AMP second messenger


G. Stanley McKnight University

of Washington,

Seattle, Washington,


Cells carefully regulate the generation and destruction of CAMP using diverse families of adenylate cyclases and phosphodiesterases. Genes for several cyclases have now been cloned, giving structural information about the enzymes and providing access to the remaining members of this family. A much larger family of phosphodiesterases has been uncovered and the regulatory properties of both the cyclases and phosphodiesterases provide diverse mechanisms to modulate intracellular CAMP. Most of the actions of CAMP are mediated through phosphorylation of substrates of the CAMP-dependent protein kinases. Recent progress has helped define the pathway between CAMP and the activation of gene transcription. Current


in Cell Biology


Using a few characterized second messenger molecules, including cyclic nucleotides, calcium, diacylglycerol, phosphoinositides, and arachidonic acid metabolites, the cell orchestrates its responses to extracellular signals and integrates cellular mechanisms. It is therefore not surprising that cyclic nucleotides can be shown to play a part in virtually every cellular function, often in concert with other second messenger pathways. Since the discovery and appreciation of CAMP as an important second messenger in intracellular signalling [ 11, the literature on cyclic nucleotides has burgeoned to such an extent that there is now an almost insurmountable mass of largely descriptive data. Progress over the past year and a half, however, while not startling in new insights, has resulted in an increased understanding of the major players in cyclic nucleotide metabolism and signailing, and in an increased emphasis on the molecular details of structure and function. The primary structures of the adenylate and guanylate cyclases that synthesize cyclic nucleotides in response to environmental signals, and of the cyclic nucleotide phosphodiesterases (PDEs) that are responsible for attenuating the signal, have been elucidated through protein chemistry and molecular genetics. Many of the effector molecules through which CAMP and cGMP act, namely the CAMP- and cGMP-dependent protein kinases, cyclic nucleotide-gated ion channels, and the cGMP-regulated PDEs, have also been sequenced and/or cloned. Although it has been appreciated for some time that multiple isoforms of each of these gene products must exist in an organism, the initial cloning of some of these isoforms promises to lead to the rapid isolation and char-

1991, 3:213-217

acterization of the complete gene families and will undoubtedly result in the discovery of novel isoforms. The emerging picture will lead to an appreciation of the diversity of enzymes and effecters that serve to determine the cell- and tissue-specilic responses mediated through the cyclic nucleotide pathways. This review focuses on the CAMP pathway because an excellent recent review on guanylate cyclases and cGMP was recently published in this journal [2*]. I will briefly describe our current understanding of the enzymes that synthesize (cyclases), degrade (phosphodiesterases), and mediate the function (kinases) of CAMP. Although CAMP can have effects on most of the cellular machinery, I wiIl focus on the cAMP-mediated regulation of gene expression, an area that has advanced rapidly over the past 2 years. Mammalian




The first cyclase that was cloned and sequenced was a bovine brain form of the calmodulin-dependent cyclase, termed type I [3**]. This enzyme can be modeled as an integral membrane protein with two large hydrophobic domains, each containing six membrane-spanning elements, and two large hydrophilic domains, which are likely to be cytoplasmic and which share sequence slmilarity with mammalian guanylate cyclases and yeast adenylate cyclases. It was originally suggested, on the basis of structural similarities with transporter proteins, that the cyclase might have some additional functions related to the export of small molecules. No demonstration of this function has yet been reported, however. More recently, new members of the cyclase gene family have been cloned, including a rat type II cyclase that is

Abbreviations C-catalytic [subunitl; CA-IX-CAMP-dependent protein kinase; CRE-cAMP response element; CRER-cAMP response element binding factor; PD&phosphodiesterase; MAP-microtubuleassociated protein; R-regulatory

@ Current



Ltd ISSN 0955-0674



Cell regulation

calmodulin independent and a rat type III ‘enzyme that is specifically expressed in olfactory cilia [ 4**1. The type III enzyme shares considerable homology with the type I gene, particularly in the hydrophilic intracellular domains. How many more adenylate cyclase genes make up this family? As the type II isozyme sets a precedent for a tissue-specific variant dedicated to a single function, olfaction, it appears probable that there are other isoforms which may also have a highly tissue-specific distribution and perhaps specialized functions. Recent studies have demonstrated a highly speciiic pattern of expression of the type I gene in rat brain using in situ hybridization [ 5.1. Because the Dmsqbbilu learning mutant, rutabaga, has a defect in the type I cyclase [6], it is particularly significant that the type I gene is highly expressed in the hippocampal formation and the neocortex (regions that are strongly implicated in memory and learning processes).




A diverse expressed

family of phosphodiesterases in animal cells


At least 20 members of the phosphodiesterase family have been characterized, and sequence information is available for 13 gene products. These enzymes can be divided into five functionally distinct families on the basis of their regulation or cyclic nucleotide specificity [7==]. The type I PDEs are stimulated by Ca2+/calmodulin and the type II enzymes are stimulated by cGIvlP. In both cases, the activity modilier (calmodulin or cGMP) binds at a site distinct from the catalytic domain and acts allosterically. The type III enzymes are inhibited by cGMP which probably competes with CAMP at the catalytic site on the enzyme rather than acting allosterically. The class IV enzymes are CAMP specific and include the Drosophila gene, dunce, and its mammalian counterparts. The dunce locus was originally identiIied by the learning-impaired phenotype characteristic of mutations in this gene [8]. Finally, the class V PDEs specifically hydrolyze cGMP and include the rod and cone isoforms that play a key role in the visual transduction system. Alternative mRNA splicing may further add to the diversity of PDEs synthesized in each of the five classes.





The protein kinases that respond to cAhlP are formed as inactive tetramers containing two regulatory subunits and two catalytic subunits. Binding of two molecules of cAMP to each regulatory subunit releases the catalytic (C)-subunit, which can then phosphoxylate intracellular targets. At present, four regulatory (R) subunit genes (RIa, RIB, RIIa, and Ri@> and three catalytic subunit genes (Ca, C/3, Cy) have been identified [9,10*]. In addition, alternative splicing of the Cg gene has been demonstrated and may give rise to a mod&d form of the Cg subunit [ ll*]. There is also the distinct possibility that other R- and Csubunit genes exist, as efforts to clone all of the members of this family have not been exhaustive.



in a tissue-specific


In general, the a-isoforms are expressed constitutively in most tissues whereas the p-isoforms are highly expressed in brain with a lower and more selective expression in other tissues [ 12.1. For example, the RI@ isoform is highly expressed in the brain but is also expressed in adipose, adrenal, bone marrow, testis, and ovary. During early embryonic development in the mouse, RIIj3 is expressed in liver (co-ordinately with globin expression) but this expression becomes repressed in late fetal and adult stages when the liver is no longer an erythrogenic organ. The RIP isoform shows a much narrower tissuespecific expression, with high expression in brain and the only detectable non-neural expression in male germ cells. The Cy gene is the most recently characterized member of the cAMP-kinase gene family and it appears to be specifically expressed in human testis [ 10). properties





As holoenzymes are formed between an R-dimer and two C-subunits and, as there are four R and three C isoforms, quite a variety of holoenzymes could be envisaged. However, because the R-subunits dimerize through interactions between the amino termini and this region is highly divergent in various R-isoforms, I would suggest that only R-subunit homodimers are likely to be stable. Even with this restriction, it is probable that at least 12 varieties of cAMP-dependent protein kinase can exist, creating diversity within this effector system. In support of this suggestion, pure proteins expressed in either bacteria or animal cells have been isolated and used to demonstrate that holoenzymes can be formed with any combination of an RI homodimer and Ca or C8. In some cases, the biochemical properties of these holoenzymes are very similar and do not provide evidence for differences in physiological roles. But, holoenzymes containing the neuronspecific RIP are activated at a 5-lo-fold lower concentration of CAMP than the RIa-containing holoenzymes, and this has led to speculation that RIP might increase the sensitivity of neurons to neurotransmitters acting through CAMP [13*1. Subcellular localization role in substrate selection

of kinase





The specific subcellular localization of a holoenzyme or free catalytic subunit is also likely to play an important role in selecting which substrates are phosphory lated and will generally lead to rapid phosphotylation of nearby targets. The RII isoforms are notable in their ability to bind with high aifinity to other cellular proteins such as microtubule-associated protein (MAP)-2 or a membrane-associated 150 kD Ca2 + -binding protein [ 14*,15*]. Evidence obtained by direct interaction of RII subunit with proteins transferred to nitrocellulose after gel electrophoresis suggest that there may be many other specific RII-binding proteins in cells. These may serve to localize the RII-containing holoenzymes proximal to specific substrates on membranes, cytoskeleton, and other cell-specilic targets. The C-subunits also have potentially interesting targeting sequences, including a myristylated amino-termi-


nal glycine [16] and a potential nuclear localization signal. Although other myristylated proteins, such as c-src, appear to require the myristic acid moiety to facilitate membrane association [ 171, there is no clue as to why the cytoplasmic C-subunit contains such a signal. Experiments to mutate the amino-terminal glycine and produce an unmyristyiated C-subunit have indicated that the myristic acid is not required for R-subunit binding or biological functions such as gene regulation or induction of steroidogenesis [ 18.1. Several studies have indicated that the C-subunit can enter the nucleus but that the holoenzyme is excluded [ 19,20*,21**]. Although the C-subunit may be small enough to diffuse passively into the nucleus, inspection of the amino acid sequence reveals a potential nuclear translocation signal starting at amino acid 188 (Ala-Lys-Arg-Val-Lys), which is identical to the nuclear translocation signal in human c-Myc [ 221. How these various signals aid the C-subunit in its ability to shuttle rapidly between cytoplasm, membranes, and nucleus, in response to an everchanging intracellular CAMP concentration, remains a mystery. Cyclic AMP regulates gene induction kinasedependent mechanism

by a

The phosphorylation of transcription factors by second messenger-stimulated pathways has become a prominent area of current studies on gene regulation. Many genes are induced in response to elevated levels of intracellular CAMP and most (but not all) of these genes have been shown to contain a CAMP response element (CRE) in their promoters [ 231. The response to cAh4P requires cAMF-dependent protein kinases (c&PKs) and is mediated by a family of transcription factors termed CAMP response element binding factor (CREB or ATF). For at least one member of the CREB family, it has been recently demonstrated that direct phosphotylation of a serine by the C-subunit of cA-PK is responsible for transcriptional activation [24=-l. The CREB transcription factor is concentrated in the nucleus and the cA-PK holoenzyme generally resides in the cytoplasm. By trapping active C-subunit in the cytoplasm, we have demonstrated that translocation of the C-subunit into the nucleus is an essential step in gene activation and that CREB cannot be phosphorylated in the cytoplasm and then shuttled to the nucleus (A Otten, Y Wang, L Wailes, E Krebs, S M&night, unpublished data). Basal promoter


can be dependent

on cyclic

Is the CRE required as a basal promoter element as well as being responsible for induction during hormonal stimulation? The evidence so far suggests that the CRE may play an integral role in basal expression and *hat if it were possible to completely eliminate the cA-PK activity in a cell, many of the genes containing CRE elements would be shut off. Several approaches have been successfully used to inhibit basal cA-PK activity in cells, including the expression of the heat-stable inhibitor peptide, PKI [25*,26] and the overexpression of a mutated RIa

AMP second


systems McKnight

subunit that has lost the ability to bind CAMP (C Clegg, M Abrahamsen, J Degen, D Morris, S McKnight, unpublished data) [ 271. The results demonstrate that not only is the cAMP-mediated gene induction lost but the basal expression of many &@-inducible genes is reduced dramatically. In agreement with these results, mutations that inactivate the CRE have been shown to abolish both basal and cAMP-induced expression [28]. Conceptually, it is easy to envisage the existence of two classes of genes: one in which the CRJZis involved in both basal and induced expression and one in which the CRE can stimulate expression but is not required for basal activity or activation through other second messenger pathways. Gene induction AMP and other

involves interactions between second messenger systems


Many cellular genes are regulated by more than one signaling pathway, and interactions between these pathways can occur at several different levels. As discussed, the generation and metabolism of CAMP can be influenced by Ca* + or cGMP by virtue of the regulatory properties of the cyclases and PDEs. In addition to the CRE discussed, regulatory elements responsive to diacyiglycerol or phorbol esters (TREs) and w-hich bind the family of Jun/Fos transcription factor complexes have been identied. Genes that have both functional elements can be stimulated through either the diacylglycerol or CAMP second messenger pathway. Whether these two pathways operate independently or not remains an unresolved question whose answer may depend on which promoter is being examined. As one example, the orthinine decarboxylase gene responds to both pathways, but the phorbol ester response requires the presence of basal cAMP-dependent protein kinase activity and is nearly completely inhibited when kinase activity is low (C Clegg et al, unpublished data). Recently, several laboratories have reported the formation of heterologous dimers between CREB-related proteins and members of the Jun family [ 29,30*]. The physiological function of the-se novel dimers remains to be studied but their formation may indicate the existence of yet another mechanism of interaction, providing additional flexibility in the control of gene expression by second messenger pathways. Propects

A major challenge over the next few years will be to understand the mechanisms by which the diverse adenylate cyclases, PDEs, and CA-PKs integrate into physiologically relevant response pathways. The potential for subtle regulatory interactions involving Ca*+ , cGMP and CAMP is obvious when one considers the regulatory properties of these enzymes. Although the biochemical properties of some of the various tissue-specilic isoforms of these enzymes clearly suggest functional differences, the physiological significance of the majority of isoforms remains to be demonstrated. It is possible that there is redundancy in the system rather than specil%ty, but it seems more likely that higher eukaryotes are maintaining the presence



Cell regulation

of multiple isoforms to exploit the increakd opportunities to regulate the pathway, References

and recommended






S. Ktwm V, Pmm W: Isoform Cp2, an Unusual of Bovine Catalytic Subunit of cAMP-Dependent ProKlnase. J Biol C!wm, 1991, 26:514@5146.

tein The exon 1 of the bovine CU gene is alternatively spliced to give rise to a CU isoform that is missing the consensus myristylation signal at the amino-terminus. This novel C isoform may be targeted to unique substrdtes.

Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstandIng interest 1.



THOMPSON DK, GARBER.S DL naling. Cuw Cpin Cell Bid




J Am


Sot 1957,

Guanylyl Cyclase 1990. 2:20&211.

79:3608. in



i review of the guanylyl cyclase receptors that have been characterized at the molecular level. 3. ..


Amino Acid Sequence: Possible Channel- or Transporterlike Structure. Science 1989, 244:15581564. Describes the first isolation of mammalIan adenylyl cylase clones from bovine brain and their sequence similarities with certain transporter proteins. The homologous Dmcophila cyclase gene maps to the rurabaga locus which, when mutated, causes a loss of calmodulindependent cyclase activity and learning delicits. 4. ..

HA, REED RR: IdentUication of a Specialized Adenylyl Cyclase That May Mediate Odorant Detection. Science



The first description of a cyclase that seems to be expressed only in sensory neuronal cilia and has enzymatic properties that diier enough from non-sensory cyclases to suggest a unique function in odor detec tion. 5.








Distinct Patterns for the Distribution of mRNA for the CalmoduU.n-Sensitive Adenylate Cyclase in Rat Brain: Expression ln Areas Associated with Learning and Memory. Neuron 1991, 6:4314i3. The type I adenylate cycla~ gene is expressed preferentially in specllk neuronal structures, including the hippocampus, cortex, and cere bellum. It is suggested that, by analogy with the rufubugu locus in Drox#ih, this cyclase may be involved in memory and learning in mammals. .










CADD G, MCKNIGHT GS: Distinct Patterns of cAMP-Dependent Protein Kinase Gene Expression in Mouse Brain. Neuran 1989, 3:71-79. All of the known isoforms of R and C subunits (with the exception of C7) are reported to be expressed in brain neurons, but the patterns of expression are distinct, suggesting a mechanism for increased functional diversity. 12.


UHIIR MD, MCKNIG~ GS: Holoenzymes of CAMPdependent Protein Kinase Containing the Neural Form of Type I Regulatory Subunit Have an Increased Sensitivity to Cyclic Nucleotides. J Biol C&m 1990, 265:19502-19506. The RI8 isoform is found to associate with either Ca or CU to form a holoenzyme that is activated at a lower threshold of CAMP, suggesting a mechanism for increasing sensitivity to neurotransmitters in neurons. 14. SCOIT JD, STOFKO RE, MCDONAUI JR, COMER JD, Vmus EA, . MANGIll JA: Type II Regulatory Subunit Dhnerization Determines the Subcellular Localization of the cAMP-Dependent Protein Kinase. J Biol Chem 1990, 265:21561-21566. The RIIa amino.terminal domain is shown to be important in determining the binding of the RI1dimer to MAP.2. This region of RII also encompasses the dimerizatlon domain and it is suggested that dimerization may be necessary to create a MAP-2 binding site.

13. .

Luo Z. SHAFIT-ZAGARCO B, ERUCHMAN J: Identification of the MAP-2 and P75-Biding Domain in the Regulatory Subunit (RI@) of Type II cAMP-dependent Protein Kinase. J Biol Uwm IWO. 265:21804-21810. Results similar to those in [14*] are obtained with the 8.isoform of RII, and the authors also demonstrate that the Same amino-terminal 50. amino.acid region is sufficient to bind both MAP-2 and P75. It is suggested that a similar recognition site on RI1 is involved.

15. .



cium/CalrncduUn Responsiveness in Adenylate Cyclase of Rutabaga, a Lbvsophflu Learning Mutant. Cell 1984, 37:205. BEAVO JA RE~FSNYDER DH: Primary Sequence of Cyclic Nucleotide Phosphodlesterase Isozymes and the Design of Selective Inhibitors. Trends Phamrucd Sci 1990, 11:15t&155. This review describes the diversity of PDEs that have been recognized so far and proposes a useful nomenclature for their classilication. 8.



S, DAVIS RL Molecular

Clones and the Corresponding of the Drosophila Dunce+

for CAMP Phosphodlesterase.


of cDNA

Genomic Coding Sequences Gene, the Structural Gene Prcc Null Acud Sci USA 1986,








Expression of Wild-type and Mutant Subunits of the CAMPDependent Protein Kinase. Cold Spring Harbor Sjnnp Quunt Bid 1988, 53111-119. 10. .

BEEBE SJ, OVEN 0, SANDBERG M, FROM A, HANSSON V, JAHNSEN T: Molecular Cloning of a Tissue-SpecPc Protein Kinase

(Cy) from Human Testis - Representing a Third Isoform for the Catalytic Subunit of cAMP-Dependent Protein Kinase.

Mel Endocrinol1990,


Describes a novel C-isofonn clone that appears to be testis-specific. The amino acid sequence has diverged from Ca and C8 by approximately 20%.

MP, Buss JE, SEFTON BM: Mutation of NHZ-terminal Glycine of p60v-sn Prevents both Mytistylation and Morphological Transformation. Proc Nat1 Acad Sci USA 1985, 82:4625-4628. KAhlps

18. .

CLECC CH, RANW, UHLER MD, MCKNIGHT GS: A Mutation in the Catalytic Subunit of Protein Kinase A Prevents Myrisrylation but Does Not Inhibit Biological Activity. J Eiol Gwm 1989, 264:2014&20146. A mutation of the aminoterminal glycine to alanine prevented myristylation of the C-subunit but the mutant was still capable of form. ing holoenzyme and activating gene expression, 19.

H, EPPENBERGER HM, DLITLY F: Rapid and ReTranslocation of the Catalytic Subunit of CAMPdependent Protein KInase Type II from the Golgi Complex to the Nucleus. EhfBO J 1985, 4:2801-2806. NIGG





CARR SA, BIEMANN K, SHOJI S, P-LEE IX. TITANI K: nTetredecanoyl is the NlQterminal Blocking Group of the Catalytic Subunit of Cyclic AMP-Dependent Protein Kinase From Bovine Cardiac Muscle. Prcc Natl Acad Sci USA 1982, 79:61286131.


7. ..


20. .

Jl, JI Y, TAYLOR SS, FERAMISCO JR: Dynamics of the Distribution of Cyclic AMP-dependent Protein Kinase in Living CeUs. Prcc Nat1 Acud Sci USA 1990. 87:959+9599. Catalytic subunit labeled with Buorescent dye was injected into cells either alone or In combination with the RI subunit. The holoenzyrne complex was excluded from the nucleus but when cells were stimulated to elevate extracellular CAMP, the C-subunit migrated to the nucleus. 21. ADAMS SR, HAROOTLINMN AT, BUECHLER YJ, TAYLOR SS, TSIEN .. RY: Fluorescence Ratio Imaging of Cyclic AMP in Single CelIs. Nature 1991, 349694-697. Reports a novel technique that allows visuaIiitIon of the changes in R- and C-subunit interaction and subcellular dIsttibutlon in Living cells. ME~NKOTH

Cyclic 22.

CHE~SKVD, FW.PH R, JONAK G: Sequence Requirements for Synthetic Peptide-mediated Translocation to the Nucleus. Mol Cell Biol 1989, 9~2487-2492.

MR. Swmo K& WAGNER JA, MANDEL G, GODDMAN RH: ldentitication of a Cyclic-AMP-Responsive Element Within the Rat Somatostatin Gene. Prcc Null Acad Sci USA 1986. 83:6682&86. 24. Go~~lu GA, MO~W MR: Cyclic AMP Stimulates Somato.. statin Gene Transcription by Phosphorylation of CREB at Wine 133. Cell 1989, 5967F680. CREB is phosphorylated in uiuo at Ser133 and mutations in this residue prevent activation of gene expression by the CA-PK pathway.



GROVEJR, AVRUCH J: Probing cAMP-Regulated Gene Expression with a Recombinant Protein Kinase Inhibitor. In The Hormonal Control Regulation of Gene Transcription edited by Cohen P, Foulkes JG [book]. Elsevier Science, 1991, in PWSS. Expression of a synthetic gene encoding the heat-stable inhibitor of C-subunit activity is used to demonstrate the role of the cA.MP.depen. dent protein kinase in both induced and basal gene transcription.

25. .


DAY RN, WAIDER JA, MAURER RA: Protein Kinase Inhibitor Gene Reduces Both Basal and Multihormone-Stimulated Prolactin Gene Transcription. J Biol them 1989, 264:431-i36.

AMP second


systems McKnight


MELLON PL Cl~cc CH, CORREUL4, McKNlcHT GS: Regulation of Transcription by Cyclic AMP-dependent Protein Kinase. Pm Natl Acud Sci USA 1989, 86:4887-@91.


DELEGEANE AM, FEIUAM) H, MELLON PL Tissue-specific Enhancer of the Human Glycoprotein Hormone a-Subunit Gene: Dependence on Cyclic AMP-inducible Elements. Mel Cell Biol 1987, 7:3994-4002.

~IACGR!XOR PF, ABATE C, CURRAN T: Direct Cloning of Leucine Zipper Proteins: Jun Binds Cooperatively to the CRE and CRE-BPl. Oncogene 1990, 5:451458. A direct interaction between Jun and a member of the CREB family of transcription factors is demonstrated. This heterodimer binds to CRE elements and may a&t gene transcription.

29. .

DM, JOMS NC: Heterodimer Formation Between CREB and Jun Proteins. Oncogene 1990, 5:295-302. Ly studies similar fo those in [29O], an association between CREB and cJun is demonstrated in uiuoakhough it appears that this complex does not activate gene transcription when it binds to the CRE. 30.


GS M&night, Department of Pharmacology, Universiry of Washington, Seattle, Washington 98195, USA


Cyclic AMP second messenger systems.

Cells carefully regulate the generation and destruction of cAMP using diverse families of adenylate cyclases and phosphodiesterases. Genes for several...
583KB Sizes 0 Downloads 0 Views