Interactive Fly, Drosophila

Calcineurin: a Calmodulin regulated phosphatase

Animals sense and adapt to variable environments by regulating appropriate sensory signal transduction pathways. Calcineurin (see Drosophila Calcinerin plays a key role in regulating the gain of sensory neuron responsiveness across multiple modalities. C. elegans animals bearing a loss-of-function mutation in TAX-6, a calcineurin A subunit, exhibit pleiotropic abnormalities, including many aberrant sensory behaviors. The tax-6 mutant defect in thermosensation is consistent with hyperactivation of the AFD thermosensory neurons. Conversely, constitutive activation of TAX-6 causes a behavioral phenotype consistent with inactivation of AFD neurons. In olfactory neurons, the impaired olfactory response of tax-6 mutants to an AWC-sensed odorant is caused by hyperadaptation, which is suppressible by a mutation causing defective olfactory adaptation. Taken together, these results suggest that stimulus-evoked calcium entry activates calcineurin, which in turn negatively regulates multiple aspects of sensory signaling (Kuhara, 2002).

Two types of cation channels, TAX-4/TAX-2 and OSM-9, are expressed in the AWC olfactory neurons. Genetic analyses suggest that odor sensing activates primary olfactory transduction through the TAX-4/TAX-2 channel, which allows calcium entry to activate AWC neurons, whereas odor-provoked calcium influx through the OSM-9 channel only affects olfactory adaptation. tax-6 animals are hyperadaptable to AWC-sensed isoamyl alcohol. Exposure to isoamyl alcohol for only 10 min is sufficient for tax-6 animals to adapt. This hyperadaptable phenotype and the partially defective olfactory response of tax-6 mutants to isoamyl alcohol are both completely suppressed by an osm-9 mutation. These results suggest that TAX-6 represses OSM-9-dependent olfactory adaptation in AWC. Taken together, two possible models are proposed for the role of TAX-6 in AWC signaling. TAX-6 could be activated by calcium entry through the primary signal transduction channel TAX-4/TAX-2 upon activation of the odorant (IAA) receptor, and the activated TAX-6 could inhibit the adaptation machinery. Alternatively, TAX-6 could be activated by the odorant (IAA)-evoked calcium influx through the OSM-9 channel, and the activated TAX-6 could negatively regulate opening of the OSM-9 channel that is required for isoamyl alcohol adaptation (Kuhara, 2002).

These models on the role of TAX-6 as a negative regulator for OSM-9-dependent olfactory adaptation in AWC might paradoxically imply that TAX-6 could be a positive regulator for TAX-4/TAX-2-dependent primary sensory signaling. If TAX-6 is a direct positive regulator of AWC primary transduction, at least partially defective olfactory responses to isoamyl alcohol could be expected in osm-9 tax-6 double mutants. It was found, however, that osm-9 tax-6 mutants show completely normal olfactory response to isoamyl alcohol. This result argues against a direct positive role of TAX-6 in AWC primary signaling. The results on osm-9 tax-6 mutants are also inconsistent with a direct negative regulatory role of TAX-6 in AWC primary signaling. If that were true, hyperattractive olfactory responses to isoamyl alcohol would be seen in osm-9 tax-6 mutants (Kuhara, 2002).

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. Interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390. Selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bind 45Ca2+ normally. Mutants BE-2 and BQ-2 interact with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390); they stimulate the phosphatase activity of the CaN A subunit. Mutant BE-1 has a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gives very little stimulation of CaN A phosphatase activity. It is concluded that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects of B subunit Ca2+ binding in stimulation of CaN A phosphatase activity (Watanabe, 1996).

The PMC1 gene in S. cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work has shown that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. Calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter is slightly diminished and a pmc1::lacZ reporter is induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. It is proposed that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions (Cunningham, 1996).

S. cerevisiae mutants that exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were classified into four complementation groups (crv1,2,3 and 4). Crv1 is allelic to cnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences of mutant crv2 are identical to those of BCK1/SLK1/SKC1/SSP31 and the sequences of mutant crv3 match those of MPK1/SLT2; both genes are involved in the MAP kinase cascade. A calcineurin-deletion mutation (delta cnb1), which by itself has no detectable effect on growth and morphology, enhances some phenotypes (slow growth and morphological abnormality) of crv2 and crv3 mutants. The phenotypes of crv2 and crv3 mutants are partially suppressed by Ca2+ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant, crv2 and crv3 mutants are defective in recovery from alpha-factor-induced growth arrest. The defect in recovery of the delta cnb1 mutant is suppressed by overexpression of MPK1. These results indicate that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth (Nakamura, 1996).

The catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) was isolated from Dictyostelium discoideum. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shows similar enzymatic properties as does the enzyme from other sources. A band of approximately 80 kDa migrates on gels and possesses an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed (Dammann, 1996).

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. Soluble rat brain phosphatases have been identified that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity have been observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A. The two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each is relatively insensitive to okadaic acid, is active only in the presence of CaCl2 and calmodulin, and contains a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcineurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A, or with cyclosporin A to inhibit calcineurin, increases apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites. These results indicate that both phosphatase 2A and calcineurin dephosphorylate sodium channels in rat brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity (Chen, 1995).

The M current regulates neuronal excitability, with its amplitude resulting from high open probability modal M channel behavior. The M current is affected by changes in intracellular calcium levels. It is proposed that internal calcium acts by regulating M channel modal gating. Intracellular application of a preactivated form of the calcium-dependent phosphatase calcineurin (CaN420) inhibits the macroscopic M current, while its application to excised inside-out patches reduces high open probability M channel activity. Addition of ATP reverses the action of CaN420 on excised patches. The change in M channel gating induced by CaN420 is different from the effect of muscarine. A kinetic model supports the proposition that shifts in channel gating induced by calcium-dependent phosphorylation and dephosphorylation control M current amplitude (Marrion, 1996).

Embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) were prepared to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appears to be normal in CNA alpha -/- mice. CNA alpha -/- T cells respond normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts have a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remain sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. Thus CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA (B. W. Zhang, 1996).

The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocks the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression are inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation does not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) is not inhibited by in vivo treatment with FK506 and that there is no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. Different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes (Wang, C. R., 1995).

A new facet of calcium signaling involves the nuclear import of the NF-AT transcription factors from their dormant position in the cytoplasm. The protein phosphatase calcineurin appears to play an essential role in activating NF-AT nuclear import, as the calcineurin inhibitors cyclosporin A and FK506 block dephosphorylation and nuclear import of NF-AT. Calcium signaling induces an association between NF-AT4 and calcineurin; these molecules are transported, as a complex, to the nucleus, where calcineurin continues to dephosphorylate NF-AT4. It is proposed that a nuclear complex of NF-AT4 and calcineurin maintains calcium signaling by counteracting a vigorous nuclear NF-AT kinase (Shibasaki, 1996).

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. Two new isoforms of the transcription factor NFAT1 are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol ester; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response (Luo, 1996).

Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters (Chin, 1998).

The myoglobin (Mb) and troponin I slow (TnIs) genes are expressed selectively in slow, oxidative skeletal muscle fibers, whereas the muscle creatine kinase (MCK) gene is expressed most abundantly in the fast, glycolytic myofiber subtype. To test whether these genes might respond differently to a calcineurin-stimulated signaling pathway, skeletal myogenic cells were transfected with reporter genes linked to well-characterized control regions from these genes, along with an expression vector encoding a constitutively active (calcium-insensitive) form of calcineurin that retains sensitivity to inhibition by cyclosporin A. Transcriptional activity of the slow-fiber-specific myoglobin and TnIs promoters is stimulated in cultured skeletal myotubes (C2C12) by active calcineurin, as measured by expression of luciferasein cotransfection assays. In contrast, activity of the fast-fiber-specific MCK promoter, or of other strong (CMV) or weak (minimal TATA element) promoters, is unaffected by activated calcineurin. The induction of the myoglobin promoter in the presence of the calcineurin expression plasmid is inhibited by cyclosporin A. This result indicates the specificity of the response, since the effect of cyclosporin A is to bind cyclophilin and form a protein complex that binds calcineurin and inhibits its protein phosphatase activity. The same relative potency of calcineurin-dependent transactivation (myoglobin and TnIs is much more potent than MCK, CMV, or TATA promoters) is observed in Sol8 myotubes, a different myogenic cell line. In contrast, forced expression of activated calcineurin had no effect on promoter activity in undifferentiated myoblasts or in 3T3 fibroblasts, demonstrating a requirement for muscle-specific factors in the calcineurin-stimulated pathway for transcriptional control of the myoglobin and TnIs promoters. Inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation (Chin, 1998).

Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. The finding that the myoglobin and TnIs promoters can be transcriptionally regulated by a calcineurin-dependent mechanism suggests the participation of NFAT transcription factors in the signaling cascade. Examination of the complete nucleotide sequences of these functionally defined transcriptional control regions (2.0 and 4.2 kb, respectively) reveals two 8-bp elements within each that match the consensus-binding sequence for NFAT transcription factors. The response to activated calcineurin of the native promoter sequences was compared to that of mutated promoters in which these putative NFAT recognition elements were ablated by site-directed mutagenesis. Disruption of putative NFAT recognition elements within both the myoglobin and TnIs promoters diminishes the response to activated calcineurin, indicating that the transactivation mechanism is likely to involve DNA binding of NFAT proteins. Transduction of the calcineurin-directed signal to the native myoglobin and TnIs promoters exhibits a saturable dose-response relationship with respect to the activated calcineurin expression plasmid; diminished reporter gene activation was evident across the entire dose range examined. Some degree of calcineurin-dependent transactivation persists after ablation of identifiable NFAT binding sites within these transcriptional control regions. Thus, either cryptic binding sites for NFAT proteins that cannot be recognized by inspection of the DNA sequence are present, or calcineurin-dependent signaling to these promoters can be driven without direct DNA binding of NFAT proteins. Nuclear localization of NFAT proteins in skeletal myocytes is under the control of calcineurin. These results identify a molecular mechanism by which different patterns of motor nerve activity promotes selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers (Chin, 1998).

Using a genetic screen in yeast, a new family of proteins conserved in fungi and animals has been identified that inhibits calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibits two independent functions of calcineurin in yeast -- the activation of the transcription factor Tcn1p and the inhibition of the H+/Ca2+ exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 binds calcineurin in vitro and inhibits its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induces Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibit phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome (Kingsbury, 2000).

Whole-cell recording in the superficial layers of the developing superior colliculus (sSC) reveals a large drop in NMDA receptor (NMDAR) current decay time synchronized across all neurons and occurring consistently between post-natal (P) day 10 and P11. Blocking the Ca2+/calmodulin-dependent phosphatase calcineurin (CaN) in the postsynaptic neuron can abolish this drop. The regulation is induced prematurely by 1-2 hr of electrical stimulation in P10 collicular slices only if CaN and NMDAR currents can be activated in the neuron. These data suggest that a long-lasting, CaN-mediated control of NMDAR kinetics is rapidly initiated by heightened activity of the NMDAR itself and demonstrate a novel developmental and tonic function of CaN that can play an important role in modulating the plasticity of the developing CNS (Shi, 2000).

It is likely that this developmental regulation of the NMDAR serves functions that are qualitatively different from desensitization in more mature brain. For example, the CaN effect on NMDARCs described here is large. During the synaptogenic period from P6-P21, NMDARC decay time decreases by ~18 ms. Close to half of this decrease is caused by the sudden appearance of CaN activity at P11. By the end of the following week, the slower incorporation of the NR2A subunit into sSC synaptic NMDARs could be responsible for the additional shortening of the NMDARC decay time. Rat eyes open on P13-P14, and the sudden increase in visual activity resulting from pattern visual could readily damage collicular neurons if the mechanism of CaN-dependent NMDARC downregulation did not exist. In addition, the rapid downregulation of NMDARC decay time on P11 probably reduces much of the amplification of synaptic function caused by NMDAR activity. This loss of amplification may account for the sustained decreases in spontaneous spiking activity reported in the sSC after P10 by other investigators, and it may also serve to abruptly limit ongoing synaptic plasticity in the sSC. In short, this developmental function of CaN appears to be an exceptionally rapid and potent homeostatic mechanism that uses an increase in Ca2+ through the NMDAR channel to tonically decrease the potency of the NMDAR posttranslationally. This could maintain a nontoxic level of intracellular free Ca2+ in the face of a sudden increase in the activity arriving at collicular neurons. It is likely that in the visual system the increase in CaN activity results from the first powerful activation of the central visual pathway by light. It is also possible that similar sudden increases in activity occur in other regions of the nervous system. This activity-dependent, tonic, CaN-mediated control system may be broadly distributed within the vertebrate CNS (Shi, 2000).

A final significant property of the developmental change in CaN activity reported here is that it is not associated with changes in total CaN protein levels. The only event necessary to initiate phosphatase activity may therefore be the activation exerted by the NMDAR itself. Nevertheless, CaN is an elaborately regulated enzyme. Thus, changes in the activity or the synaptic localization of AKAP proteins, FKBP or cyclophorin family members, DARPP-32, CHP, or Cabin 1 may be mediating an additional level of control. Alternatively, or in addition, a prolonged depression of kinase activity could contribute to both the onset and stability of the CaN effect in sSC NMDARCs. The prolonged CaN activation at sSC synapses may also arise from a fundamentally different mechanism, such as protection of the phosphatases Fe-Zn active center from oxidation by superoxide dismutase. Regardless of the precise mechanism through which the prolongation of synaptic CaN activity is exerted, these developmental data suggest an interaction that may be retained in the mature brain. Prolonged activation of synaptic CaN could protect neurons from excitotoxicity in the face of seizure, ischemia, trauma, or disease-induced tonic increases in NMDAR activity. Thus, interventions that amplify or maintain this response may prove useful in the clinical treatment of a variety of neurological dysfunctions (Shi, 2000).

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV). Skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occur despite the fact that CnA* muscles display threefold higher calcineurin activity and enhance dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events (Dunn, 2000).

Regarding the potential identity of contractile activity-dependent signal transduction events, there is mounting evidence that calcineurin must interact with parallel calcium-sensitive signaling pathways in order to fully activate downstream target genes. For instance, calcineurin synergizes with phorbol ester-dependent pathways to stimulate the IL-2 promoter in T lymphocytes and the expression of atrial natriuretic factor in cardiomyocytes. Similarly, calcineurin acts in conjunction with CaM-dependent kinase IV to fully activate the myoglobin promoter in cultured skeletal myocytes and the Nur77 promoter in T lymphocytes. Moreover, retroviral-mediated gene transfer of CnA* induces skeletal myogenesis in vitro only in the presence of extracellular Ca2+. Additionally, there is evidence that MAP kinase pathways are activated in response to increased contractile activity and play a role in regulation of the slow fiber phenotype. In this context, MEF2 is an enticing candidate as an integrator of calcineurin and other activation-linked signal transduction pathways, since this transcription factor is both dephosphorylated by calcineurin and phosphorylated by various CaM kinases, ERK5, p38, and PKC (Dunn, 2000 and references therein).

An alternative possibility is that calcineurin signaling may converge with other activity-linked pathways via the association of GATA with NFAT. Indeed, activation of calcineurin promotes the association of these two transcription factors via the dephosphorylation of NFATc1 and increased expression of GATA-2 under conditions of skeletal myocyte growth. Consistent with findings from hypertrophic myocytes, this protein is upregulated in the plantaris in response to muscle overload, but not lowered by CsA treatment, suggesting that this transcription factor may be important for growth but not necessarily a gene target of calcineurin. The fact that GATA is also known to associate with MEF2, and that fiber hypertrophy is observed only when NFATc1 and MEF2 are dephosphorylated and GATA-2 increases, leads to the idea that NFAT, MEF2, and GATA proteins act in synergy to transactivate target genes that lead to fiber growth in response to OV. Future studies should help identify the particular permutations of these transcription factors involved in the activation of slow fiber-specific genes versus those modulating adult fiber size (Dunn, 2000 and references therein).

The threshold for hippocampal-dependent synaptic plasticity and memory storage is thought to be determined by the balance between protein phosphorylation and dephosphorylation mediated by the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance, the effect of genetically inhibiting calcineurin on plasticity and memory was examined. Using the doxycycline-dependent rtTA system to express a calcineurin inhibitor reversibly in the mouse brain, it has been found that the transient reduction of calcineurin activity facilitates LTP in vitro and in vivo. This facilitation is PKA dependent and persists over several days in vivo. It is accompanied by enhanced learning and strengthened short- and long-term memory in several hippocampal-dependent spatial and nonspatial tasks. The LTP and memory improvements are reversed fully by suppression of transgene expression. These results demonstrate that endogenous calcineurin constrains LTP and memory (Malleret, 2001).

The main finding of this study is that the regulated inhibition of the phosphatase CN leads to enhanced LTP both in vitro and in vivo, and to improved learning and memory storage. The parallel in time course of the increased persistence of LTP in awake animals and of the memory improvement strongly suggest a correlation between the duration of LTP and memory storage. Improved cognitive performance was observed both on spatial and nonspatial hippocampal-dependent tasks, consistent with the multipurpose role of the hippocampus in human declarative memory. Moreover, with different tasks, different temporal components of memory were improved. Thus, the complex object recognition task, involving brief training sessions, multiple objects, spatial transfer, and object change, elicit a weak form of memory that is strengthened at early and intermediate time points by the CN inhibitor, but does not persist longer in mutants than in controls. By contrast, a more robust form of memory elicited by a more intense training is maintained and persists for over a week longer in mutants expressing the CN inhibitor when compared to controls (Malleret, 2001).

Facilitated learning and memory was also observed in the Morris water maze and was evident not only with traditional measurements of spatial performance, such as escape latency, but also with more specific aspects of performance such as the precision of navigation. These measures suggest that mutant mice expressing the CN inhibitor retain spatial information more efficiently than controls. The persistent memory for the first platform position associated with the efficient learning of a second platform position suggests an overall enhanced capacity for memory storage with the CN inhibitor. Further, the rapid adaptation to spatial changes observed in mutants expressing the CN inhibitor on both the Morris water maze and the object exploration task suggest increased cognitive flexibility, a process that depends on the hippocampus (Malleret, 2001).

One of the molecular mechanisms allowing transmitted signals to persist or decay is thought to be the balance between phosphatase and kinase activity. Much evidence suggests that PKA and CN specifically regulate this balance and thereby serve as a gate for LTP. In the current study, evidence in support of this model is provided by demonstrating that shifting the endogenous balance away from calcineurin activity positively modulates synaptic plasticity in a PKA-dependent manner. Further, the data indicate that altering CN activity transiently in the adult brain is sufficient to positively or negatively control synaptic plasticity and memory storage. The effects observed suggest that CN is essential both for early events of plasticity and memory and for downstream pathways that contribute to persistent changes in plasticity and memory storage (Malleret, 2001).

Mechanistically, early and transient forms of plasticity and memory are known to rely on the covalent modification of pre-existing proteins while long-term forms require activation of transcription factors such as CREB, and protein synthesis. One possible mechanism for the facilitatory effect of the CN inhibitor may be a decrease in the activity of PP1, a protein phosphatase positively regulated by CN through dephosphorylation of inhibitor-1 (I-1). PP1 inhibition has been shown to promote the induction of LTP, whereas increased PP1 activity, produced by genetic suppression of I-1, has been shown to affect certain forms of LTP in some hippocampal regions. Since PP1 is effective in modulating CaMKII, a kinase critical for the transmission of postsynaptic signals required for the induction of LTP, it is possible that increased CaMKII activity mediated by lower PP1 activity facilitates the induction of LTP. Raising the signal for the induction of LTP, through genetic upregulation of NMDA-R function, has been shown to enhance LTP, learning, and memory. The findings suggest that LTP and memory enhancements can be similarly achieved by relieving a constraint downstream of the NMDA-R and that this constraint is exercised by CN (Malleret, 2001).

The effect of the CN inhibitor on long-lasting changes in plasticity and memory may be mediated by modulation of transcriptional control. Thus, the prolonged maintenance of LTP and of memory may arise from augmented CREB transcriptional activity via reduced CREB dephosphorylation by PP1. In this context, it is important to note that unlike the phenotypes observed in Drosophila mutants expressing active CREB, the CN inhibitor does not convert labile memory into long-lasting memory. It is, however, able to strengthen or prolong different phases of memory, suggesting that CN inhibition modulates rather than mediates memory processes (Malleret, 2001).

The PKA and CN pathways may also interact antagonistically at sites other than CREB. For example, CN can inhibit specific isoforms of adenylyl cyclase required for PKA activation. Similarly, PKA and CN can regulate, in opposite ways, phosphorylation sites on key proteins in synaptic transmission, such as the NMDA-R or AMPA receptor. The effect of inhibiting CN may also occur through processes additional to or independent of the cAMP pathway. For instance, CN inhibitor may modulate Ca2+-dependent kinases such as CaMKII or PKC through control of intracellular Ca2+ mobilization by regulation of inositol 1,4,5-triphosphate receptors. Finally, the PKA/CN gate most likely represents only one of several activator/suppressor mechanisms regulating plasticity and memory (Malleret, 2001).

Several genetic approaches have been used to study the molecular mechanisms of hippocampal functions such as memory. Standard genetic techniques, however, have suffered from the limitation that the genetic modification is permanent. Here, the usefulness of the rtTA system for such studies has been demonstrated by showing that inducible and reversible transgene expression allows temporary improvement of complex cognitive functions and of brain plasticity. The ability to achieve such reversible improvements in the adult animal demonstrates that no permanent changes in neuronal circuits are involved and that the effects result specifically from molecular and biochemical changes elicited by a reduction in CN activity. In this context, the rtTA system could be further exploited to assess the timing of the requirement of CN in such processes, for instance in various stages of memory storage such as memory consolidation or retrieval since CN has been suggested to be involved in both processes. Transgene expression was found in several brain structures and, therefore, the contribution of other structures, in addition to the hippocampus, to the enhancement of long-term memory, cannot be excluded. Overall, however, these results may provide a clear target for potential treatment of learning and memory disorders (Malleret, 2001).

Calmodulin and the myristoylated alanine-rich C kinase substrate (MARCKS)

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, the gene was disrupted in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. It is concluded that MARCKS is necessary for normal mouse brain development and postnatal survival (Stumpo, 1995).

MARCKS and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin regulated by PKC phosphorylation. The kinetics of PKC-mediated phosphorylation and the calmodulin binding properties of intact, recombinant MARCKS and MRP were investigated and compared with previous studies of synthetic peptides spanning the PKC phosphorylation site/calmodulin binding domains (PSCBD) of these proteins. Both MARCKS and MRP were high affinity substrates for the catalytic fragment of PKC, and their phosphorylation occurs with positive cooperativity. Affinities are similar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of MRP and its synthetic PSCBD peptide yield identical patterns of tryptic phosphopeptides; as in the case of MARCKS, all of the PKC phosphorylation sites in MRP lie within the 24-amino acid PSCBD. Sequence analysis of tryptic phosphopeptides reveals that the first and third (but not the second) serines in the MRP PSCBD are phosphorylated by PKC. Both MARCKS and MRP bind dansyl-calmodulin with high affinity, with a Kapp of 4.6 and 9.5 nM, respectively. Phosphorylation of MARCKS and MRP by PKC disrupt the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic PSCBD peptides with respect to PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties (Verghese, 1994).

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), have shown that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. The kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique is described in this study. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is ~10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin decreases markedly the lifetime of the peptide on vesicles. These results suggest that Ca2+-CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. It is thought that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175) (Arbuzova,1997).

Ionomycin stimulates membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator phorbol ester. However, while phorbol ester (as expected) powerfully stimulates the phosphorylation of the PKCs' 85-kDa MARCKS protein, ionomycin unexpectedly does not. Instead, ionomycin reduces the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevents phorbol-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin requires external Ca2+ because they are both abolished by removing Ca2+ from the culture medium. It is thought that Ca2+/calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes that have been stimulated by raising the external Ca2+ concentration. In the present experiments, calmodulin prevents MARCKS phosphorylation by phorbol-stimulated PKCs in glioma cell lysates, and this blockade is lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. Physiologically more significant is the fact that pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevents ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and phorbol-stimulated cells. The effect of ionomycin on MARCKS phosphorylation is not due to the stimulation of the Ca2+/calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, does not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent phorbol-stimulated PKCs from phosphorylating MARCKS depends on whether ionomycin was added before, with, or after phorbol treatment. Maximum blockade occurs when ionomycin is added before phorbol but is less effective when added with or after phorbol. These results indicate that Ca2+/calmodulin can profoundly affect PKCs' signaling at the substrate level (Chakravarthy, 1995).

Calmodulin interaction with miscellaneous proteins

Continued: see Calmodulin: Evolutionary homologs part 3/4 | part 4/4 | return to part 1/4

Calmodulin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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