Oxana M. Tsygankova

Valproic acid (VPA), a well-established therapy for seizures and bipolar disorder, has recently been shown to inhibit histone deacetylases (HDACs). Similar to more widely studied HDAC inhibitors, VPA can cause growth arrest and induce differentiation of transformed cells in culture. Whether this effect of VPA is through inhibition of HDACs or modulation of another target of VPA has not been tested. We have used a series of VPA analogs to establish a pharmacological profile for HDAC inhibition. We find that VPA and its analogs inhibit multiple HDACs from class I and class II (but not HDAC6 or HDAC10) with a characteristic order of potency in vitro. These analogs also induce hyperacetylation of core histones H3 and H4 in intact cells with an order of potency that parallels in vitro inhibition. VPA and VPA analogs induce differentiation in hematopoietic cell lines in a p21-dependent manner, and the order of potency for induction of differentiation parallels the potencies for inhibition in vitro, as well as for acetylation of histones associated with the p21 promoter, supporting the argument that differentiation caused by VPA is mediated through inhibition of HDACs. These findings provide additional evidence that VPA, a well-tolerated, orally administered drug with extensive clinical experience, may serve as an effective chemotherapeutic agent through targeting of HDACs.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. Cyclic adenosine 3′,5′-monophosphate (cAMP) is produced as an intracellular second messenger in response to a variety of extracellular signals, including hormones, growth factors, and neurotransmitters. cAMP regulates a wide range of important biological processes, which, alongside cell metabolism, include cell division, growth, differentiation, secretion, memory, and neoplastic transformation. For many years, major intracellular effects of cAMP in mammalian cells were believed to be mediated by cAMP-dependent protein kinase (PKA). 1The abbreviations used are: PKAcAMP-dependent protein kinaseCcAMP-dependent protein kinase catalytic subunitRcAMP-dependent protein kinase regulatory subunitEpacexchange protein directly activated by cAMPGEFguanine nucleotide exchange factorPI3Kphosphatidylinositol 3-kinasedibutyryl cAMPN6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphatePBSphosphate-buffered salinePDKphosphoinositide-dependent kinaseHEKhuman embryonic kidneyEGFPepidermal growth factor proteinWRTWistar rat thyroidTSHthyrotropin 1The abbreviations used are: PKAcAMP-dependent protein kinaseCcAMP-dependent protein kinase catalytic subunitRcAMP-dependent protein kinase regulatory subunitEpacexchange protein directly activated by cAMPGEFguanine nucleotide exchange factorPI3Kphosphatidylinositol 3-kinasedibutyryl cAMPN6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphatePBSphosphate-buffered salinePDKphosphoinositide-dependent kinaseHEKhuman embryonic kidneyEGFPepidermal growth factor proteinWRTWistar rat thyroidTSHthyrotropin The regulation of PKA is achieved via a unique three-component signaling system in which PKA is composed of two separate subunits, the catalytic (C) and regulatory (R) subunits that interact to form an inactive holoenzyme complex (1.Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar). Although phosphorylation of Thr197 in the activation loop of the C subunit is necessary for the maturation and optimal catalytic activity of PKA (2.Steinberg R.A. Cauthron R.D. Symcox M.M. Shunton H. Mol. Cell. Biol. 1993; 13: 2332-2341Crossref PubMed Google Scholar, 3.Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Crossref PubMed Scopus (191) Google Scholar), unlike most other kinases whose activity is regulated by dynamic phosphorylation/dephosphorylation of the activation loop this phosphorylation step does not seem to be a regulatory mechanism for PKA in vivo. Once phosphorylated, PKA is fully active in its catalytic potential and the Thr197 phosphate does not turn over readily (4.Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (133) Google Scholar). The activation of PKA is achieved by binding of the second messenger cAMP to the R subunit, which consequently induces a conformational change in the R subunit and leads to the dissociation of the holoenzyme into its constituent subunits (1.Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar). The free active C subunit can then affect a range of diverse cellular events by phosphorylating an array of cytoplasmic and nuclear protein substrates, including enzymes and transcription factors (5.Zetterqvist Ö. Z. Ragnarsson U. Engstrom L. Kemp B.E. Peptides and Protein Phosphorylation. CRC Press Inc., Boca Raton, FL1990: 1-41Google Scholar). cAMP-dependent protein kinase cAMP-dependent protein kinase catalytic subunit cAMP-dependent protein kinase regulatory subunit exchange protein directly activated by cAMP guanine nucleotide exchange factor phosphatidylinositol 3-kinase N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate phosphate-buffered saline phosphoinositide-dependent kinase human embryonic kidney epidermal growth factor protein Wistar rat thyroid thyrotropin cAMP-dependent protein kinase cAMP-dependent protein kinase catalytic subunit cAMP-dependent protein kinase regulatory subunit exchange protein directly activated by cAMP guanine nucleotide exchange factor phosphatidylinositol 3-kinase N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate phosphate-buffered saline phosphoinositide-dependent kinase human embryonic kidney epidermal growth factor protein Wistar rat thyroid thyrotropin The effect of cAMP on certain cellular functions has been shown to be dependent on cell-type and biological responses (6.Grave L.M. Lawrance Jr., J.C. Trends Endocrinol. Metab. 1990; 7: 43-50Abstract Full Text PDF Scopus (44) Google Scholar). For example, in PC12 cells, Swiss 3T3 cells, and thyrocytes, cAMP activates MAP kinases, potentiates the effects of growth factors on differentiation and gene expression, and/or stimulates cell growth and promotes the G1 to S phase transition in the cell cycle (7.Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6217Abstract Full Text PDF PubMed Google Scholar, 8.Vaillancourt R.R. Gardner A.M. Johnson G.L. Mol. Cell. Biol. 1994; 14: 6522-6530Crossref PubMed Scopus (149) Google Scholar, 9.Withers D.J. Bloom S.R. Rozengurt E. J. Biol. Chem. 1995; 270: 21411-21419Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10.Medina D.L. Santisteban P. Eur. J. Endocrinol. 2000; 143: 161-178Crossref PubMed Scopus (92) Google Scholar). In contrast, cAMP inhibits the proliferation of many cells, including fibroblasts (Rat1 and NIH 3T3), smooth muscle cells, and cells transformed by oncogenes such as ras (11.Burgering B.M.T. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar, 12.Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (823) Google Scholar, 13.Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (404) Google Scholar, 14.Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar). Despite extensive studies, the molecular mechanism underlying the cell type-specific effects of cAMP remains elusive. The growth inhibitory effect of cAMP is believed to be mediated partly through activation of PKA, which interferes with Ras/MAPK signaling pathways (15.Qiu W. Zhuang S. von Lintig F.C. Boss G.R. Pilz R.B. J. Biol. Chem. 2000; 274: 31921-31929Abstract Full Text Full Text PDF Scopus (92) Google Scholar). Recent studies suggested that PI3K activity may be required for cAMP-stimulated cell proliferation in thyroid cells (16.Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar, 17.Ciullo I. Diez-Roux G. Di Domenico M. Migliaccio A. Avvedimento E.V. Oncogene. 2001; 20: 1186-1192Crossref PubMed Scopus (88) Google Scholar). Interestingly, the effects of cAMP on PI3K/PKB pathways are also cell type-specific and correlate well with the mitogenic effects of cAMP (16.Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar). In cells in which cAMP is mitogenic, cAMP stimulates PKB phosphorylation and membrane ruffling. Furthermore, cAMP effects on PKB and membrane ruffling are PKA independent (16.Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar). These findings indicate that multiple cAMP-mediated pathways exist and only some are PKA dependent. Therefore, the recently discovered cAMP receptor Epac (exchange protein directly activated by cAMP) or cAMP-GEF (cAMP-regulated guanine nucleotide exchange factor) may represent an important piece of the puzzle that is critical to our understanding of cAMP-mediated cell signaling. Epac contains a cAMP-binding domain that is homologous to the R subunit of PKA and a guanine exchange factor (GEF) domain (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar, 19.Kawasaki H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). Epac cAMP with and a guanine as an for the of H. Y. T. Y. M. Cell. Full Text PDF PubMed Scopus Google and on the of its to Chardin P. I. A. Oncogene. 1998; Scholar), is activated in response to an in intracellular cAMP in additional to many other D.L. E.G. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, H. R.D. Cell. Full Text Full Text PDF Scopus Google Scholar, J.L. Rooij J. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Although PKA can its C L.A. H. L.A. G.M. J. Google Scholar), the that the PKA phosphorylation can be activated by Epac in response to cAMP (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar). of the PKA phosphorylation in does not its biological functions H. EMBO J. 1999; PubMed Scopus Google Scholar). These cAMP-mediated activation of may be independent of phosphorylation by PKA. It is most that the cAMP-mediated signaling mechanism is complex believed and many cAMP-mediated effects that were previously to through PKA are in fact also by Epac. Therefore, is to of cAMP-mediated signaling to include the of Epac. The of Epac many the mechanism of cAMP-mediated signaling. PKA and Epac are in many (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar, 19.Kawasaki H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar), an in intracellular cAMP to the activation of and other potential cAMP as It is conceivable that while PKA through a of signaling Epac may signaling and the net cellular effects of cAMP are by the of Therefore, the cellular effect of cAMP can upon the cellular abundance and distribution of Epac and PKA. Our is that is a dynamic of cellular and targeting of Epac and PKA which, with the dynamic in the of are the that the cell type-specific cAMP this we examined the specific effects of Epac and PKA on the PI3K/PKB that has been to cell type-specific responses to cAMP. Our study for the that Epac and PKA are activated by a second messenger can opposing effects in regulating such as Therefore, the net outcome of cAMP signaling on PKB activation may be dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. a J. L. Bos The by a and a The into the of mammalian by a and a The into the of in which the gene and the the we the and that the Epac were by were that PKB and PKB were and were The were and N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate cAMP) were embryonic kidney cells were were Epac were by Epac The were an and further by Epac the were in of by Epac protein in cells were in with The were in a with The the cells were into to and with were Wistar thyroid cells were in in of and in were for the activation were with cAMP for Activation by cells with For kinase cells were with or to were by directly the cells into The of a of the domain of as Rooij J. Bos J.L. Oncogene. 14: PubMed Scopus Google Scholar). cells were to in in for and with for in the cells were in a and The cell with of with of domain and for with in the were in of of protein were a and further with specific Protein of cell with the protein and of protein by for to by were to membrane a cell and a were in in to binding by with for PKB PKB and were then with and with or for in were with for and to independent were for PKB activity an a were to the as well as to the of the the subcellular of and cells were with an in with a in well and for the cells were in in The were with on and in were the an with and were a the specific roles of Epac and PKA in the PKB signaling we examined the effects of on activation of PKB in and cells that been with Epac. shown in cells of PKA of Epac cell and cells were probed by PKA catalytic subunit and Epac protein with the molecular of to the of Epac readily in the Furthermore, of Epac in cells not affect the protein of PKA with PKB activity in cells as by phosphorylation of and whose phosphorylation is critical for the kinase activity of PKB S.R. P. Biol. 7: Full Text Full Text PDF PubMed Google Scholar, L. H. McCormick F. P. J. Science. 1998; PubMed Scopus Google Scholar). in to that of the cells an in phosphorylation of the PKB and of PKB phosphorylation by cAMP with a specific PKA that the inhibitory effect of cAMP on PKB is mediated by PKA. also the cAMP-mediated PKB activation in cells the inhibitory effect of PKA on PKB The of PKB activation in cells as with that of the cells the PKB phosphorylation of and cells were on the In to monitoring the phosphorylation of PKB and we also the cellular PKB kinase activity kinase activity the our this is the that Epac and PKA the PKB signaling in the between the roles of Epac and PKA on PKB activation, we examined the effects of on PKB phosphorylation in and cells as a of shown in PKB activation by the in the cells and while the inhibitory effect of PKA on PKB and PKB activity in and PKB activity in the of to and PKB in the of were to the PKB PKB activity in the of to and PKB in the of were to the PKB PKB activity in cells is that in PKB in and with and were the The PKB activity in the of to and PKB in the of were to the PKB The PKB activity in cells is that in in a new PKB in and with and were the used a cell cAMP T. G. PubMed Scopus Google as a for Epac and PKA to the effect of in our shown in cAMP the PKB activation in cells while the activation of PKB in cells in a to that of our that of cAMP to or activation of PKB in cells and cells, that the PKB activation in cells in the of is the of activation of Epac by a of a critical in the domain of Epac is in J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Taylor S.S. J. Biol. Chem. Full Text PDF PubMed Google and directly with the phosphate of cAMP Y. L. Taylor S.S. Science. 1995; 269: PubMed Scopus Google Scholar). this leads to the of nucleotide binding activity and biological activity of Epac H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). cells the Epac, cells in a to the cells in response to in of PKB activity to that the cAMP-mediated PKB activation the of Epac activation and required a is the only for Epac activation to PKB activation, we the of in and and shown in the of in cells the of the and not the of in well with our that the of Epac in cells is the of Epac In contrast, cells of with is with the that Epac is to in the of and Epac is of further in response to cAMP (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar, 19.Kawasaki H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). with further the of in These that activation is in PKB activation of is required for PKB activation, we a in cells to the in activation of of the of Epac to PKB in cells in response to intracellular cAMP. activated PKB the fact that in cells in our and not in cells, the effect of may be that is of Epac and that activation is required for PKB activation in proper subcellular targeting of Epac is required for cAMP-mediated PKB activation and to further that the effect of cAMP on PKB phosphorylation in cells to the of a Epac, we into the cells a whose been Epac is inactive be to the subcellular Epac exerts its shown in the protein, protein the cell while the Epac a the of the Epac is for proper cellular targeting of Epac. cells the were with of PKB as shown in cells, cAMP the phosphorylation of PKB in with the inhibitory effect of as we in the cells domain also that of in the and of stimulation cAMP binding activity and its activity can be regulated by cAMP as the Epac in with A. Cool R.H. Rooij J. Bos J.L. Wittinghofer A. J. Mol. Biol. 2001; PubMed Scopus Google Scholar), with our of the targeting domain of the Epac to its proper subcellular activates the that is necessary for PKB activation in response to intracellular cAMP These further that Epac is for the effects of cAMP on PKB in cells and proper targeting of Epac is for the cAMP-mediated activation of PKB in vivo. a of the activation of PKB is dependent upon the of PI3K in many S.R. P. Biol. 7: Full Text Full Text PDF PubMed Google Scholar, L. H. McCormick F. P. J. Science. 1998; PubMed Scopus Google Scholar), stimulation of PKB can also be achieved in a Hemmings Van Obberghen E. PubMed Scopus Google Scholar, Hemmings Van Obberghen E. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). PKB activation is dependent on PI3K we examined the effect of a specific PI3K on cAMP-mediated activation of PKB in can be in and phosphorylation of of PKB in cells Epac by of the cells with were with These indicate that PKB activation by by a that of Epac is the thyroid as has been shown by that the of Epac is that human H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). cell Wistar rat thyroid cells by Epac a protein Epac is in In the of Epac in with a to that of the cell Interestingly, unlike the cells, cells to the including thyrotropin in a to that of cAMP activated the PKB Furthermore, a active in cells to a in PKB activation in response to In to directly regulating many important cellular processes, cAMP an array of intracellular signaling pathways such the MAP kinase pathways (7.Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6217Abstract Full Text PDF PubMed Google Scholar, B.M.T. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The PI3K/PKB signaling is a in of cell and proliferation J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, S.R. A. 1999; 13: PubMed Scopus Google Scholar). cAMP has been in PKB of intracellular cAMP can to inhibitory X. Y. Jr., Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, S. J. Eur. J. 2000; PubMed Scopus Google Scholar, S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google or Hemmings Van Obberghen E. PubMed Scopus Google Scholar, Hemmings Van Obberghen E. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, J. 1999; Google effects on PKB activity. the that Epac in this is cAMP can or PKB is conceivable that Epac and PKA, as may mediate PKA inhibits PKB activation while may as a of PKB in response to cAMP. this we Epac into cells that of Epac we probed the effects of on activation of PKB in and with PKB activity in cells as by phosphorylation of and cells not of Epac, our that the inhibitory effect of cAMP on PKB is most mediated through PKA. is further by the that of PKB phosphorylation in cells by cAMP with the PKA studies further that the inhibitory effect of cAMP on PKB is mediated by PKA. Our that through PKA, inhibits the PKB activity in cells is with the recent by S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google that activation of PKB activity in a in a wide variety of cell including inhibitory effect of cAMP on PKB activation is mediated by the between PKB and its in the membrane S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google Scholar). In to the inhibitory effects of cAMP on PKB in cells, our study that cAMP activated PKB in Epac These that the cAMP activates Epac and PKA, can effects on PKB The cellular effects of Epac on PKB activation can be mediated by a or Although we the that Epac activates PKB through a our suggests that activation of is necessary for PKB activation as of a form of the effect of Epac in of Epac leads to a activation of in activation of to Epac and activity of M. 1993; Google Scholar), is most to the PKB activation the of PKB activity is only in cells that only a specific of is important for PKB a specific Epac further this of the of Epac the to be to proper subcellular is to PKB through activation of specific cellular activation of is an important mechanism for in regulation. It has been previously that guanine nucleotide exchange factor that a domain with Epac, can the membrane of while the the cellular X. T. Y. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). on our a for cAMP-mediated PKB regulation is shown in the two intracellular cAMP Epac and PKA, mediate the opposing effects of cAMP on PKB the through PKA, can PKB recent study by S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google that of PKB by PKA is It further suggested that PKA inhibits PKB activity by PI3K kinase activity and consequently PKB its the membrane S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google Scholar). The mechanism of PI3K is not and It is that PKA directly PI3K and consequently its kinase activity. It has also been proposed that PKA regulates activation through phosphorylation A. L.A. Meinkoth J.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). the other our study that cAMP can also PKB through an signaling in a of activation of PKB our understanding of the cAMP-mediated signaling pathways. Our of opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is conceivable that the net of the cellular effects of cAMP may be dependent upon the dynamic of Epac and PKA and specific subcellular distribution in a Our that activates PKB in cells that of Epac our that Epac activates while PKA inhibits PKB in response to cAMP and the cellular effects of cAMP are of the Epac and PKA signaling pathways. Furthermore, our that the effects of Epac and PKA activation on a specific may provide an additional regulatory mechanism for intracellular cAMP signaling. the complex and important that cAMP in regulating cell differentiation, and is that other cAMP-mediated signaling pathways may also L. Bos The for the Epac and for in

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.

Ras mutations are frequent in thyroid tumors, the most common endocrine malignancy. The ability of Ras to transform thyroid cells is thought to rely on its mitogenic activity. Unexpectedly, acute expression of activated Ras in normal rat thyroid cells induced a DNA damage response, followed by apoptosis. Notably, a subpopulation of cells evaded apoptosis and emerged with features of transformation, including the loss of epithelial morphology, dedifferentiation, and the acquisition of hormone- and anchorage-independent proliferation. Strikingly, the surviving cells showed marked chromosomal instability. Acutely, Ras stimulated replication stress as evidenced by the induction of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR) activity (Chk1 phosphorylation) and of gammaH2A.X, a marker of DNA damage. Despite the activation of a checkpoint, cells continued through mitosis in the face of DNA damage, resulting in an increase in cells harboring micronuclei, an indication of defects in chromosome segregation and other forms of chromosome damage. Cells that survived exposure to Ras continued to exhibit replication stress (ATR activation) but no longer exhibited gammaH2A.X or full activation of p53. When rechallenged with Ras or DNA-damaging agents, the surviving cells were more resistant to apoptosis than parental cells. These data show that acute expression of activated Ras is sufficient to induce chromosomal instability in the absence of other signals, and suggest that Ras-induced chromosomal instability arises as a consequence of defects in the processing of DNA damage. Hence, abrogation of the DNA damage response may constitute a novel mechanism for Ras transformation.