Regulation of phosphorylation at the postsynaptic density during different activity states of Ca/calmodulin-dependent protein kinase II

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), the most abundant kinase at the postsynaptic density (PSD), is expected to be involved in activity-induced regulation of synaptic properties. CaMKII is activated when it binds calmodulin in the presence of Ca²⁺ and, once autophosphorylated on T-286/7, remains active in the absence of Ca²⁺ (autonomous form). In the present study we used a quantitative mass spectrometric strategy (iTRAQ) to identify sites on PSD components phosphorylated upon CaMKII activation. Phosphorylation in isolated PSDs was monitored under conditions where CaMKII is: (1) mostly inactive (basal state), (2) active in the presence of Ca²⁺, and (3) active in the absence of Ca²⁺. The quantification strategy was validated through confirmation of previously described autophosphorylation characteristics of CaMKII. The effectiveness of phosphorylation of major PSD components by the activated CaMKII in the presence and absence of Ca²⁺ varied. Most notably, autonomous activity in the absence of Ca²⁺ was more effective in the phosphorylation of three residues on SynGAP. Several PSD scaffold proteins were phosphorylated upon activation of CaMKII. The strategy adopted allowed the identification, for the first time, of CaMKII-regulated sites on SAPAPs and Shanks, including three conserved serine residues near the C-termini of SAPAP1, SAPAP2, and SAPAP3. Involvement of CaMKII in the phosphorylation of PSD scaffold proteins suggests a role in activity-induced structural re-organization of the PSD.     

Identification of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II in the postsynaptic density by protein sequencing and mass spectrometry.

Previously we detected more than 28 PSD proteins to be phosphorylated by CaM kinase II, and identified 14 protein substrates (Yoshimura, Y., Aoi, T., Yamauchi, T., Mol. Brain Res. 81, 118-128, 2000). In the present study, the remaining substrates were analyzed by protein sequencing and mass spectrometry. We found 6 proteins not previously known to be substrates of CaM kinase II, namely PSD95-associated protein, SAP97, TOAD-64, TNF receptor-associated protein, insulin-receptor tyrosine kinase 58/53 kDa substrate, and homer 1b.     

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.     

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ

NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca²⁺/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca²⁺/CaM exposure strongly suppresses kinase activation and conformational change by Ca²⁺/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.     

CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy

Ca2+/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca2+ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-kappaB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIdelta3 (AdCaMKIIdelta3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIdelta3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIdelta3-induced hypertrophic responses. Overexpression of CaMKIIdelta3 increased the kappaB-dependent promoter/luciferase activity and induced IkappaBalpha degradation. Coinfection with AdCaMKIIdelta3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIdelta3- and phenylephrine-induced NF-kappaB activation, respectively. Expression of a degradation resistant mutant of IkappaBalpha inhibited CaMKIIdelta3-induced hypertrophic responses. These results indicate that CaMKIIdelta3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-kappaB signal transduction pathway.     

Regulation of phosphorylation at Ser of GluN2B receptor in the postsynaptic density

Neuronal N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) that plays essential roles in excitatory synaptic transmission is regulated by phosphorylation. However, the kinases and phosphatases involved in this regulation are not completely known. We show that the GluN2B subunit of NMDAR is phosphorylated at Ser¹³⁰³ by protein kinase C (PKC) and is dephosphorylated by protein phosphatase 1 (PP1), but not protein phosphatase 2A (PP2A) in isolated postsynaptic density (PSD). Although PSD is known to harbor PKC, PP1 and PP2A, their ability to regulate phosphorylation of GluN2B-Ser¹³⁰³ would depend on the accessibility of GluN2B-Ser¹³⁰³ to these proteins. Since PSD preparation is likely to maintain the organization of its component proteins as inside neurons, accessibility of kinases and phosphatases to GluN2B-Ser¹³⁰³in vivo would be addressed by experiments using this system. Using an antibody specific for the phosphorylated state of GluN2B-Ser¹³⁰³ we demonstrate that PP1 is the major phosphatase in rat brain PSD that can dephosphorylate the GluN2B-Ser¹³⁰³ endogenous to PSD. We also show that PKC present in PSD can phosphorylate GluN2B-Ser¹³⁰³. The events reported here might be important in regulating GluN2B-Ser¹³⁰³ phosphorylation in vivo.     

Nuclear Translocation of Calcium/Calmodulin-dependent Protein Kinase IIδ3 Promoted by Protein Phosphatase-1 Enhances Brain-derived Neurotrophic Factor Expression in Dopaminergic Neurons

We have reported previously that dopamine D₂ receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser³³² by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D₂ receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser³³² by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons.     

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

The influence of the signal dynamics of activated form of IKK on NF-kappaB and anti-apoptotic gene expressions: a systems biology approach

NF-kappaB activation plays a crucial role in anti-apoptotic responses in response to the apoptotic signaling during tumor necrosis factor (TNF)-alpha stimulation. TNF-alpha induces apoptosis sensitive to the hepatitis B virus (HBV) infected cells, despite sustained NF-kappaB activation. Our results indicate that the HBV infection induces sustained NF-kappaB activation, in a manner similar to the TNF-alpha stimulation. However, these effects are not merely combined. Computational simulations show that the level of form of the IKK complex activated by phosphorylation (IKK-p) affects the dynamic pattern of NF-kappaB activation during TNF-alpha stimulation in the following ways: (i) the initial level of IKK-p determines the incremental change in IKK-p at the same level of TNF-alpha stimulation, (ii) the incremental change in IKK-p determines the amplitudes of active NF-kappaB oscillation, and (iii) the steady state level of IKK-p after the incremental change determines the period of active NF-kappaB oscillation. Based on experiments, we observed that the initial level of IKK-p was upregulated and the active NF-kappaB oscillation showed smaller amplitudes for a shorter period in HepG2.2.15 cells (HBV-producing cells) during TNF-alpha stimulation, as was indicated by the computational simulations. Furthermore, we found that during TNF-alpha stimulation, NF-kappaB-regulated anti-apoptotic genes were upregulated in HepG2 cells but were downregulated in HepG2.2.15 cells. Based on the previously mentioned results, we can conclude that the IKK-p-level changes induced by HBV infection modulate the dynamic pattern of active NF-kappaB and thereby could affect NF-kappaB-regulated anti-apoptotic gene expressions. Finally, we postulate that the sensitive apoptotic response of HBV-infected cells to TNF-alpha stimulation is governed by the dynamic patterns of active NF-kappaB based on IKK-p level changes.     

Exploring the dominant role of Cav1 channels in signalling to the nucleus

Calcium is important in controlling nuclear gene expression through the activation of multiple signal-transduction pathways in neurons. Compared with other voltage-gated calcium channels, Ca_V1 channels demonstrate a considerable advantage in signalling to the nucleus. In this review, we summarize the recent progress in elucidating the mechanisms involved. Ca_V1 channels, already advantaged in their responsiveness to depolarization, trigger communication with the nucleus by attracting colocalized clusters of activated CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Ca_V2 channels lack this ability, but must work at a distance of >1 μm from the Ca_V1-CaMKII co-clusters, which hampers their relative efficiency for a given rise in bulk [Ca²⁺]_i (intracellular [Ca²⁺]). Moreover, Ca²⁺ influx from Ca_V2 channels is preferentially buffered by the ER (endoplasmic reticulum) and mitochondria, further attenuating their effectiveness in signalling to the nucleus.     

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.     

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca²⁺-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.     

Expression differences of miRNAs and genes on NF-κB pathway between the healthy and the mastitis Chinese Holstein cows

In order to discover the variation of microRNAs and genes associated with NF-κB signaling pathway between the healthy and the mastitis Chinese Holstein cows, Illumina Deep Sequencing and qRT-PCR are applied to detect 25 kinds of miRNAs (miR-16, miR-125b, miR-15, miR-29a, miR-23b, miR-146, miR-301a, miR-181b, let-7, miR-30b, miR-21, miR-223, miR-27b, miR-10a, miR-143, etc.) expression levels in blood samples and 14 genes (RelA, RelB, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBε, IκBζ, Bcl-3, IKKα, IKKβ, IKKγ/NEMO) relative expression levels in nine tissues. The total number of miRNAs is declining, and RelA, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBζ, Bcl-3, and IKKα expressions are rising in mastitis individuals. So, we suppose that NF-κB pathway is active in mastitis individuals as a result of the decrease inhibition of miRNAs. While in healthy ones, the NF-κB pathway is inactive, because of the miRNAs enhanced inhibition action. However, the specific regulatory mechanism of miRNAs on NF-κB pathway in mastitis Holstein cows needs further investigation. Moreover, due to obvious expression differences, some miRNAs, especially miR-16 and miR-223, may be used as new markers for the dairy mastitis prognosing.     

TGF-β-activated kinase 1 mediates mechanical stress-induced IL-6 expression in osteoblasts

Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca²⁺ influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca²⁺ chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca²⁺ influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.     

NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha

Osteogenesis associated with persistent inflammation or infection exists in a broad range of conditions including rheumatoid arthritis and traumatic bone fracture. The poor outcomes of these conditions will benefit from more effective treatments. Here we investigated the molecular mechanisms and tested NEMO-binding domain peptide as a new approach of circumventing TNF-α inhibition of osteoblast differentiation. Our results showed: TNF-α markedly decreased BMP-2-induced alkaline phosphatase activity in the multipotent myoblast C2C12 cells in a dose dependent manner; stepwise experiments demonstrated that BMP-2-induced Smad1 activity was abrogated by addition of exogenous TNF-α or overexpression of NF-κB, and it was significantly elevated by overexpression of IκBα, an inhibitor of NF-κB; Western blotting showed that TNF-α markedly decreased the amount of phospho-Smad1 in BMP-2-activated C2C12 cells, but it did not alter Smad1 mRNA abundance as measured by real-time PCR; addition of a functional cell-permeable NEMO-binding domain (NBD) peptide antagonized NF-κB activity and ameliorated TNF-α inhibition of osteoblast differentiation. Taken together, our study reveals for the first time that NF-κB activation inhibits osteoblast differentiation by attenuating Smad1 activity and application of NBD peptide ameliorates this inhibitory effect. This could lead to new therapeutic drugs that circumvent the inflammatory inhibition of osteogenesis for treatment of traumatic open fractures with infection, rheumatoid arthritis and other bone loss disorders.     

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Nuclear factor κB (NF-κB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.     

Investigation of neuronal cell type-specific gene expression of Ca2+/calmodulin-dependent protein kinase II

The promoter activity of the rat Ca²⁺/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5′-flanking region of α and β isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5′-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types. Published: April 12, 2002