Involvement of calcium/calmodulin-dependent protein kinase kinase in meiotic maturation of pig oocytes

Calcium (Ca(2+))/calmodulin-dependent protein kinase kinase (CaMKK) is a novel member of Ca(2+)/calmodulin-dependent protein kinase (CaMK) family, whose physiological roles in regulating meiotic cell cycle needs to be determined. We showed by Western blot that CaMKK was expressed in pig oocytes at various maturation stages. Confocal microscopy was employed to observe CaMKK distribution. In oocytes at the germinal vesicle (GV) or prometaphase I (pro-MI) stage, CaMKK was distributed in the nucleus, around the condensed chromatin and the cortex of the cell. At metaphase I (MI) stage, CaMKK was concentrated in the cortex of the cell. After transition to anaphase I or telophase I stage, CaMKK was detected around the separating chromosomes and in the cortex of the cell. At metaphase II (MII) stage, CaMKK was localized to the cortex of the cell, with a thicker area near the first polar body (PB1). Treatment of pig cumulus-enclosed oocytes with STO-609, a membrane-permeable CaMKK inhibitor, resulted in the delay/inhibition of the meiotic resumption and the inhibition of first polar body emission. The correlation between CaMKK and microfilaments during meiotic maturation of pig oocytes was then studied. CaMKK and microfilaments were colocalized from MI to MII during porcine oocyte maturation. After oocytes were treated with STO-609, microfilaments were depolymerized, while in oocytes exposed to cytochalasin B (CB), a microfilament polymerization inhibitor, CaMKK became diffused evenly throughout the cell. These data suggest that CaMKK is involved in regulating the meiotic cell cycle probably by interacting with microfilaments in pig oocytes.     

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)alpha autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCalpha and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCalpha activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on -aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (I) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and Imus), respectively in the Mg2+-free external solution containing 1 mol/L glycine at a holding potential (VH) of 40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 mol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 mol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 mol/L) or La3+ (30 mol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.     

Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association

Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta subunits were analyzed using light scattering to determine a mechanism for self-association. Under identical reaction conditions, only alphaCaM-kinase II holoenzymes self-associated. Self-association was detected at a remarkably low enzyme concentration (0.14 microM or 7 microg/mL). Light scattering revealed two phases of self-association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2-3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self-association required activation by Ca(2+)/CaM and was strongly dependent on pH. Self-association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and inhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II potently prevented self-association, whereas the peptide substrate syntide-2 did not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A model for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit-interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM-kinase II previously reported in models of ischemia.     

High throughput screening,docking, and molecular dynamics studies to identify potential inhibitors of human calcium/calmodulin-dependent protein kinase IV

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is associated with many diseases including cancer and neurodegenerative disorders and thus being considered as a potential drug target. Here, we have employed the knowledge of three-dimensional structure of CAMKIV to identify new inhibitors for possible therapeutic intervention. We have employed virtual high throughput screening of 12,500 natural compounds of Zinc database to screen the best possible inhibitors of CAMKIV. Subsequently, 40 compounds which showed significant docking scores (?11.6 to ?10.0?kcal/mol) were selected and further filtered through Lipinski rule and drug likeness parameter to get best inhibitors of CAMKIV. Docking results are indicating that ligands are binding to the hydrophobic cavity of the kinase domain of CAMKIV and forming a significant number of non-covalent interactions. Four compounds, ZINC02098378, ZINC12866674, ZINC04293413, and ZINC13403020, showing excellent binding affinity and drug likeness were subjected to molecular dynamics simulation to evaluate their mechanism of interaction and stability of protein-ligand complex. Our observations clearly suggesting that these selected ligands may be further employed for therapeutic intervention to address CAMKIV associated diseases.

Communicated by Ramaswamy H. Sarma     

Calcium/calmodulin-dependent protein kinase II expression in motor neurons: Effect of axotomy

Although Ca²⁺/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIα and IIβ are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIβ antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIα, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIα immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIα is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIα as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIβ down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIα appears to support axonal regrowth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 796–810, 1997     

Phosphorylation and activation of tryptophan hydroxylase 2: identification of serine-19 as the substrate site for calcium, calmodulin-dependent protein kinase II

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. TPH was once thought to be a single-gene product but it is now known to exist in two isoforms. TPH1 is found in the periphery and pineal gland whereas TPH2 is expressed specifically in the CNS. Both TPH isoforms are known to be regulated by protein kinase-dependent phosphorylation and the sites of modification of TPH1 by protein kinase A have been identified. While TPH2 is activated by calcium, calmodulin-dependent protein kinase II (CaMKII), the sites at which this isoform is modified are not known. Treatment of wild-type TPH2 with CaMKII followed by mass spectrometry analysis revealed that the enzyme was activated and phosphorylated at a single site, serine-19. Mutagenesis of serine-19 to alanine did not alter the catalytic function of TPH2 but this mutant enzyme was neither activated nor phosphorylated by CaMKII. A phosphopeptide bracketing phosphoserine-19 in TPH2 was used as an antigen to generate polyclonal antibodies against phosphoserine-19. The antibodies are highly specific for phosphoserine-19 in TPH2. The antibodies do not react with wild-type TPH2 or TPH1 and they do not recognize phophoserine-58 or phosphoserine-260 in TPH1. These results establish that activation of TPH2 by CaMKII is mediated by phosphorylation of serine-19 within the regulatory domain of the enzyme. Production of a specific antibody against the CaMKII phosphorylation site in TPH2 represents a valuable tool to advance the study of the mechanisms regulating the function of this important enzyme.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (I_gaba and I_Mus), respectively in the Mg²⁺-free external solution containing 1 μmol/L glycine at a holding potential (V _H ) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of I_gaba; (ii) when the neurons were incubated in a Ca²⁺-free bath or pre-loaded with a membrane-permeable Ca²⁺ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on I_GAba disappeared. Cd²⁺ (10 μmol/L) or La³⁺ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of I_gaba by NMDA application; (iii) the suppression of I_GAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca²⁺-dependent and CaMKII is involved in the process of the Ca²⁺-dependent inhibition.     

莱茵衣藻丝/苏氨酸蛋白激酶突变株蓝光响应的表征

为研究莱茵衣藻丝/苏氨酸蛋白激酶(silk/threonine protein kinase, STK)介导藻细胞蓝光响应的分子机制,本文对蓝光胁迫下莱茵衣藻STK突变株系crstk11(AphvIII盒反向插入stk11基因编码区)进行表型鉴定及转录组分析。表型鉴定显示,正常光(白光)下,野生型株CC5325与突变株crstk11的生长和色素含量差异较小;蓝光抑制了crstk11藻细胞生长和叶绿素合成,但显著促进类胡萝卜素积累。转录组分析显示,蓝光处理4 d,突变株(STK4) vs.野生型(wild type, WT4)共检测到差异表达基因(differential expression genes, DEGs) 860条(559个上调,301个下调)。高蓝光处理8 d,STK8 vs. WT8共获得1 088个DEGs (468个上调,620个下调)。KEGG富集分析发现,与CC5325相比,crstk11蓝光响应基因主要参与胞内光合作用催化活性、碳代谢和色素合成等。其中,上调基因包括psaA、psaB和psaC,psbA、psbB、psbC、psbD、psbH和psbL,pet...     

The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKII delta using a wheat cell-free system. The cell-free mixture expressing CaMKII delta was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKII delta phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKII delta phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKII delta may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases.     

A novel serine/threonine kinase gene,Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects. © 1996 Wiley-Liss, Inc.     

Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin

Recently, a novel type of calcium-dependent protein kinase (CDPK) that requires neither calmodulin nor phospholipids for activation, has been described in plants. We have isolated a cDNA clone for carrot CDPK by probing a library of somatic embryo cDNAs with oligonucleotides corresponding to highly conserved regions of protein kinases. The product of this gene overexpressed in Escherichia coli reacted strongly with monoclonal antibodies to soybean CDPK. The deduced amino acid sequence of carrot CDPK reveals two major functional domains. An N-terminal catalytic domain with greatest homology to calcium/calmodulin-dependent protein kinase type II from rat brain is coupled to a C-terminal calcium-binding domain resembling calmodulin. These features of the primary sequence explain how CDPK binds calcium and suggest a model for CDPK regulation based on similarities to animal calcium/calmodulin-dependent protein kinases.     

BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and palmitoylation, and interacts with alpha, but not beta, subunit of Ca/calmodulin-dependent protein kinase II

We cloned a rat BAALC 1-6-8 isoform cDNA (GenBank Accession No. AB073318) that encoded a 22-kDa protein, and identified endogenous BAALC 1-6-8 protein in the brain. The gene was expressed widely in the frontal part of the brain, and the protein was localized to the synaptic sites and was increased in parallel with synaptogenesis. The protein interacted with the alpha, but not beta, subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIalpha). The interaction occurred between the N-terminal 35-amino-acid region of BAALC 1-6-8 protein and the C-terminal end of the regulatory domain of CaMKIIalpha, which contains alpha isoform-specific sequence. Thus, the interaction may be CaMKIIalpha-specific. We also found that BAALC 1-6-8 protein, as well as CaMKIIalpha, was localized to lipid rafts and that both myristoylation and palmitoylation of BAALC 1-6-8 N-terminal portion were required for targeting of the protein into lipid rafts. These findings suggest that BAALC 1-6-8 protein play a synaptic role at the postsynaptic lipid raft possibly through interaction with CaMKIIalpha.     

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E₂ (PGE₂) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE₂ provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca⁺⁺ channel blockers, verapamil and nifedipine, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂ as did the phorbol ester PMA, which activates Ca⁺⁺-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE₂ did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE₂ secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE₂ does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂ receptor signalling pathways. Taken together, our results suggest that PGE₂ modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.     

ACE I/D and MTHFR C677T polymorphisms are significantly associated with type 2 diabetes in Arab ethnicity: A meta-analysis

In this meta-analysis study, SNPs were investigated for their association with type 2 diabetes (T2D) in both Arab and Caucasian ethnicities. A total of 55 SNPs were analyzed, of which 11 fulfilled the selection criteria, and were used for analysis. It was found that TCF7L2 rs7903146 was significantly associated with a pooled OR of 1.155 (95%C.I. = 1.059–1.259), p < 0.0001 and I² = 78.30% among the Arab population, whereas among Caucasians, the pooled OR was 1.45 (95%C.I. = 1.386–1.516), p < 0.0001 and I² = 77.20%. KCNJ11 rs5219 was significantly associated in both the populations with a pooled OR of 1.176(1.092–1.268), p < 0.0001 and I² = 32.40% in Caucasians and a pooled OR of 1.28(1.111–1.475), p = 0.001 among Arabs. The ACE I/D polymorphism was found to be significantly associated with a pooled OR of 1.992 (95%C.I. = 1.774–2.236), p < 0.0001 and I² = 83.20% among the Arab population, whereas among Caucasians, the pooled OR was 1.078 (95%C.I. = 0.993–1.17), p = 0.073 and I² = 0%. Similarly, MTHFR C677T polymorphism was also found to be significantly associated among Arabs with a pooled OR of 1.924 (95%C.I. = 1.606–2.304), p < 0.0001 and I² = 27.20%, whereas among Caucasians, the pooled OR was 0.986 (95%C.I. = 0.868–1.122), p = 0.835 and I² = 0%. Meanwhile PPARG-2 Pro12Ala, CDKN2A/2B rs10811661, IGF2BP2 rs4402960, HHEX rs7923837, CDKAL1 rs7754840, EXT2 rs1113132 and SLC30A8 rs13266634 were found to have no significant association with T2D among Arabs. In conclusion, it seems from this study that both Arabs and Caucasians have different SNPs associated with T2D. Moreover, this study sheds light on the profound necessity for further investigations addressing the question of the genetic components of T2D in Arabs.