Localization of phospho-tyrosine489-beta-adducin immunoreactivity in the hypothalamic tanycytes and its involvement in energy homeostasis.

beta-Adducin is a cytoskeletal protein that interacts with the actin filaments to suppress actin polymerization and facilitate actin-spectrin binding. We have previously shown that beta-adducin is phosphorylated by Fyn at tyrosine489 in the rat brain and bound to its Src-homology 2 domain. In the present study, we examined the immunohistochemical localization of the tyrosine489-phosphorylated form of beta-adducin (pY489-beta-adducin) in the rat brain. Among brain regions, highest immunoreactivity was located in the hypothalamic tanycytes that are of glial origin lining around the third cerebral ventricle. Their immunoreactive processes extended into the arcuate nucleus, ventromedial hypothalamus and the median eminence. In addition, the pY489-beta-adducin immunoreactivity in the tanycytes was enhanced after fasting for 36-48 h, being associated with a morphological change of the DARPP-32-immunoreactivity. Intraperitoneal injection of 2-deoxy-d-glucose also enhances pY489-beta-adducin immunoreactivity in the tanycytes, along with increased food intake. These results suggest that tyrosine phosphorylation of beta-adducin in the tanycytes is involved in hypothalamic regulation of food intake and energy homeostasis.

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein.

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix alpha4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.

A WD40 repeat protein regulates fungal cell differentiation and can be replaced functionally by the mammalian homologue striatin.

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus Sordaria macrospora showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca2+-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.

The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain.

A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.

A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination.

Calmodulin (CaM), a ubiquitous multifunctional calcium sensor in all eukaryotes, mediates calcium action by regulating the activity/function of many unrelated proteins. Although calcium and CaM are known to play a crucial role in pollen germination and pollen tube growth, the proteins that mediate their action have not been identified. We isolated three closely related CaM-binding proteins (NPG1, NPGR1, and NPGR2) from Arabidopsis. NPG1 (No Pollen Germination1) is expressed only in pollen, whereas the NPG-related proteins (NPGR1 and NPGR2) are expressed in pollen and other tissues. The bacterially expressed NPG1 bound three isoforms of Arabidopsis CaM in a calcium-dependent manner. To analyze the function of NPG1, we performed a reverse genetics screen and isolated a mutant in which NPG1 is disrupted by a T-DNA insertion. Segregation and molecular analyses of the NPG1 knockout mutant and a cross with a male sterile mutant indicate that the mutated NPG1 is not transmitted through the male gametophyte. Expression of NPG1 in the knockout mutant complemented the mutant phenotype. Analysis of pollen development in the knockout mutant by light microscopy showed normal pollen development. Pollen from NPG1 mutant in the quartet background has confirmed that NPG1 is dispensable for pollen development. However, germination studies with pollen from the mutant in the quartet background indicate that pollen carrying a mutant allele does not germinate. Our genetic, histological, and pollen germination studies with the knockout mutant line indicate that NPG1 is not necessary for microsporogenesis and gametogenesis but is essential for pollen germination.

Transcriptional program of apoptosis induction following interleukin 2 deprivation: identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor.

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca(2+) concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca(2+) increase with the cytoplasmic Ca(2+) chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca(2+) is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca(2+) levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca(2+) concentration.

CaMBOT: profiling and characterizing calmodulin-binding proteins.

Calmodulin (CaM) is an essential calcium-binding protein that binds to and activates a diverse population of downstream targets (calmodulin-binding proteins; CaMBPs) that carry out its critical signalling functions. In spite of the central importance of CaM in Ca(2+)-mediated signal transduction pathways in all eukaryotes, many CaMBPs remain to be identified and characterized. SDS-PAGE followed by gel overlay with recombinant, metabolically radiolabelled CaM (Calmodulin-binding Overlay Technique, CaMBOT) is a valuable method for following behavioural, developmental, forensic and physiological changes in total CaMBP populations and to identify candidate CaMBPs for further study. CaMBOT has also been adapted to isolate cDNAs encoding novel CaMBPs in various organisms. Recently, the method was used to examine the CaMBP complement encoded by the Arabidopsis genome and to identify a new family of transcription activators. To add to its diversity, CaMBOT may be useful for finding target proteins for work on phytoremediation and for the screening of pharmaceuticals and toxic agents that, directly or indirectly, affect CaM and its target proteins. This review discusses all of these topics and the role of CaMBOT in characterizing a functional unit of the proteome-proteins regulated by calmodulin.

Hypertension-linked decrease in the expression of brain gamma-adducin.

Gene profiling data coupled with adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of adducins in neurotransmission at the cellular level. All three adducin subunits (alpha, beta, and gamma) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the gamma-adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in gamma-adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize gamma-adducin gene expression. A hypertension-linked decrease of gamma-adducin was confirmed by demonstrating a decrease in gamma-adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a gamma-adducin-specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous gamma-adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in gamma-adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters.

The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast.

Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes.

Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins.

cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases are Mucor racemosus Ras2 and Ras3, to which they show approximately 48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

A facilitatory effect on the induction of long-term potentiation in vivo by chronic administration of antisense oligodeoxynucleotides against catalytic subunits of calcineurin.

A rise in Ca2+ concentration at postsynaptic sites provides an initial step in inducing both the long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus. LTP induction requires the activation of Ca(2+)-sensitive protein kinases following the rise in Ca2+. By contrast, the activity of protein phosphatase(s) appears to be critical to induce LTD. Here we demonstrate that inhibition of the synthesis of calcineurin A alpha and A beta, catalytic subunits of Ca2+/calmodulin- (CaM) dependent protein phosphatase, reduces the threshold of induction for commissural-CA1 LTP in anesthetized rats. In rats administered antisense oligodeoxynucleotides (ODNs) against calcineurin A alpha and A beta intraventricularly for 7 days, a brief tetanic stimulation to the CA3 region, which in the control case was below threshold for the induction of LTP, now produced a long-lasting increase in both the EPSP slope and the amplitude of population spike recorded from the commissural-CA1 pathway. Western blot analysis of calcineurin showed that the threshold reduction was accompanied by a selective decrease in the protein levels in the hippocampus. Thus our study provides direct evidence that calcineurin per se has an antagonizing role in LTP induction. Complementary experiments with the selective calcineurin inhibitor, FK506, also showed the reduction of LTP threshold in a dose-dependent manner. These results, together with previous studies, support the hypothesis that the quantitative phosphorylation level of critical intracellular proteins determines whether the synaptic efficacy will increase or decrease after the activity-dependent rise in postsynaptic Ca2+.

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

cAMP inhibits bud growth in a yeast strain compromised for Ca2+ influx into the Golgi.

Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes. Here we show that yeast cells partially depleted of PKA activity (due to a tpkw mutation) and bearing a lesion in a Golgi-localized Ca2+ pump (Pmr1), arrest division with a small bud. The bud morphology of the arrested tpk1w pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary. This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since the tpk1w pmr1 double mutants retain viability. The growth defect of the tpk1w pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca2+. Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of the pmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels. One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast.

Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5'[gamma-thio]triphosphate.

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

Calcineurin activation protects T cells from glucocorticoid-induced apoptosis.

In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway.