A45: 8, 8, 10. Values are given in ascending order of incubation times. (b) Cells were harvested 20 min after exposure to the indicated concentrations of FSK. Typical immunoblots are shown. Relative levels of phosphoCREB (above basal values), expressed as the mean of three independent experiments, were HEK293: 1, 21, 26; HEK-A75: 7, 80, 265. Values are presented in ascending order of FSK concentration.
the exact location and composition of PKAII–anchor protein complexes were not determined; phosphorylation and activation of CREB was not addressed and data documenting positive regulation of an endogenous, cAMP- and PKA-regulated target gene (for example cfos) was lacking. Our present results show that assembly of AKAP75–PKAII complexes in cortical cytoskeleton strongly promotes CREB activation via phosphorylation at Ser 133 and a high level of expression of mRNA encoding a key transcription factor for immediate early genes, c-Fos. Regulation of AKAP75 content by hormones or developmental factors could provide a novel mechanism for regulating the sensitivity of cells to agents that activate adenylate cyclase.
Materials and methods Cell culture, transfections, assay for c-fos mRNA, protein kinase assay, and western analysis were carried out by standard methods [6,11–13,15,16] and are given in more detail in the Supplementary material published with this paper on the internet.
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