Type II cAMP-dependent Protein Kinase-deficientDrosophila Are Viable but Show Developmental, Circadian, and Drug Response Phenotypes

Abstract

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII EP(2)2162. Extracts from pka- RII EP(2)2162 flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII EP(2)2162 shows 2-fold increased basal PKA activity and ∼40% of normal cAMP-inducible PKA activity.pka-RII EP(2)2162 is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol. We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII EP(2)2162. Extracts from pka- RII EP(2)2162 flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII EP(2)2162 shows 2-fold increased basal PKA activity and ∼40% of normal cAMP-inducible PKA activity.pka-RII EP(2)2162 is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol. cAMP-dependent protein kinase type II cAMP-dependent protein kinase type II PKA regulatory subunit protein kinase A-anchoring proteins Berkeley Drosophila Genome Project cAMP is a widely used second messenger that participates in a plethora of cellular, neural, and developmental processes. Neural functions of cAMP signaling include roles in a number of plastic responses, including learning and memory (1.Schwartz J.H. Greenberg S.M. Annu. Rev. Neurosci. 1987; 10: 459-476Crossref PubMed Scopus (93) Google Scholar, 2.Yin J.C. Wallach J.S. Del Vecchio M. Wilder E.L. Zhou H. Quinn W.G. Tully T. Cell. 1994; 79: 49-58Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 3.Davis R.L. Cherry J. Dauwalder B. Han P.L. Skoulakis E. Mol. Cell. Biochem. 1995; 149-150: 271-278Crossref PubMed Scopus (129) Google Scholar, 4.Connolly J.B. Roberts I.J. Armstrong J.D. Kaiser K. Forte M. Tully T. O'Kane C.J. Science. 1996; 274: 2104-2107Crossref PubMed Scopus (414) Google Scholar, 5.Goodwin S.F. Del Vecchio M. Velinzon K. Hogel C. Russell S.R. Tully T. Kaiser K. J. Neurosci. 1997; 17: 8817-8827Crossref PubMed Google Scholar, 6.Mayford M. Kandel E.R. Trends Genet. 1999; 15: 463-470Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), synaptic transmission (7.Davis G.W. DiAntonio A. Petersen S.A. Goodman C.S. Neuron. 1998; 20: 305-315Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 8.Pothos E.N. Przedborski S. Davila V. Schmitz Y. Sulzer D. J. Neurosci. 1998; 18: 5575-5585Crossref PubMed Google Scholar), circadian rhythmicity (9.Majercak J. Kalderon D. Edery I. Mol. Cell. Biol. 1997; 17: 5915-5922Crossref PubMed Scopus (71) Google Scholar), modulation of photoreceptor responses to light (10.Chyb S. Hevers W. Forte M. Wolfgang W.J. Selinger Z. Hardie R.C. J. Neurosci. 1999; 19: 8799-8807Crossref PubMed Google Scholar), and modulation of responses to alcohol (11.Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar,12.Pandey S.C. Mol. Neurobiol. 1998; 17: 1-15Crossref PubMed Scopus (71) Google Scholar) and other drugs of abuse (13.Nestler E.J. Aghajanian G.K. Science. 1997; 278: 58-63Crossref PubMed Scopus (1140) Google Scholar, 14.Brandon E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar). A key intermediate in cAMP signaling is cAMP-dependent protein kinase, PKA1 (15.Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (956) Google Scholar). PKA acts as a key sensor of cellular cAMP levels and mediates the flow of signals to downstream effector pathways by phosphorylating their targets. PKA is a heterotetrameric holoenzyme composed of two catalytic subunits (C2) and a homodimer of regulatory subunits (R2). The R2 homodimer not only inhibits the catalytic activity of the C subunits but also stabilizes C subunits against proteolytic degradation in the PKA heterotetramer. Binding of cAMP to the R subunits of the holoenzyme results in dissociation of R and C subunits, which are then catalytically active. These general features of PKA signaling are shared by most eukaryotic organisms. PKA can be formed from several types of C and R subunits that can endow the PKA heterodimer with different signaling and subcellular localization properties. The three subtypes of C subunits (Cα, Cβ, and Cγ) have virtually identical kinetic and physiological properties (15.Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (956) Google Scholar). By contrast, the different R subunit types, RI and RII, exhibit distinct cAMP binding affinities and are differentially localized in cells, leading to PKA holoenzymes termed PKAI or PKAII, respectively. PKAI holoenzyme is predominantly cytoplasmic, whereas the majority of PKAII associates with cellular structures and organelles (16.Scott J.D. Pharmacol. Ther. 1991; 50: 123-145Crossref PubMed Scopus (314) Google Scholar). This subcellular localization is largely mediated by anchoring of RII subunits to protein kinase A-anchoring proteins (AKAPs). RII subunits bind to AKAPs with nanomolar affinity (17.Carre I.A. Edmunds Jr., L.N. J. Biol. Chem. 1992; 267: 2135-2137Abstract Full Text PDF PubMed Google Scholar), whereas RI subunits only bind weakly to AKAPs (18.Burton K.A. Johnson B.D. Hausken Z.E. Westenbroek R.E. Idzerda R.L. Scheuer T. Scott J.D. Catterall W.A. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11067-11072Crossref PubMed Scopus (118) Google Scholar). Adding to this complexity, higher vertebrates express two types of RI and RII genes that have different spatial patterns of expression (19.Cadd G. McKnight G.S. Neuron. 1989; 3: 71-79Abstract Full Text PDF PubMed Scopus (195) Google Scholar). In Drosophila, genetic and molecular approaches have led to the identification of a type I R subunit (pka-RI) and three genes for catalytic subunit isoforms, pka-C1 (also known asDC0), pka-C2, and pka-C3 (20.Kalderon D. Rubin G.M. Genes Dev. 1988; 2: 1539-1556Crossref PubMed Scopus (136) Google Scholar). Both RI and RII PKA subunits have also been detected biochemically. PKA-RI is detectable during larval stages and early and mid-pupation (21.Foster J.L. Guttman J.J. Hall L.M. Rosen O.M. J. Biol. Chem. 1984; 259: 13049-13055Abstract Full Text PDF PubMed Google Scholar), although its transcripts can be detected in the adult mushroom bodies of the brain (5.Goodwin S.F. Del Vecchio M. Velinzon K. Hogel C. Russell S.R. Tully T. Kaiser K. J. Neurosci. 1997; 17: 8817-8827Crossref PubMed Google Scholar). Consistent with this expression pattern, apka-RI mutant shows learning defects (5.Goodwin S.F. Del Vecchio M. Velinzon K. Hogel C. Russell S.R. Tully T. Kaiser K. J. Neurosci. 1997; 17: 8817-8827Crossref PubMed Google Scholar). PKA-RII is expressed much more abundantly than PKA-RI in adultDrosophila (21.Foster J.L. Guttman J.J. Hall L.M. Rosen O.M. J. Biol. Chem. 1984; 259: 13049-13055Abstract Full Text PDF PubMed Google Scholar), and its expression is largely restricted to the nervous system, where it is also preferentially expressed in mushroom bodies (22.Muller U. J. Neurobiol. 1997; 33: 33-44Crossref PubMed Scopus (69) Google Scholar). The Drosophila gene encoding PKA-RII has not been previously identified. From the analysis of pka-C1 and other cAMP signaling genes such as amnesiac (amn, a PACAP-like adenylate cyclase-activating peptide), pka-RI (5.Goodwin S.F. Del Vecchio M. Velinzon K. Hogel C. Russell S.R. Tully T. Kaiser K. J. Neurosci. 1997; 17: 8817-8827Crossref PubMed Google Scholar), rutabaga(rut, Ca2+/CaM-responsive adenylate cyclase), and dunce (dnc, cAMP phosphodiesterase), there is functional evidence for the involvement of cAMP signaling in learning and memory (3.Davis R.L. Cherry J. Dauwalder B. Han P.L. Skoulakis E. Mol. Cell. Biochem. 1995; 149-150: 271-278Crossref PubMed Scopus (129) Google Scholar), ovarian development (23.Lane M.E. Kalderon D. Genes Dev. 1993; 7: 1229-1243Crossref PubMed Scopus (143) Google Scholar), of circadian locomotor rhythmicity (9.Majercak J. Kalderon D. Edery I. Mol. Cell. Biol. 1997; 17: 5915-5922Crossref PubMed Scopus (71) Google Scholar), and modulation of the sensitivity to ethanol (11.Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) in In PKA signaling functions in the widely used signaling in Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the roles for PKAII signaling have been to In of the gene to locomotor defects and to repeated E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar). These phenotypes be to the loss of the but there are in other RI and RII subunits that also the is in K. E.P. K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In the pka-RI mutant shows a learning but this not be the of this mutant PKA-RI expression (5.Goodwin S.F. Del Vecchio M. Velinzon K. Hogel C. Russell S.R. Tully T. Kaiser K. J. Neurosci. 1997; 17: 8817-8827Crossref PubMed Google Scholar). the Drosophila pka-RII gene and that virtually its expression are We used these to the involvement of PKAII in processes in The EP(2)2162 Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar) from the Berkeley Drosophila Genome Project genetic and as analysis as in (21.Foster J.L. Guttman J.J. Hall L.M. Rosen O.M. J. Biol. Chem. 1984; 259: 13049-13055Abstract Full Text PDF PubMed Google Scholar) with protein from flies by the flies in A and for The protein of by the A of of protein with of in the or of in a of of for The by of of of a The and A of of protein from in and to the The with in for and with by D. for three with the with for and then with three the the used to the and to the of cAMP binding activity by the in A. K. H. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of of protein with of in a of of C for and the with C. The of A of of protein with in a of of with of in the of cAMP and of PKA for of to by the of and with of kinase activity the by PKA of to the of H. K.A. D. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). and the as flies to of where is and is light by in in a locomotor activity type and mutant flies in a The of flies during by from flies that to the of the activity used for ethanol from a in the of two The have a number of the to of flies to and the number of flies the In the identical in in the of flies are to the of a in the as in (11.Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). A of of protein to the of alcohol and for and This to ethanol by ethanol by the cocaine as previously C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the for sensitization to the in pka- RII higher of cocaine The second the RII EP(2)2162 flies with a and the with and and with and as by the for the that the been of the of We identified a encoding a Drosophila of a type II regulatory subunit of cAMP-dependent protein kinase (PKA-RII) in the the protein of known PKA-RII These identified a in which in its The a protein that to PKA-RII proteins and the previously identified Drosophila PKA-RI The most are in and that two and in is a PKA autophosphorylation by in a unique of type II PKA regulatory subunits O.M. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). also that have been identified by from Drosophila PKA-RII H. T. Biochem. 1997; PubMed Scopus Google Scholar), by in PKA-RII from the and and to the are the that is localized in the of in The also led to the identification of a of the to the of in a that of This is and with behavioral or developmental the of this the expression of PKA-RII the autophosphorylation of PKA-RII in from type and EP(2)2162 in the to RII autophosphorylation that is by cAMP and C subunit dissociation Rosen O.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The autophosphorylation to the of and proteins The which is in a is reduced by in the EP(2)2162 and the of the protein is increased that only the PKA-RII that is to be in its and in its in Drosophila (21.Foster J.L. Guttman J.J. Hall L.M. Rosen O.M. J. Biol. Chem. 1984; 259: 13049-13055Abstract Full Text PDF PubMed Google Scholar), this that EP(2)2162 not express a functional PKA-RII. Consistent with by analysis RII is reduced in EP(2)2162 In the EP(2)2162 significantly reduced cAMP binding activity attributable to the of a cellular protein Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the in EP(2)2162 the normal expression of PKA-RII a if not of which to as pka-RII EP(2)2162. against the Drosophila to show pka-RII genes not pka-RII EP(2)2162 the in which PKAII activity is We that the PKA-RII a significantly than the by A is in with molecular of and molecular of K. K.A. K. Proc. Natl. Acad. Sci. U. S. A. 79: PubMed Scopus Google Scholar). This that the properties of PKA-RII proteins that the are these organisms. In loss of PKA regulatory subunits by genetic to of cAMP-inducible PKA activity to proteolytic degradation of PKA C subunits K. E.P. K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and of the other PKA regulatory subunits E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar). the of loss of PKA-RII PKA activity PKA activity in the protein from type EP(2)2162 flies In the of the basal of PKA activity is significantly increased in EP(2)2162 with in C subunits to loss of RII in the EP(2)2162 shows only ∼40% of the activity in type The of PKA activity by cAMP is in from type flies and in EP(2)2162 with the of cAMP attributable to the loss of this cAMP signaling defects in have previously been for a PKA catalytic subunit mutant (23.Lane M.E. Kalderon D. Genes Dev. 1993; 7: 1229-1243Crossref PubMed Scopus (143) Google Scholar, M.E. Kalderon D. Dev. 1995; PubMed Scopus Google Scholar), pka-RII EP(2)2162 show phenotypes of EP(2)2162 flies show defects in and These defects include cells, or a of and number of the of the These phenotypes are but not to in In pka-C1 the that have the two M.E. Kalderon D. Dev. 1995; PubMed Scopus Google Scholar), but in pka-RII EP(2)2162 have with also to have than EP(2)2162 also have where and the normal number of is and these are not from the by The and pka-RII EP(2)2162 phenotypes that PKAII is a to PKA signaling during ovarian PKA is also in the of locomotor circadian rhythmicity in Drosophila, downstream of the circadian in (9.Majercak J. Kalderon D. Edery I. Mol. Cell. Biol. 1997; 17: 5915-5922Crossref PubMed Scopus (71) Google Scholar). We locomotor rhythmicity of pka-RII EP(2)2162. of pka-RII EP(2)2162 flies normal circadian rhythmicity in with the locomotor of pka-C1 (9.Majercak J. Kalderon D. Edery I. Mol. Cell. Biol. 1997; 17: 5915-5922Crossref PubMed Scopus (71) Google Scholar), a PKA catalytic subunit pka-RII EP(2)2162 to but the rhythmicity In of flies a normal of is of type and show normal rhythmicity A and not evidence for the involvement of cAMP in the ethanol has been (11.Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). in the which is to a of cAMP sensitivity to ethanol as by increased of We a that of ethanol sensitivity of two of flies in by for flies to to the of a which ethanol is of this Consistent with the the (11.Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), these show than type or not In contrast, pka-RII EP(2)2162 shows to ethanol with than type flies that the is not to the ethanol or the of ethanol in the of pka-RII EP(2)2162 and type are in and EP(2)2162 and type not In PKA signaling is a key responses to of We the responsiveness of pka-RII EP(2)2162 to cocaine, a in flies C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The RII EP(2)2162 to the of cocaine is much than that of type flies and other higher of cocaine are to responses, which are of cocaine in type of cocaine to sensitization in pka-RII EP(2)2162 whereas type flies show sensitization as to the second cocaine that the phenotypes are to the EP(2)2162 multiple and by the EP(2)2162 against their and and pka-RII show of the behavioral and developmental defects that are in pka-RII EP(2)2162 not of the in to the pka-RII gene and behavioral phenotypes RII EP(2)2162 is a severely if not a II PKA is the in adult Drosophila (21.Foster J.L. Guttman J.J. Hall L.M. Rosen O.M. J. Biol. Chem. 1984; 259: 13049-13055Abstract Full Text PDF PubMed Google Scholar, U. J. Neurobiol. 1997; 33: 33-44Crossref PubMed Scopus (69) Google Scholar), the pka-RII gene has not been We a unique pka-RII gene and a PKA-RII protein that is in in a We have not detected other PKA-RII genes in that most of A EP(2)2162 of the autophosphorylation activity, of that pka-RII as a of of this show phenotypes that are more than the of these show These are the in which PKAII activity is for roles of PKAII PKA activity in pka-RII EP(2)2162 shows reduced as be for loss of this cAMP regulatory This in cAMP is to two these show increased basal PKA activity in the of cAMP to to in C subunits that are not with R pka-RII EP(2)2162 show reduced PKA activity to type in the of In loss of R subunits to of C subunits, more to proteolytic degradation E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar, K. E.P. K. Idzerda R.L. McKnight G.S. J. Biol. 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Consistent with these EP(2)2162 shows locomotor the lack of PKAII not the in pka-RII EP(2)2162 a for the in This is to the of which show locomotor but protein (9.Majercak J. Kalderon D. Edery I. Mol. Cell. Biol. 1997; 17: 5915-5922Crossref PubMed Scopus (71) Google Scholar). the that PKA is in the locomotor and that PKAII is a in this We the Berkeley for the EP(2)2162 and for that its We for and and McKnight and the of for the We Kalderon for the of