Title: “Tell Me Where Is Calcium Bred”: Clarifying the Roles of Nuclear Calcium
Abstract: The first European conference on Ca2+ signaling in the cell nucleus was held at Baia Paraellios, Calabria, Italy, during October 4–8, 1997. This conference was European only in name as the organizers (O. Bachs, E. Carafoli, P. Nicotera, and L. Santella) succeeded in assembling for the first time a group of distinguished international scientists holding divergent views for an enlightening dialog on the various aspects of Ca2+ signaling in the cell nucleus. Ca2+ signaling is an area of intense research, and the basic mechanisms of cytosolic Ca2+ signals are beginning to be understood. It is now accepted that calcium signals do exist in the cell nucleus. But this has not always been so evident. The nuclear envelope (NE) consists of an inner and an outer membrane separated by the perinuclear space. The perinuclear space is continuous with the lumen of the endoplasmic reticulum (ER). The inner and outer membranes join periodically at the nuclear pore complex (NPC). It has long been assumed that the NPC forms a large channel for the free diffusion of ions and macromolecules. In this view, the flow of calcium between nucleus and cytoplasm would appear unrestricted. This idea has been challenged over the years. One of the convincing arguments for autonomous regulation of nuclear Ca2+ was the finding that the nuclear membrane possesses a Ca2+-ATPase. Receptors for Ca2+ signaling molecules such as inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate have also been located on the nuclear membranes (14Humbert J.P Matter N Artault J.C Köppler P Malviya A.N Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signaling by inositol 1,4,5-trisphosphate.J. Biol. Chem. 1996; 271: 478-485Crossref PubMed Scopus (260) Google Scholar). Among various reasons for the controversy regarding nuclear Ca2+ homeostasis, the most prominent concerns the methods used for quantitating Ca2+ concentrations. Although our current understanding of cellular Ca2+ homeostasis has benefited considerably from the availability of Ca2+-sensitive fluorescent dyes, the use of these indicators in the case of the nucleus has posed problems. Some of these difficulties include dye compartmentalization and alteration in dye sensitivity due to binding with different proteins in various cell compartments. Potential artifacts have been identified using both conventional and confocal imaging techniques. The development of selectively targeted photoprotein aequorin derivatives, although providing an important new tool, has failed to resolve the question of whether nuclear Ca2+ is independently regulated from the cytosolic Ca2+. Despite these limitations, the field has benefited from the use of Ca2+-sensitive fluorescent indicators. In particular it has shifted the emphasis from the long-term determination of Ca2+ level to the study of short-term delays in the transmission of the cytosolic Ca2+ waves to the nucleus. Cytosolic Ca2+ waves depend upon the coordinated recruitment of elementary subcellular release units in order to spread across the entire cell. Individually, these elementary Ca2+ release events dissipate rapidly: they are of short duration and remain spatially restricted unless they become functionally coupled. But the interior of the nucleus is devoid of organelles that might serve to store and release Ca2+. This raises the question of the origin of nuclear Ca2+ signals. The use of high-speed confocal line-scanning microscopy revealed that nuclear Ca2+ waves emanate at the nucleus/cytosol border (8Fox J.L Burgstahler A.D Nathanson M.H Mechanism of long-range Ca2+ signaling in the nucleus of isolated rat hepatocytes.Biochem. J. 1997; 326: 491-495Crossref PubMed Scopus (35) Google Scholar). Thus, rather than allowing passive diffusion, the NE may in fact serve as a barrier (1Al-Mohanna F.A Caddy K.W.T Bolsover S.R The nucleus is insulated from large cytosolic calcium changes.Nature. 1994; 367: 745-750Crossref PubMed Scopus (227) Google Scholar) to cytosolic calcium changes and function as the Ca2+ storage site that sustains propagation of Ca2+ signals through the nucleoplasm. Ultimately, studies on nuclear Ca2+ become relevant only in the light of specific functions. Numerous Ca2+-binding proteins and Ca2+-regulated nuclear processes have been identified. These topics were debated during the Baia Paraellios Conference, including Ca2+ transporting system of the nuclear envelope, cross-talk between the cytosolic and nuclear Ca2+ pools, nuclear pore complexes, Ca2+ dependent nuclear functions, and the possible role of nuclear Ca2+ in pathology. Evidence in support of the independent regulation of nuclear Ca2+ has centered on the existence of a mechanism for generating Ca2+ signals in the nucleus. Inositol 1,4,5-trisphosphate receptor (IP3R), ryanodine receptor (RYR), and inositol 1,3,4,5-tetrakisphophate receptor (IP4R) have been identified on the nuclear membrane, confirming that the NE serves as a pool for Ca2+. In the cytosol, Ca2+ signals are produced by the release of Ca2+ from intracellular storage sites, mainly the ER, by the second messenger inositol 1,4,5-trisphosphate (InsP3). Ca2+ mobilization can also be mediated by another second messenger, cyclic adenosine diphosphate ribose (cADP ribose), acting upon a second class of intracellular ER release channels, the RYR. Similarly, it was established in 1990 that isolated nuclei are endowed with functional InsP3 receptors (23Malviya A.N Rogue P Vincendon G Stereospecific inositol 1,4,5-[32P] trisphosphate receptor-mediated calcium release from the nucleus.Proc. Natl. Acad. Sci. USA. 1990; 87: 9270-9274Crossref Scopus (170) Google Scholar). The introduction into the nucleus of IP3 or cADP-ribose evokes the release of Ca2+ accumulated into the NE, which is primarily directed toward the nucleoplasm (9Gerasimenko O.V Gerasimenko J.V Tepikin A.V Petersen O.H ATP-dependent accumulation and inositol trisphosphate or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope.Cell. 1995; 80: 439-444Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 12Hennager D.J Welsh M.J DeLisle S.J Changes in either cytosolic or nucleoplasmic inositol 1,4,5-trisphosphate levels can control nuclear calcium concentration.J. Biol. Chem. 1995; 270: 4959-4962Crossref Scopus (96) Google Scholar, 34Santella L Kyozuka K Effects of 1-methyladenine on nuclear Ca2+ transients and meiosis resumption in starfish oocytes are mimicked by the nuclear injection of inositol 1,4,5-trisphosphate and cADP-ribose.Cell Calcium. 1997; 22: 11-20Crossref Scopus (74) Google Scholar). Furthermore, IP3R and RYR have been located on the inner nuclear envelope (9Gerasimenko O.V Gerasimenko J.V Tepikin A.V Petersen O.H ATP-dependent accumulation and inositol trisphosphate or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope.Cell. 1995; 80: 439-444Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 14Humbert J.P Matter N Artault J.C Köppler P Malviya A.N Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signaling by inositol 1,4,5-trisphosphate.J. Biol. Chem. 1996; 271: 478-485Crossref PubMed Scopus (260) Google Scholar). The localization, though it may not be exclusive, also lends strength to the concept of an autonomous regulation of nuclear Ca2+ signals. In this scenario, IP3 or cADP ribose binding to their receptors on the inner nuclear membrane are expected to open the second messenger-regulated Ca2+-release channel causing the generation of nuclear Ca2+ signals into the nucleoplasm. This was the essence of the talks by A. N. Malviya (CNRS, Strasbourg), O. H. Petersen (Physiology, University of Liverpool), and L. Santella (Cell Biology, University of Naples). Santella presented data showing immunogold staining of RYRs both on the inner and outer nuclear membranes (34Santella L Kyozuka K Effects of 1-methyladenine on nuclear Ca2+ transients and meiosis resumption in starfish oocytes are mimicked by the nuclear injection of inositol 1,4,5-trisphosphate and cADP-ribose.Cell Calcium. 1997; 22: 11-20Crossref Scopus (74) Google Scholar). Also, based on patch-clamp studies, D. E. Clapham (Children's Hospital, Harvard Medical School, Boston) showed IP3Rs located on the outer nuclear membrane. Further support for the release of calcium from NE to nucleoplasm came from the presentation of N. Divecha (NCI, Amsterdam), who described the presence, inside the nucleus, of systems required for generating the second messenger IP3. In the cytosol, the generation of IP3 is the focal point for intracellular Ca2+ signaling. IP3 is produced by the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). Two interconnected pools of PIP2 have been identified in the nucleus, one probably present in the nuclear membrane and the other located internally, either as a part of a proteolipid complex, or as part of the recently identified invaginations of the nuclear envelope, which appear to invade the inner matrix of the nucleus. Other components required for driving the nuclear inositide cycle were shown to be present in the nucleus (6Divecha N Banfic H Irvine R.F Inositides and the nucleus and inositides in the nucleus.Cell. 1991; 74: 405-407Abstract Full Text PDF Scopus (207) Google Scholar), including phospholipase Cβ1, which can hydrolyze PIP2 to generate nuclear IP3 and diacylglycerol. How is the nuclear calcium pool filled? An ATP-mediated nuclear Ca2+-transporter (nuclear Ca2+-ATPase or NCA) was identified in 1982 by Kulikova (17Kulikova O.G Savostyanov G.A Belyavtseva L.M Razumovskaya N.I Study of ATPase activity and ATP-dependent accumulation of 45Ca2+ in skeletal muscle nuclei effects of denervation and electrical stimulation.Biokhimia. 1982; 47: 1216-1221Google Scholar) and confirmed by Orrenius (26Nicotera P McConkey D.J Jones D.P Orrenius S ATP stimulates Ca2+ uptake and incrases the free Ca2+ concentration in isolated rat liver nuclei.Proc. Natl. Acad. Sci. USA. 1989; 86: 453-457Crossref Scopus (256) Google Scholar). NCA is located on the outer nuclear membrane (14Humbert J.P Matter N Artault J.C Köppler P Malviya A.N Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signaling by inositol 1,4,5-trisphosphate.J. Biol. Chem. 1996; 271: 478-485Crossref PubMed Scopus (260) Google Scholar). The NCA is inhibited by thapsigargin and is insensitive to DBHQ (2,5-di(t-butyl)-1,4-benzohydroquinone), whereas the ER Ca2+ pump is sensitive to both thapsigargin and DBHQ. Whether this difference reflects the direct interaction of DBHQ with the pump or is related to the different lipid environment (ER and nucleus), is an open question (18Lanini L Bachs O Carafoli E The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum.J. Biol. Chem. 1992; 267: 11548-11552Abstract Full Text PDF Google Scholar). Malviya also presented data based on confocal microscopic observations and biochemical studies, showing that nuclear Ca2+ pump activity is stimulated by cyclic AMP–dependent protein kinase (PKA) phosphorylation. Malviya discussed yet another pathway for filling the nuclear calcium pool. Nuclear calcium uptake (16Köppler P Matter N Malviya A.N Evidence for stereospecific inositol 1,3,4,5-[3H]tetrakisphosphate binding sites on rat liver nuclei.J. Biol. Chem. 1993; 268: 26248-26252Abstract Full Text PDF Google Scholar) is mediated by inositol 1,3,4,5-tetrakisphosphate (IP4). The concentration of free calcium in the uptake medium bathing the nuclei determines the "choice" between ATP or IP4−mediated nuclear calcium entry. IP4−mediated nuclear calcium uptake becomes operative above 1 μM free calcium concentration in the uptake medium. IP4−mediated calcium uptake remains enigmatic, particularly its energetics. In the cytosol, the role of IP4 is also poorly understood. The nuclear IP4R is a 74 kDa protein that is totally different from other IP4R so far documented. Signal termination requires extrusion of Ca2+ out of the nucleoplasm. It was suggested that this may occur by exit through the NPC. E. Carafoli (Biochemistry, ETH, Zurich) expressed concern about nuclear pores being the exclusive way of getting calcium out of the nucleoplasm. An alternative mechanism may be the presence of Ca2+-ATPase on the inner nuclear membrane. Additional studies are required to settle this issue (33Santella L Carafoli E Calcium signaling in the cell nucleus.FASEB J. 1997; 11: 1091-1109Crossref Scopus (193) Google Scholar). The existence of NPCs perforating the two membranes of the NE raises the obvious question of the relationship between Ca2+ concentration changes in the cytosol ([Ca2+]c) and the nucleus ([Ca2+]n). Indeed, the large diameter of NPCs would suggest that cytosolic Ca2+ can diffuse freely into the nucleus, in particular during wave passing. However, differences between [Ca2+]c and [Ca2+]n have been observed, particularly after cell stimulation. Single cell imaging with Ca2+-sensitive fluorescent indicators employing conventional or confocal microscopy is widely used for measuring nucleo-cytoplasmic Ca2+ gradients. These dyes accumulate essentially in the cytosol, yet some of them do have access to the nucleus by diffusion through the NPC. Depending on the cell system studied, the method used, or the physiological state, the concentration of Ca2+ in the nucleus has been found to be lower, higher, or equal to that of the cytoplasm. S. Bolsover (Physiology, University College London) described that when cells loaded with fluorescent calcium indicators are stimulated, the amplitude of the resulting fluorescence change is often greater in the nucleus than in the cytosol. In contrast, B. Himpens (Physiology, University of Leuven) detected significant delays in the amplitude and duration of the nucleocytoplasmic calcium gradient (13Himpens B Smedt H Casteels R Relationship between [Ca2+] changes in nucleus and cytosol.Cell Calcium. 1994; 16: 239-246Crossref PubMed Scopus (49) Google Scholar). However, M. Bootman (Department of Zoology, University of Cambridge) indicated that global calcium waves may penetrate the nucleus without any appreciable delay. One reason for these discrepancies concerns the cell-specific nature of nuclear–cytoplasmic Ca2+ exchanges. The stage of cell development (i.e., proliferation or differentiation) may also play a role. But the main source of concern over the significance of the differences in nuclear and cytosolic fluorescence has centered mainly upon possible artifacts. Bolsover pointed out that, first, even if the degree to which the dye becomes calcium loaded is the same in both cytosol and nucleus, the signal may be larger in the nucleus because of a difference in the fluorescence properties of the indicator. Second, a similar calcium increase in the two locations may produce a greater loading of nuclear dye, due to a greater affinity of nuclear dye for calcium. And third, although the calcium concentration increase in the nucleoplasm and cytosol may be identical, the overall calcium change estimated by cytoplasmic dye may be smaller because the dye also determines the calcium concentration in membrane-bound organelles. These views were also shared by W. T. Mason (Neurobiology, Babraham Institute, Cambridge). The development of recombinant aequorin derivatives selectively targeted to the nucleus or to the cytosol by Pozzan and collaborators has provided an important new tool (3Brini M Murgia M Pasti L Picard D Pozzan T Rizzuto R Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin.EMBO J. 1993; 12: 4813-4819Crossref PubMed Scopus (175) Google Scholar). However, this technique has also produced conflicting data. Using aequorin targeted to the nucleus with parts of the glucocorticoid receptor sequence, R. Rizzuto (Biochemistry, University of Padova) found no delay in the transmission of cytosolic calcium signals to the nucleus. In contrast A. K. Campbell (Biochemistry, University of Wales, Cardiff) showed that under certain conditions significant rises in cytosolic Ca2+ do not result in the elevation of nuclear Ca2+ (2Badminton M.N Campbell A.K Rembold M Differential regulation of nuclear and cytosolic Ca2+ in HeLa cells.J. Biol. Chem. 1996; 271: 31210-31214Crossref Scopus (89) Google Scholar). Discussions among the participants led to the conclusion that both fluorescent indicators as well as photoprotein aequorin have proved inadequate in monitoring the cross-talk between cytosolic and nuclear calcium. Finally, everyone agreed that we need to develop an alternative method for monitoring calcium concentrations in various cell compartments. Hope was expressed that the newly introduced "cameleons" by the groups of Roger Tsien (25Miyawaki A Llopis J Heim R McCaffery J.M Adams J.A Ikura M Tsien R.Y Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.Nature. 1997; 388: 882-887Crossref PubMed Scopus (2613) Google Scholar) and Anthony Persechini (32Romoser V.A Hinkle P.M Persechini A Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence.J. Biol. Chem. 1997; 272: 13270-13274Crossref PubMed Scopus (345) Google Scholar) would allow significant steps forward to understand how the distribution of cytosolic calcium changes influences the amplitude and duration of nuclear calcium. Results from electrophysiological studies argue more consistently against the idea of unrestricted diffusion of Ca2+ between the cytosol and the nucleoplasm. This was first suggested by the work of Loewenstein and Kanno in 1963 indicating the existence of ionic gradients across the NE. More recent studies, based on patch-clamping of isolated nuclei, also suggested that the NE is not freely permeable to small ionic species. Patch-clamping of NPCs is very different in principle from the much more established work on cell surface membranes (normal ion channels span one membrane whereas the NPCs span two). The observed ionic conductances are attributed to NPCs. Patch-clamping data, discussed by J. Bustamante (Physiology, University of Sao Paulo), Clapham, and Santella, showed giga-ohm resistance, i.e., absolute sealing of the envelope. Clearly, unless one claims that the patch-clamping procedure had altered the permeability of the NPC, sealing them tightly, the electrophysiological data are incompatible with the concept of free Ca2+ permeability of the pores. Bustamante also presented data showing opening and closing of pores resembling the ion channel conductances seen in mitochondria, ER, or SR. Another important parameter that has emerged in recent studies concerns the distance from the NE at which cytosolic calcium signals can influence nuclear calcium. Bootman, based on studies on HeLa cells, showed that cytosolic Ca2+ puffs generated at a mean distance of 4–6 μm from the NE were almost instantly transmitted to the nucleus, whereas Ca2+ puffs produced at distance greater than 6 μm from the nucleus were not transmitted (20Lipp P Thomas D Berridge M.J Bootman M.D Nuclear calcium signaling by individual cytoplasmic calcium puffs.EMBO J. 1997; 16: 7166-7173Crossref Scopus (160) Google Scholar). In the case of pancreatic acinar cells, Petersen showed that calcium signals are generated far away from the nucleus and therefore Ca2+ rises in the cytosol are not transmitted to the nucleoplasm. The spatial organization of ER and NE Ca2+ stores was also addressed. Very recently37Subramanian K Meyer T Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores.Cell. 1997; 89: 963-971Abstract Full Text Full Text PDF Scopus (195) Google Scholar distinguished between various possible topologies of ER and NE Ca2+ stores and demonstrated that the lumen of ER and NE of resting cells is a continuous space not compartmentalized by mechanical barriers, indicating that free calcium concentration in the lumen of ER and NE can equilibrate throughout the cell by rapid diffusion. Based upon these findings, Petersen described experiments, using Ca2+-sensitive dyes in intact pancreatic acinar cells, aimed at characterizing the mechanism by which the concentration of Ca2+ inside the NE store is regulated. The Ca2+ concentration in the ER lumen ([Ca2+]L) was in the range 100–300 μM under basal conditions. Stimulation with acetylcholine (ACh) resulted in a rapid loss of Ca2+ from the store, and removal of ACh allowed reuptake of Ca2+ into the ER. Simultaneous recordings of the cytosolic Ca2+ concentration ([Ca2+]i) indicated that removal of ACh led to a rapid return of [Ca2+]i to the normal resting value at a time when [Ca2+]L was still far from having been fully restored. This means that a major part of the Ca2+ refilling of the ER/NE store occurs at the normal resting [Ca2+]i. It was also shown that the rate of Ca2+ reuptake into the store is a function of [Ca2+]L (when [Ca2+]L increases, the Ca2+ reuptake rate decreases). As the ER and NE lumen equilibrate quickly, the Ca2+ concentration inside the NE is therefore also regulated by a negative feedback exerted on the inside of the ER membrane. It is not known whether the lumen of the ER and NE Ca2+ stores are subcompartmentalized or whether the lumenal connectivity within and between the ER and NE could be regulated by particular signaling processes. Evidently, the NPCs are central to the transport of Ca2+ in and out of the nucleus (28Panté N Aebi U Towards the molecular dissection of protein import into nuclei.Curr. Opin. Cell. Biol. 1996; 8: 397-406Crossref Scopus (99) Google Scholar). So their 3-D architecture (28Panté N Aebi U Towards the molecular dissection of protein import into nuclei.Curr. Opin. Cell. Biol. 1996; 8: 397-406Crossref Scopus (99) Google Scholar) and their permeability were discussed in great detail in a number of presentations, including the EMBO Lecture delivered by U. Aebi (Biozentrum, Basel). The NPC reveals a tripartite architecture consisting of a basic framework made of eight multidomain spokes embracing a central pore, which is sandwiched between a cytoplasmic ring, from which eight kinked fibrils radiate, and a nuclear ring, capped with a basket assembled from eight filaments joined distally into a 30–40 nm diameter terminal ring. The central pore harbors a gated channel, which has also been called a "plug" or "transporter." The nature of the central plug depicted in electron microscopy-based projection images of the NPC remains unknown. Whether it represents the central channel that is gated, or whether it is merely transported material in transit is a subject of ongoing debate. According to Aebi, contribution to the mass of this central plug stems from the terminal ring of the nuclear basket which, in projection, superimposes onto the central pore. Furthermore, by EM one can often see through the central pore into the nuclear basket when visualizing the NPC from its cytoplasmic face. The highlight of Aebi's presentation was recent work from his laboratory concerning the regulation of NPC permeability by Ca2+. Atomic force microscopic (AFM) imaging of NPCs prepared under conditions that minimize denaturation using physiological buffers, reveals a distinct asymmetry of the two surfaces with a "dome-like" appearance of the nuclear face and a cytoplasmic side characterized by a "donut-like" aspect with a pronounced central pore. The AFM appearance of these unfixed NPCs kept "alive" in physiological buffers was modulated by Ca2+, showing a reversible Ca2+-mediated opening and closing of the nuclear baskets. Similar data were presented by Clapham showing that permeability of the NPC is modulated by the calcium pool in the NE lumen (29Perez-Terzic C Pyle J Jaconi M Stehno-Bittel L Clapham D.E Conformational states of nuclear pore complex induced by depletion of nuclear Ca2+ stores.Science. 1996; 273: 1875-1877Crossref PubMed Scopus (163) Google Scholar). H. Oberleithner (Physiology, University of Würzburg) also described AFM imaging of NPCs, showing dramatic changes in their structure in response to ATP and Ca2+. L. Vann (Cell Biology and Anatomy, Johns Hopkins University, Baltimore) presented results involving Xenopus egg extracts, showing that the assembly of NPCs was blocked by the Ca2+ chelator BAPTA. The study of nuclear Ca2+ derives its importance from the study of Ca2+-regulated processes in the nucleus. Three major Ca2+-dependent nuclear functions were discussed at this meeting, the regulation of gene transcription, protein import, and apoptosis. Evidence is accumulating that gene expression can be influenced not only by cytoplasmic Ca2+ but also by nuclear Ca2+. Numerous Ca2+-binding proteins have been identified in the nucleus, and prominent among these is calmodulin. A. R. Means (Pharmacology and Cancer Biology, Duke University, Durham) reminded the audience that just a few years ago the very existence of a nuclear calmodulin pool was vigorously debated, whereas now it has become a central theme of nuclear signal transduction. Other members of the EF-hand family of calcium-binding proteins such as calreticulin (4Camacho P Lechleiter J.D Calreticulin inhibits repetitive intracellular Ca2+ waves.Cell. 1995; 82: 765-771Abstract Full Text PDF Scopus (199) Google Scholar) have been localized to the cell nucleus. Calbindin-D28k has also been found in nerve cell nuclei. Calbindin-D28k does not possess a classical nuclear localization sequence (NLS). However, its molecular mass is 28 kDa, and most proteins that are imported into the nucleus through NLS-dependent mechanisms are larger than 30 kDa. Thus, it may use an alternative route to enter the nucleus, such as the pathway regulated by intralumenal calcium that operates for relatively small proteins. Nucleolin is yet another Ca2+-binding multifunctional protein that is subject to regulation by nuclear Ca2+, as was discussed by B. Abrenica (Faculty of Dentistry, University of Manitoba, Winnipeg). The nature of the targets for Ca2+ and calmodulin in the nucleus was also adressed. In the central nervous system (CNS), one of these targets is the transcription factor CREB (CRE-binding protein). CREB is phosphorylated on a specific residue (Ser133) within 1 min after Ca2+ entry by synaptic activation of N-methyl-D-aspartate (NMDA) receptors and L-type Ca2+-channels. K. Deisseroth (Molecular and Cell Physiology, University of Stanford) described experiments showing that under these conditions calmodulin is the messenger that carries the Ca2+ signal into the nucleus (5Deisseroth K Bito H Tsien R.W Signaling from synapse to nucleus postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity.Neuron. 1996; 16: 89-101Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar). Nuclear calmodulin can be a limiting factor in CREB-Ser133 phosphorylation (CREB is also phosphorylated on Ser133 in response to increased levels of cAMP). There are multiple CREB-kinase candidates, one of which is Ca2+/calmodulin-dependent protein kinase (CaM kinase). Specific isoforms of CaM kinase have been localized in the nucleus, including type II and type IV. It has been shown that the δB isoform of CaM kinase II is targeted to the nucleus via an NLS (35Srinivasan M Edman C.F Schulman H Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus.J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (235) Google Scholar). It was initially thought that both nuclear isoforms of CaM kinase were mediators of CREB Ser133 phosphorylation. However, recent data indicate that CaM kinase IV only phosphorylates the activating Ser133, whereas CaM kinase II also phosphorylates inhibitory Ser142. In the CNS, nuclear calmodulin appears essential in the rapid synaptic control of CaM kinase-dependent CREB phosphorylation. The complexity of the regulation of CREB phosphorylation is further illustrated by the discovery that CaM kinase activity is not dependent solely on Ca2+ and calmodulin. Several groups have identified factor(s) in brain extracts that stimulate CaM kinase I and IV. This has led to the discovery of a CaM kinase kinase (CaMKKα) that phosphorylates CaM kinase on the activation loop. Means described a novel CaMKK, referred to as CaMKKβ, which activates CaM kinase IV. The activity of the CaMKKs is itself Ca2+- and calmodulin-dependent. CaMKKβ is also activated by intramolecular autophosphorylation. CaM kinase IV can phosphorylate members of the CREB/ATF family of transcription factors other than CREB, namely CREMτ (during spermatogenesis). Another example is provided by the CCAAT enhancer element-binding protein β (C/EBPβ). There is evidence that nuclear CaM kinase can phosphorylate C/EBPβ at a site within its leucine zipper, which results in potentiation of its transcriptional activity. CaM kinase phosphorylation may regulate the CREB-mediated transcription response through modulation of differential dimerization. An added level of complexity is provided by the transcriptonal coactivator proteins CBP or its close relative p300. Indeed, upon phosphorylation on Ser133, CREB fixes CBP/p300. Other Ca2+-regulated serine/threonine kinases have been identified in the nucleus. For instance, rat liver nuclei contain PKC type β, which is a Ca2+-dependent isoform (31Rogue P Labourdette G Masmoudi A Yoshida Y Huang F.L Huang K.-P Zwiller J Vincendon G Malviya A.N Rat liver nuclei protein kinase C is the isozyme type II.J. Biol. Chem. 1990; 265: 4161-4165Abstract Full Text PDF Google Scholar). T. Millward (FMI, Basel) described Ndr, a novel serine/threonine kinase activated not only by calmodulin but by protein S100B. Ndr kinase is a member of a subfa