Calmodulin-dependent Protein Kinase IV Research Articles

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway.

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating APN is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of APN through the Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. APN expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by the detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on APN promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV-dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also canceled the effect of HDL on APN gene expression. These results suggest that HDL has an important role to improve the function of adipocytes by activating hSR-BI/CLA-1, and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

Flunarizine inhibits osteoclastogenesis by regulating calcium signaling and promotes osteogenesis.

Many bone diseases such as osteoporosis and periodontitis are caused by hyperactivation of osteoclasts. Calcium (Ca2+ ) signals are crucial for osteoclast differentiation and function. Thus, the blockade of Ca2+ signaling may be a strategy for regulating osteoclast activity and has clinical implications. Flunarizine (FN) is a Ca2+ channel antagonist that has been used for reducing migraines. However, the role of FN in osteoclast differentiation and function remains unknown. Here, we investigated whether FN regulates osteoclastogenesis and elucidated the molecular mechanism. FN inhibited osteoclast differentiation along with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and attenuated osteoclast maturation and bone resorption. FN inhibition of osteoclast differentiation was restored by ectopic expression of constitutively active NFATc1. FN reduced calcium oscillations and its inhibition of osteoclast differentiation and resorption function was reversed by ionomycin, an ionophore that binds Ca2+ . FN also inhibited Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV) and calcineurin leading to a decrease in the cAMP-responsive element-binding protein-dependent cFos and peroxisome proliferator-activated receptor-γ coactivator 1β expression, and NFATc1 nuclear translocation. These results indicate that FN inhibits osteoclastogenesis via regulating CaMKIV and calcineurin as a Ca2+ channel blocker. In addition, FN-induced apoptosis in osteoclasts and promoted osteogenesis. Furthermore, FN protected lipopolysaccharide- and ovariectomy-induced bone destruction in mouse models, suggesting that it has therapeutic potential for treating inflammatory bone diseases and postmenopausal osteoporosis.

The Influence of Thyroid Hormone on Ca2+ Signaling Pathways During Embryonal Development.

Thyroid hormones influence brain development through the regulation of gene expression. Ca2+-dependent gene expression is a major pathway controlled by the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), which in turn is induced by the thyroid hormone T3, as also demonstrated in a mouse embryonic stem cell line. In addition, T3 controls the expression of neurexin, synaptotagmin2 (SYT2), synaptotagmin-related gene1 (SRG1), and a number of other genes involved in neurotransmitter release in a Ca2+-dependent manner. It has been noticed that the development of dopaminergic neurons by evoking significant calcium entry occurs through TRPC calcium channels. It was also demonstrated that the T3-mediated development of an early neuronal network is characteristic for depolarizing GABAergic neurons concomitant with intracellular calcium transients. An important aspect of T3-dependent regulation of gene expression in the developing brain is its modulation by the transcription activator COUP-TF1. Regulation of alternative splicing by CaMKIV is another important aspect for embryonal neural development since it can lead to the expression of PMCA1a, the neuronal-specific isoform of the plasma membrane calcium pump. Maternal hypothyroidism or CaMKIV deficiency can have a severe influence on fetal brain development.

Attenuated β-adrenergic response in calcium/calmodulin-dependent protein kinase IV-knockout mice.

In the present study, we examined the importance of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of cardiac function using genetically modified CaMKIV-null mice. RT-PCR analysis revealed decreased expression of voltage-dependent calcium channels in the cardiac myocytes of CaMKIV-null mice compared with wild-type mice. CaMKIV-null mice showed shortened QT time on electrocardiograms. Pharmacological analysis revealed decreased responsiveness to the β-adrenergic blocker propranolol in CaMKIV-null mice, whereas the plasma norepinephrine level was not affected. CaMKIV-null mice showed decreased baroreflex on electrocardiograms. Heart rate variability analysis showed unstable R-R intervals, a decreased low frequency power/high frequency power (LF/HF) ratio, and increased standard deviation of the normal to normal R-R intervals (SDNN) in CaMKIV-null mice, suggesting decreased responsiveness to β-adrenergic stimulation in CaMKIV-null mice. Atrial contraction analysis and cardiac action potential recording showed a decreased response to the β-adrenoceptor agonist isoproterenol in CaMKIV-null mice. Furthermore, fluorescence imaging in a CRE-hrGFP assay revealed a decreased response to isoproterenol in CaMKIV-null cardiac myocytes. Taken together, our data strongly suggest a significant effect of CaMKIV gene ablation on cardiac β-adrenergic signal transduction.

Promotion of Dendritic Differentiation of Cerebellar Purkinje Cells by Ca2+/calmodulin-dependent Protein Kinase IIα, IIβ and IV and Possible Involvement of CREB Phosphorylation

Cerebellar Purkinje cells develop the most elaborate dendritic trees among neurons in the brain. To examine the role of Ca2+/calmodulin-dependent protein kinase (CaMK) IIα, IIβ and IV in the dendritic differentiation of Purkinje cells, we introduced siRNA against these CaMKs into Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. Single-cell electroporation enables us to transfer siRNA into specific cells within a heterogeneous cell population. In addition, we can easily and reliably transfer multiple types of siRNA into a cell simply by loading them together in one micropipette. Any one of the siRNA against CaMKIIα, IIβ and IV (single knockdown) or any combinations of two of the siRNA against these CaMKs (double knockdown) had no significant effects on the dendritic differentiation of Purkinje cells. However, the combination of all three siRNA against these CaMKs (triple knockdown) inhibited the branching of Purkinje cell dendrites. Furthermore, the triple knockdown reduced the phosphorylation of CREB in Purkinje cells. These findings suggest the promotion of dendritic differentiation of Purkinje cells by CaMKIIα, IIβ and IV and the possible involvement of phosphorylation of CREB as a common substrate of these CaMKs.

Calmodulin-dependent signalling pathways are activated and mediate the acute inflammatory response of injured skeletal muscle.

There is a close relationship between skeletal muscle physiology and Ca2+ /calmodulin (CaM) signalling. Despite the effects of Ca2+ /CaM signalling on immune and inflammatory responses having been extensively explored, few studies have investigated the role of CaM pathway activation on the post-injury muscle inflammatory response. In this study, we investigated the role of CaM-dependent signalling in muscle inflammation in cardiotoxin induced myoinjuries in mice. The Ca2+ /calmodulin-dependent protein kinase II (CaMII), Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV), and nuclear factor of activated T cells (NFAT) pathways are likely to be simultaneously activated in muscle cells and in infiltrating lymphocytes and to regulate the immune behaviours of myofibres in an inflammatory environment, and these pathways ultimately affect the outcome of muscle inflammation. Calcium/calmodulin (Ca2+ /CaM) signalling is essential for immune and inflammatory responses in tissues. However, it is unclear if Ca2+ /CaM signalling interferes with muscle inflammation. Here we investigated the roles of CaM-dependent signalling in muscle inflammation in mice that had acute myoinjuries in the tibialis anterior muscle induced by intramuscular cardiotoxin (CTX) injections and received intraperitoneal injections of either the CaM inhibitor calmidazolium chloride (CCL) or CaM agonist calcium-like peptide 1 (CALP1). Multiple inflammatory parameters, including muscle autoantigens and toll-like receptors, mononuclear cell infiltration, cytokines and chemokines associated with peripheral muscle inflammation, were examined after the injury and treatment. CALP1 treatment enhanced intramuscular infiltration of monocytes/macrophages into the damaged tibialis anterior muscle and up-regulated mRNA and protein levels of muscle autoantigens (Mi-2, HARS and Ku70) and Toll-like receptor 3 (TLR3), and mRNA levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), Monocyte chemoattractant protein-1 (MCP1), Monocyte chemoattractant protein-3 (MCP3) and Macrophage inflammatory protein-1(MIP-1α) in damaged muscle. In contrast, CCL treatment decreased the intramuscular cell infiltration and mRNA levels of the inflammatory mediators. After CALP1 treatment, a substantial up-regulation in Ca2+ /calmodulin-dependent protein kinase II (CaMKII), Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV) and nuclear factor of activated T cells (NFAT) activity was detected in CD45+ cells isolated from the damaged muscle. More pro-inflammatory F4/80+ Ly-6C+ cells were detected in CD45-gated cells after CALP1 treatment than in those after CCL treatment or no treatment. Consistently, in interferon-γ-stimulated cultured myoblasts and myotubes, CALP1 treatment up-regulated the activities of CaMKII, CaMKIV and NFAT, and levels of class I/II major histocompatibility complexes (MHC-I/II) and TLR3. Our findings demonstrated that CaM-dependent signalling pathways mediate the injury-induced acute muscle inflammatory response.

Redox regulation of Ca2+/calmodulin-dependent protein kinase IV via oxidation of its active-site cysteine residue

We have recently reported that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) is inactivated by reactive sulfur species via polysulfidation of the active-site Cys residue. Here, we show that hydrogen peroxide (H2O2) limit CaMKIV activity at the same active-site Cys residue through oxidation and downstream signaling in cells. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), which induces its full activity. In vitro incubation of CaMKIV with H2O2 resulted in reversible inhibition of CaMKK-induced phospho-Thr196 and the consequent inactivation of CaMKIV. In contrast, mutated CaMKIV (C198V) was refractory to the H2O2-induced enzyme inhibition. In transfected cells expressing CaMKIV, Ca2+ ionophore-induced CaMKIV phosphorylation at Thr196 was decreased upon treatment with H2O2, whereas cells expressing mutant CaMKIV (C198V) were resistant to H2O2 treatment. Modification of free thiol with N-ethylmaleimide revealed that Cys198 in CaMKIV is a target for S-oxidation. Additionally, the Ca2+ influx-induced phospho-Thr196 of endogenous CaMKIV was also inhibited upon treatment with H2O2 in Jurkat T-lymphocytes and cerebellar granule cells. Phosphorylation of cyclic AMP response element-binding protein (CREB) at Ser133, which is downstream of CaMKIV, was also decreased upon treatment with H2O2. Thus, our results indicate that oxidation stress regulates cellular function by decreasing the activity of CaMKIV through Cys198 oxidation.

Calcium/Calmodulin-Dependent Protein Kinase IV (CaMKIV) Mediates Acute Skeletal Muscle Inflammatory Response

The objective of this study is to investigate the role of Calmodulin-dependent protein kinase IV (CaMKIV) in Cardiotoxin (CTX)-induced mice muscle inflammation. CTX injection i.m. was performed to induce B6 mice acute tibialis anterior (TA) muscle injury. The mice were then injected i.p. with the recombinant CaMKIV protein or its antagonist KN-93. Immunoblotting was used to assess Calmodulin (CaM) and CaMKIV levels. Immunofluorescence was used to detect intramuscular infiltration or major histocompatibility complex (MHC)-I expression in damaged muscle. The extent of infiltration was evaluated by fluorescent intensity analysis. Cytokines/chemokines levels were determined by qPCR. CaMKIV gene knockdown in C2C12 cells was performed in order to evaluate the effects of CaMKIV on immuno-behavior of muscle cells. CTX administration induced a strong up-regulation of CaM and p-CaMKIV levels in infiltrated mononuclear cells and regenerated myofibers. In vivo adding of the recombinant CaMKIV protein enhanced intramuscular infiltration of monocytes/macrophages in damaged muscle and increased the number of proinflammatory Ly-6C+F4/80+ macrophage cells. CaMKIV protein treatment induced a striking up-regulation of mRNA levels of IL-1, IL-6, MCP-1, and MCP-3 in CD45+ cells sorted from damaged muscle; increased the infiltration of CD8+ T cells; and induced the up-regulation of MHC-I in partial regenerated myofibers, which was rarely observed in muscle damage alone. Additionally, CaMKIV protein treatment diminished the regulatory T cells (Tregs) number and led to the damaged TA muscle repair delay. In vitro CaMKIV gene knockdown reversed IFN-γ-induced up-regulation of MHC-I/II and TLR3 in the differentiated C2C12 myotubes. CaMKIV can act as an immunostimulation molecule and enhances the acute muscle inflammatory responses.

Reactive sulfur species inactivate Ca2+/calmodulin-dependent protein kinase IV via S-polysulfidation of its active-site cysteine residue.

Reactive sulfur species (RSS) modulate protein functions via S-polysulfidation of reactive Cys residues. Here, we report that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na2S n (n = 2-4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr196 and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na2S n -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys198 in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr196 and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr196 was decreased upon treatment with either Na2S4 or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr196 of endogenous CaMKIV was also inhibited by treatment either with Na2S4 or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S-polysulfidation of its Cys198 during the response to ER stress.

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca2+ concentration with a simultaneous increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca2+/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca2+/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca2+/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

Memory Improvement by Yokukansankachimpihange and Atractylenolide III in the Olfactory Bulbectomized Mice

Alzheimer’s Disease (AD) shows cognitive dysfunction as core symptoms and Behavioral and Psychological Symptoms of Dementia (BPSD). Since acetylcholine nerve system derived from septum is collapsed in the AD patients, we have used Olfactory Bulbectomized (OBX) mice whose cholinergic system is largely impaired in the septum. Recently, Yokukansankachimpihange (YKH), a traditional Japanese Kampo medicine has used for BPSD in addition to improve cognitive dysfunction in AD patients. However the essential components for cognition and BPSD improvement and their mechanism are largely unknown. In present study, we found that Atractylenolide III (Aen-III), one of the components of YKH, improved cognitive deficits and depression in the OBX mice. OBX mice were orally administered with Aen-III (1.0 and 3.0 mg/kg) and YKH extracts daily for 18 days. Like YKH extracts, the Aen-III treatments ameliorated cognitive deficits and depression-like behavior observed in OBX mice. Importantly, Aen-III administration significantly restored the decreases in Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and cyclic AMP response element binding protein (CREB). The restoration of CaMKII and CaMKIV signaling is closely related to the increased BDNF levels. Furthermore, ATP reduction in OBX mice was rescued by Aen-III (3.0 mg/kg) and YKH (1000 mg/kg) treatment. In summary, Aen-III as a component of YKH ameliorates cognitive dysfunctions and depression via restoring CaMKII and CaMKIV signaling.

Structure guided design of potential inhibitors of human calcium-calmodulin dependent protein kinase IV containing pyrimidine scaffold.

Calmodulin dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family and considered as an encouraging target for the development of novel anticancer agents. The interaction and binding behavior of three designed inhibitors of human CAMKIV, containing pyrimidine scaffold, was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. In silico docking studies were performed to screen several compounds containing pyrimidine scaffold against CAMKIV. Molecular docking calculation predicted the binding of these ligands in active-site cavity of the CAMKIV structure correlating such interactions with a probable inhibition mechanism. Finally, three active pyrimidine substituted compounds (molecules 1-3) have been successfully synthesized and characterized by (1)H and (13)C NMR. Molecule 3 is showing very high binding-affinity for the CAMKIV, with a binding constant of 2.2×10(8), M(-1) (±0.20). All three compounds are nontoxic to HEK293 cells up to 50 μM. The cell proliferation inhibition study showed that the molecule 3 has lowest IC50 value (46±1.08 μM). The theoretical and experimental observations are significantly correlated. This study reveals some important observations to generate an improved pyrimidine based compound that holds promise as a therapeutic agent for the treatment of cancer and neurodegenerative diseases.

Ca(2+)/Calmodulin-Dependent Protein Kinase IV Promotes Interplay of Proteins in Chromatoid Body of Male Germ Cells.

The chromatoid body is a granule-like structure of male germ cells, containing many proteins and RNAs, and is important for spermatogenesis. However, the molecular mechanisms for the formation and function of the chromatoid body are still elusive. Here, we report that Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) accumulates in the chromatoid body by immunofluorescence staining, indicating that CaMKIV is a new component of the chromatoid body. Furthermore, we find that CaMKIV can interplay with the other components of the chromatoid body by immunoprecipitation: mouse VASA homologue (MVH), mouse homologue of PIWI, PIWIL1 (MIWI), and kinesin KIF17b. Importantly, interplay between KIF17b and MVH or MIWI can be potentially regulated by CaMKIV. These results imply that CaMKIV plays a role in maintenance the structure of chromatoid body by regulating the associations of proteins in it.

Function of Ca2+ -/calmodulin-dependent protein kinase IV in Ca2+ -stimulated neuronal signaling and behavior.

The activity of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is sensitive to activity-dependent changes in the level of intracellular Ca(2+). Following neuronal stimulation, the activation of CaMKIV may trigger synaptic modifications and transcriptional responses, both of which are involved in regulating cognitive and emotional behavior. Here, we used CaMKIV knockout (KO) neurons and mice to examine the function of CaMKIV in Ca(2+)-stimulated intracellular signaling and animal behavior, respectively. Following NMDA receptor activation or membrane depolarization, the up-regulation of CREB (cAMP responsive element binding protein) and its target gene Bdnf (brain-derived neurotrophic factor) was intact in cortical neurons obtained from CaMKIV KO mice. CaMKIV KO mice displayed severe impairment in contextual fear memory but normal locomotor activity and anxiety level in the contextual training chamber. Although CaMKIV KO mice showed normal memory in the standard passive avoidance task, they were defective in learning the temporal dissociative passive avoidance task. As indicated by the light/dark test and marble-burying test data, CaMKIV KO mice showed less anxiety and normal perseveration. In the voluntary wheel-running test, CaMKIV KO mice showed normal running time and distance but higher maximal running speed. Our results demonstrate the function of CaMKIV in regulating different forms of fear memory, anxiety, and certain aspect of motor function.

A Calmodulin Interacting Protein Iqcg Is Required for Definitive Hematopoiesis in Zebrafish

AbstractAbstract 764We previously identified a novel fusion gene NUP98-IQCG in a bi-phenotypic acute T-lymphoid/myeloid leukemia patient. However, the function of IQCG has never been demonstrated. By taking advantage of the zebrafish as a model organism, we investigated the role of IQCG in hematopoietic development and found that the definitive hematopoiesis was severely impaired in the iqcg deficient embryos. The hematopoietic stem cell (HSC) population as well as the number of multilineage differentiated hematopoietic cells was reduced. Moreover, the number of phosphorelated histone3 positive HSC was decreased, which indicated a compromised proliferation in iqcg deficient HSC.Since IQCG protein contains an IQ calmodulin binding motif (IQ motif), we next examined if IQCG protein can interact with calmodulin. Co-immunoprecipitation assay results showed that both human and zebrafish IQCG proteins have the ability to precipitate calmodulin. Deletion of IQ motif disrupted this interaction. The isothermal titration calorimetry results indicated that the IQ motif of IQCG protein have a lower association constant with calcium-loaded calmodulin than apo-calmodulin. We next crystallized the complex of calmodulin and IQ motif of IQCG in two circumstances-with and without calcium. Determination of these two crystal structures indicated different interaction details between them. These results suggest that IQCG may be involved in calcium signaling in HSC.Previous studies reported that calmodulin dependent protein kinase IV (CaMKIV) involves in HSC maintenance and survival. So we wondered if CaMKIV acts downstream of IQCG. Indeed, whole-mount immunofluorescence data showed that the level of phosphorelated CaMKIV markedly reduced in iqcg morphants compared with controls. Reactivation of CaMKIV by co-injection with constitutively activated CaMKIV (CACaMKIV) mRNA rescued the HSC number in iqcg deficient embryos. Furthermore, embryos injected with camkIV morpholino phenocopied the iqcg morphants.In summary, we demonstrate an important role of the novel NUP98 partner gene IQCG in definitive hematopoietic development. IQCG may control the HSC proliferation through interacting with calmodulin and acting upstream of CaMKIV, which suggests a novel pathway in definitive hematopoietic development. Disclosures:No relevant conflicts of interest to declare.

Decreased levels of Ca2+-calmodulin-dependent protein kinase IV in the testis as a contributing factor to reduced fertility in male Crybb2−/− mice

βB2-crystallin (Crybb2), a member of the βγ-crystallin superfamily, in conjunction with α-crystallin, constitute the major proteins of the mammalian eye lens. Crybb2 is also expressed outside the lens, and certain related functions in these tissues have been reported. In the present study, in order to define the physiological role of Crybb2, we generated mice with a targeted deletion of the Crybb2 gene. Surprisingly, fertility was markedly reduced in male homozygous knockout mice compared to wild-type (WT) mice. Further experiments were performed to explore the underlying mechanism of subfertility in male Crybb2⁻/⁻ mice. Our results showed that Crybb2 was mainly expressed in the spermatogonia from the testes of mice with the WT C57BL/C genetic background. The testes of 4-week-old Crybb2⁻/⁻ mice were significantly hyperplastic, and no significant difference was found within 3 weeks postpartum. Additionally, there was a marked increase in the proliferation and apoptosis of germ cells, and the biological defects of these cells correlated with the decreased Bcl-2 levels, which correlated with the reduction of Ca²⁺-calmodulin-dependent protein kinase IV (CaMKIV) in the testis. These results suggest that the reduced fertility of Crybb2⁻/⁻ male mice may result from the disordered proliferation and apoptosis of germ cells in the testis, possibly due to reduced CaMKIV from the loss of Crybb2.

Reversible inactivation of Ca2+/calmodulin-dependent protein kinase IV by oxidation of the active-site cysteine residue

Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) is serine/threonine-specific protein kinase that is involved in many signaling cascades, and plays a key role in memory. CaMKIV is activated via binding to Ca 2+ /CaM complex and phosphorylation by upstream kinase, CaM kinase. In the present study, we investigate direct mechanism of inactivation of CaMKIV by oxidative stress. We show that CaMKIV is directly inhibited by its sulfenic acid formation at the Cys198. In situ studies demonstrated that treatment of CaMKIV with hydrogen peroxide (H 2 O 2 ) results in inactivation of the enzyme, with a concomitant sulfenic acid formation of CaMKIV at Cys198. The methodology of detection of sulfenic acid formation is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Mutagenesis studies confirmed that sulfenic acid formation of Cys198 is both necessary and sufficient for the inhibition of CaMKIV by H 2 O 2 . In transfected cells expressing CaMKIV, treatment with H 2 O 2 caused a reversible decrease in CaMKIV activity. Cells expressing mutant CaMKIV (C198V) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKIV via its sulfenic acid formation at Cys198 is an important mechanism in processing calcium signal transduction in cells.

A Conserved Serine of Heterogeneous Nuclear Ribonucleoprotein L (hnRNP L) Mediates Depolarization-regulated Alternative Splicing of Potassium Channels

Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in response to membrane depolarization and Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.

CRP1, a Protein Localized in Filopodia of Growth Cones, Is Involved in Dendritic Growth

The cysteine-rich protein (CRP) family is a subgroup of LIM domain proteins. CRP1, which cross-links actin filaments to make actin bundles, is the only CRP family member expressed in the CNS with little known about its function in nerve cells. Here, we report that CRP1 colocalizes with actin in the filopodia of growth cones in cultured rat hippocampal neurons. Knockdown of CRP1 expression by short hairpin RNA interference results in inhibition of filopodia formation and dendritic growth in neurons. Overexpression of CRP1 increases filopodia formation and neurite branching, which require its actin-bundling activity. Expression of CRP1 with a constitutively active form of Cdc42, a GTPase involved in filopodia formation, increases filopodia formation in COS-7 cells, suggesting cooperation between the two proteins. Moreover, we demonstrate that neuronal activity upregulates CRP1 expression in hippocampal neurons via Ca²⁺ influx after depolarization. Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein mediate the Ca²⁺-induced upregulation of CRP1 expression. Furthermore, CRP1 is required for the dendritic growth induced by Ca²⁺ influx or CaMKIV. Together, these data are the first to demonstrate a role for CRP1 in dendritic growth.

Characterization of the CaMKKβ–AMPK signaling complex

The AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis, and is a potential target for treatment of metabolic diseases as well as cancer. AMPK can be phosphorylated and activated by the tumor suppressor LKB1 or the Ca2+/CaM-dependent protein kinase kinase β (CaMKKβ). We previously identified a physical complex between CaMKKβ and AMPK (Anderson, K. A., Ribar, T. J., Lin, F., Noeldner, P. K., Green, M. F., Muehlbauer, M. J., Witters, L. A., Kemp, B. E., and Means, A. R. (2008) Cell Metabolism 7, 377–388). Here we expand our analysis of the CaMKKβ–AMPK signaling complex and show that whereas CaMKKβ can form a complex with and activate AMPK, CaMKKα cannot. In addition, we show that CaMKKβ and AMPK associate through their kinase domains, and CaMKKβ must be in an active conformation in order to bind AMPK but not to associate with an alternative substrate, Ca2+/Calmodulin-dependent protein kinase IV (CaMKIV). Our results demonstrate that CaMKKβ and AMPK form a unique signaling complex. This raises the possibility that the CaMKKβ–AMPK complex can be specifically targeted by small molecule drugs to treat disease.