Calmodulin-dependent Kinase Pathway Research Articles

Targeted Intron Retention and Excision for Rapid Gene Regulation in Response to Neuronal Activity

Activity-dependent transcription has emerged as a major source of gene products that regulate neuronal excitability, connectivity, and synaptic properties. However, the elongation rate of RNA polymerases imposes a significant temporal constraint for transcript synthesis, in particular for long genes where new synthesis requires hours. Here we reveal a novel, transcription-independent mechanism that releases transcripts within minutes of neuronal stimulation. We found that, in the mouse neocortex, polyadenylated transcripts retain select introns and are stably accumulated in the cell nucleus. A subset ofthese intron retention transcripts undergoes activity-dependent splicing, cytoplasmic export, and ribosome loading, thus acutely releasing mRNAs in response to stimulation. This process requires NMDA receptor- and calmodulin-dependent kinase pathways, and it is particularly prevalent for long transcripts. We conclude that regulated intron retention in fully transcribed RNAs represents a mechanism to rapidly mobilize a pool of mRNAs in response to neuronal activity.

The di-peptide Trp-His activates AMP-activated protein kinase and enhances glucose uptake independently of insulin in L6 myotubes

The di-peptide Trp-His (WH) has vasorelaxant and anti-atherosclerotic functions. We hypothesized that WH has multiple biological functions and may aid AMP-activated protein kinase (AMPK) activation and affect the glucose transport system in skeletal muscle.First, we examined whether WH or His-Trp (HW) can activate AMPKα. Treatment of L6 myotubes with WH or HW significantly increased phosphorylation of AMPKα. WH activated AMPK independently of insulin and significantly increased glucose uptake into L6 myotubes following translocation of glucose transporter 4 (Glut4) to the plasma membrane. This activation was induced by the LKB1 pathway but was independent of changes in intracellular Ca2+ levels and the Ca2+/calmodulin-dependent kinase pathway. L6 myotubes express only one type of oligopeptide transporter, peptide/histidine transporter 1 (PHT1, also known as SLC15a4), and WH is incorporated into cells and activates AMPKα following PHT1-mediated cell uptake.These findings indicate that (1) WH activates AMPK and insulin independently enhances glucose uptake following translocation of Glut4 to the plasma membrane, (2) activation of AMPKα by WH is mediated by the LKB1 pathway, without altering the Ca2+-dependent pathway, and (3) L6 myotubes express only one type of peptide transporter (PHT1; SLC15a4), which incorporates WH into cells to activate AMPKα.

Identification and Inhibitory Properties of a Novel Ca2+/Calmodulin Antagonist

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

Kinase Networks Integrate Profiles of N-Methyl-d-aspartate Receptor-mediated Gene Expression in Hippocampus

The postsynaptic N-methyl-d-aspartate (NMDA) receptor activates multiple kinases and changes the phosphorylation of many postsynaptic proteins organized in signaling networks. Because the NMDA receptor is known to regulate gene expression, it is important to examine whether networks of kinases control signaling to gene expression. We examined the requirement of multiple kinases and NMDA receptor-interacting proteins for gene expression in mouse hippocampal slices. Protocols that induce long-term depression (LTD) and long-term potentiation (LTP) activated common kinases and overlapping gene expression profiles. Combinations of kinases were required for induction of each gene. Distinct combinations of kinases were required to up-regulate Arc, Npas4, Egr2, and Egr4 following either LTP or LTD protocols. Consistent with the combinatorial data, a mouse mutant model of the human cognition disease gene SAP102, which couples ERK kinase to the NMDA receptor, showed deregulated expression of specific genes. These data support a network model of postsynaptic integration where kinase signaling networks are recruited by differential synaptic activity and control both local synaptic events and activity-dependent gene expression.

The Role of NFAT in Osteoclast Formation

Osteoclasts are cells of monocyte-macrophage origin that degrade bone matrix. Receptor activator of NF-kappaB ligand (RANKL) induces osteoclast formation in the presence of macrophage-colony-stimulating factor (M-CSF) and costimulatory signals. RANKL induces activation of the TNF receptor-associated factor 6 (TRAF6) and c-Fos pathways, which lead to the osteoclast-specific event, that is, autoamplification of nuclear factor of activated T cells (NFAT)c1, the master transcription factor for osteoclast differentiation. Autoamplification of NFATc1 is dependent on the calcium signaling of immunoglobulin-like receptors associated with immunoreceptor tyrosine-based activation motif (ITAM)-harboring adaptors. In addition to the calcineurin-NFATc1 axis, calcium signaling activates the calmodulin-dependent kinase pathway, which also plays a critical role in osteoclast formation. Such advances in the understanding of the molecular mechanism of osteoclast differentiation are expected to lead to novel therapeutic approaches to bone diseases.

Roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation of cultured adult mouse skeletal muscle fibers

Two Ca2+-dependent signaling pathways, mediated by the Ca2+-activated phosphatase calcineurin and by the Ca2+-activated kinase Ca2+/calmodulin-dependent kinase (CaMK), are both believed to function in fast-to-slow skeletal muscle fiber type transformation, but questions about the relative importance of the two pathways still remain. Here, the differential gene expression during fast-to-slow fiber type transformation was studied using cultured adult flexor digitorum brevis (FDB) fibers and a custom minimicroarray system containing 21 fiber type-specific marker genes. After 3 days of culture, unstimulated fibers showed a generally slower gene expression profile; 3 days of electric field stimulation of cultured FDB fibers with a slow fiber-type pattern transformed the fibers to an even slower gene expression profile. Unstimulated FDB fibers overexpressing constitutively active calcineurin featured a slower gene expression profile, except four genes, indicating that transformation occurred, but was incomplete with activation of the calcineurin pathway alone. In both unstimulated FDB fibers and slow-type electrically stimulated FDB fibers, blocking of CaMK pathway with KN93 generated a faster gene expression profile compared with the negative control KN92, indicating that CaMK pathway functions during the transformation induced by both unstimulated culturing and slow fiber-type electrical stimulation. Moreover, neither the calcineurin nor the CaMK pathway alone could maximally activate the transformation, and coordination of the two pathways is required to accomplish a complete fast-to-slow fiber type transformation.

Upregulation of substance P receptor expression by calcitonin gene-related peptide, a possible cooperative action of two neuropeptides involved in airway inflammation

Airway hyperresponsiveness (AHR) is present in almost all patients with symptomatic asthma, yet its mechanism is not well understood. Airway inflammation is thought to be an important underlying mechanism involved in causing AHR. Recent studies indicate release of neuropeptides from C-fiber endings plays a pivotal role in airway inflammation. Substance P (SP) is a critical neurotransmitter of sensory C-fiber and a well-known effector of inflammatory response. However, roles of other neuropeptides and interaction among these neuropeptides in airway inflammation and AHR were largely unknown. Calcitonin gene-related peptide (CGRP), another intrapulmonary neuropeptide that functions as a potent vasodilator and neutrophils activator, is released from the same C-fiber ending as SP is released. By using an ozone-stressing animal model, previously we had demonstrated that CGRP might be involved in the development of AHR in rabbits. To extend the functional roles of SP, and to explore the possible interactive roles of SP and CGRP in airway inflammation, we examined expressions of SP, SP receptor (neurokinin 1, or NK-1R) and CGRP in vivo and ex vivo. We exposed guinea pigs at intervals to inhalation of ozone to induce airway inflammation. Animals were sacrificed at different time points; SP, SP receptor and CGRP expression were determined during the onset and progression of airway inflammation by radioimmunoassay, immunohistochemistry and in situ hybridization. Our data showed that after exposure to ozone, the concentration of SP in lung homogenate and the number of SP-immunoreactive cell bodies in lung slides increased within 24 h, peaked on day 2, and then decreased slowly. Interestingly, CGRP expressions exhibited a similar temporal and spatial pattern, and there was a strong correlation between SP expression and CGRP expression, indicating a possible cooperative action of these two neuropeptides. We also noted an increased expression of SP receptor NK-1R in the development of airway inflammation. In order to test the hypothesis that CGRP as a coexisting neurotransmitter with SP can regulate the expression of NK-1R in the lung, and contribute to the SP-mediated inflammatory response, we used in vitro lung tissue culture to determine the effect of CGRP on NK-1R expression. We found that NK-1R expression was induced by CGRP incubation at both mRNA and protein levels, and the induction was attenuated by additions of the inhibitors of Protein Kinase A (PKA) pathway, Calmodulin-dependent Kinase pathway, and Tyrosine Protein Kinase pathway. In conclusion, our data provide compelling evidence that SP and CGRP are involved in the development of airway inflammation. The interaction between SP and CGRP is likely to contribute to the pathogenesis of AHR and other lung inflammatory diseases.

Vasopressin-dependent myogenic cell differentiation is mediated by both Ca2+/calmodulin-dependent kinase and calcineurin pathways.

Arg8-vasopressin (AVP) promotes the differentiation of myogenic cell lines and mouse primary satellite cells by mechanisms involving the transcriptional activation of myogenic bHLH regulatory factors and myocyte enhancer factor 2 (MEF2). We here report that AVP treatment of L6 cells results in the activation of calcineurin-dependent differentiation, increased expression of MEF2 and GATA2, and nuclear translocation of the calcineurin target NFATc1. Interaction of these three factors occurs at MEF2 sites of muscle specific genes. The different kinetics of AVP-dependent expression of early (myogenin) and late (MCK) muscle-specific genes correlate with different acetylation levels of histones at their MEF2 sites. The cooperative role of calcineurin and Ca2+/calmodulin-dependent kinase (CaMK) in AVP-dependent differentiation is demonstrated by the effect of inhibitors of the two pathways. We show here, for the first time, that AVP, a "novel" myogenesis promoting factor, activates both the calcineurin and the CaMK pathways, whose combined activation leads to the formation of multifactor complexes and is required for the full expression of the differentiated phenotype. Although MEF2-NFATc1 complexes appear to regulate the expression of an early muscle-specific gene product (myogenin), the activation of late muscle-specific gene expression (MCK) involves the formation of complexes including GATA2.

Sending signals from the synapse to the nucleus: Possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth

The ability to learn and form memories depends on specific patterns of synaptic activity and is in part transcription dependent. However, the signal transduction pathways that connect signals generated at synapses with transcriptional responses in the nucleus are not well understood. In the present report, we discuss three signal transduction pathways: the Ca(2+)/calmodulin-dependent kinase (CaMK) pathway, the Ras/ERK pathway, and the SAPK pathways that might function to couple synaptic activity to long-term adaptive responses, in part through the regulation of new gene expression. Evidence suggests that these pathways become activated in response to stimuli that regulate synaptic function such as the influx of extracellular Ca(2+) and certain neurotrophin growth factors such as brain-derived neurotrophic factor. Once activated, the CaMK, Ras/ERK, and SAPK pathways lead to the phosphorylation and activation of transcription factors in the nucleus such as the cyclic AMP response element binding protein (CREB). Genes regulated by CREB or other transcription factor targets of the CaMK, Ras/ERK, and SAPK pathways could mediate important adaptive responses to changes in synaptic activity such as changes in synaptic strength and the regulation of neuronal survival and death.

Parathyroid Hormone-related Peptide Is Produced by Cultured Cerebellar Granule Cells in Response to L-type Voltage-sensitive Ca2+ Channel Flux via a Ca2+/Calmodulin-dependent Kinase Pathway

Parathyroid hormone (PTH)-related peptide (PTHrP) is expressed in the adult mammalian brain, but its function is unknown. Here we show that PTHrP and the PTH/PTHrP receptor are products of cerebellar granule cells in primary culture. Granule cells maintained under depolarizing conditions (25 mM K+) make and release PTHrP. Further, PTHrP-(1-36) stimulates cAMP accumulation in granule neurons in a dose-dependent manner with half-maximal activation at approximately 16 nM. Granule cell PTHrP mRNA is activity-dependent, and the pathway of regulation depends absolutely on the flux of Ca2+ ions through the L-type voltage-sensitive Ca2+ channel and the Ca2+/calmodulin kinase cascade. PTHrP is therefore a neuropeptide whose regulation depends upon L-type voltage-sensitive Ca2+ channel activity, and the gene is expressed under conditions that promote granule cell survival.

Rapid increase of the human IFN-gamma receptor phosphorylation in response to human IFN-gamma and phorbol myristate acetate. Involvement of different serine/threonine kinases.

Abstract Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-IFN-gamma. Both pathways involved the activation of a serine/threonine kinase and therefore we have investigated the possibility of hu-IFN-gamma-R phosphorylation by these kinases. PMA, an activator of protein kinase C, induced a rapid increase of the receptor phosphorylation in Raji cells, whereas the Ca2+ ionophore A23187 did not. PMA-induced hu-IFN-gamma-R phosphorylation was not associated with any effect on expression or inactivation of the receptor. PMA alone did not mimic the hu-IFN-gamma effect in Raji cells as measured by induction of IP-10 gene expression, a high specific marker of hu-IFN-gamma response. But the protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine, reduced this IFN-gamma-induced expression. However, H7 and staurosporine treatment as well as protein kinase C depletion suppressed PMA-induced receptor phosphorylation, whereas constitutive and hu-IFN-gamma-induced phosphorylation remained unchanged. Our results suggest that the serine/threonine kinase involved in the hu-IFN-gamma-R phosphorylation induced by IFN-gamma is different from protein kinase C.