CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II by Brain Gangliosides

Abstract: Purified rat brain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca²⁺/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca²⁺-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC₅₀=0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca²⁺/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Ischemia-Induced Loss of Brain Calcium/Calmodulin-Dependent Protein Kinase II

Forebrain ischemia in gerbils, produced by brief bilateral carotid occlusion, induced the dramatic loss of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) as determined by both kinase activity assays and western blot analysis. In cortex and hippocampus, cytosolic CaM-kinase II was completely lost within 2-5 min of ischemia. Particulate CaM-kinase II was more stable and decreased in level approximately 40% after 10 min of ischemia followed by 2 h of reperfusion. CaM-kinase II in cerebellum, which does not become ischemic, was not affected. The rapid loss of CaM-kinase II within 2-5 min was quite specific because cytosolic cyclic AMP kinase and protein kinase C in hippocampus were not affected. These data indicate that cytosolic CaM-kinase II is one of the most rapidly degraded proteins after brief ischemia. Because the multifunctional CaM-kinase II has been implicated in the regulation of numerous neuronal functions, its loss may destine the neuronal cell for death.     

Calcium/Calmodulin-dependent Protein Kinase Kinase 2: Roles in Signaling and Pathophysiology

Many cellular Ca(2+)-dependent signaling cascades utilize calmodulin (CaM) as the intracellular Ca(2+) receptor. Ca(2+)/CaM binds and activates a plethora of enzymes, including CaM kinases (CaMKs). CaMKK2 is one of the most versatile of the CaMKs and will phosphorylate and activate CaMKI, CaMKIV, and AMP-activated protein kinase. Cell expression of CaMKK2 is limited, yet CaMKK2 is involved in regulating many important physiological and pathophysiological processes, including energy balance, adiposity, glucose homeostasis, hematopoiesis, inflammation, and cancer. Here, we explore known functions of CaMKK2 and discuss its potential as a target for therapeutic intervention.     

Regulatory Properties of Calcium/Calmodulin-Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.     

Polyamine Regulation of the Microtubule-Associated Protein Kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.     

Effects of Cyclic Nucleotides and Calcium/Calmodulin on Protein Phosphorylation in the CNS of Hirudo medicinalis

Protein phosphorylation plays an important role in the regulation of neural functions. We have studied the phosphorylation of proteins in homogenates of segmental ganglia of the leech Hirudo medicinalis. We describe a number of proteins whose phosphorylation is dependent on calcium/calmodulin or cyclic nucleotides. Most of the proteins whose phosphorylation is increased in the presence of calcium seem to be substrates for cyclic nucleotide-dependent protein kinases. Only two of the phosphoproteins described appear to be specific substrates for calcium/calmodulin protein kinase(s), and at least six phosphoproteins appear to be specific substrates for cyclic nucleotide-dependent kinase(s). The leech nervous system, with large and identifiable neurons, provides a good tool for studies of neural functions, such as learning. The results are discussed in the context of the role of protein phosphorylation on learning processes.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Ca2+ and Calmodulin-Dependent Phosphorylation of Endogenous Synaptic Vesicle Tubulin by a Vesicle-Bound Calmodulin Kinase System

Endogenous synaptic vesicle alpha- and beta-tubulin were shown to be the major substrates for a Ca2+-calmodulin-regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two-dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+-calmodulin kinase. Evidence is presented indicating that the Ca2+-calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. alpha-Tubulin and beta-tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH-L and DPH-M. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+-activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization-dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle alpha- and beta-tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.     

Phosphorylation of P400 Protein by Cyclic AMP-Dependent Protein Kinase and Ca2+/Calmodulin-Dependent Protein Kinase II

Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin-Binding Subunit of a Calmodulin-Dependent Protein Kinase

The major postsynaptic density protein (mPSDp), comprising greater than 50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin-dependent phosphorylation as well as a calmodulin-binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin-binding subunit (p) of tubulin-associated calmodulin-dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the sigma-subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the rho- and sigma-subunits of TACK were established by the following criteria: (1) identical apparent molecular weights; (2) identical calmodulin-binding properties; (3) manifestation of Ca2+-calmodulin-stimulated autophosphorylation; (4) identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two-dimensional gels; (6) homologous two-dimensional tryptic peptide maps; and (7) similar phosphoamino acid-specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin-binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane-bound form in the PSD.     

Roles of Calcium/Calmodulin-Dependent Kinase II in Long-Term Memory Formation in Crickets

Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca²⁺/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca²⁺ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca²⁺ signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.     

Calcium/Diacylglycerol-Dependent Protein Kinase and Its Major 87-Kilodalton Protein Substrate Are Differentially Distributed in Rat Basal Ganglia

When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol-dependent protein kinase (protein kinase C) and an 87-kilodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chemical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87-kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal ganglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87-kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87-kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.     

Role of Calmodulin and Protein Kinase C in Astrocytic Cell Volume Regulation

We investigated the role of Ca(2+)-dependent protein kinases in the regulation of astrocytic cell volume. Calmodulin (CaM) antagonists were used to inhibit CaM and thus Ca2+/CaM-dependent protein kinase. The effect of these inhibitors as well as activators and inhibitors of protein kinase C (PKC) on astrocytic volume was measured in response to hypoosmotic stress and under isoosmotic conditions. In conditions of hypoosmolarity, CaM antagonists had no effect on swelling, but inhibited the regulatory volume decrease. PKC activation facilitated the swelling induced by hypoosmotic stress. PKC inhibitors induced cell shrinkage and inhibited the initial phase of regulatory volume decrease, whereas PKC down-regulation caused pronounced swelling and partial inhibition of regulatory volume decrease. In isoosmotic conditions, CaM antagonists and PKC activation did not affect astrocytic volume, but PKC inhibitors caused shrinking and PKC down-regulation led to swelling of these cells. These studies indicate the importance of Ca(2+)-dependent protein kinases in the regulation of astrocytic cell volume.     

Calcium/Ganglioside-Dependent Protein Kinase Activity in Rat Brain Membrane

The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high-molecular-weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low-molecular-weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+-ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1 X GD1a was the most potent activator tested. Asialo-GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+-dependent, ganglioside-stimulated phosphorylation was qualitatively similar to the pattern for calmodulin-dependent phosphorylation. However, while calmodulin-dependent kinase activity was inhibited with an IC50 of 10 microM trifluoperazine, ganglioside-stimulated kinase was inhibited with an IC50 of 200 microM trifluoperazine. These results indicate that gangliosides have complex effects on membrane-associated kinase activities and suggest that Ca2+-ganglioside complexes are potent stimulators of membrane kinase activity.     

Calcium/Calmodulin-Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that α-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Calmodulin and Ca2+- and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.     

Site Mutation in the Rat μ-Opioid Receptor Demonstrates the Involvement of Calcium/Calmodulin-Dependent Protein Kinase II in Agonist-Mediated Desensitization

Abstract: The rat μ-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the μ agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase II (CaM kinase II) on μ-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the μ-opioid receptor. The increase of desensitization could not be observed with a μ-opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser²⁶¹ and Ser²⁶⁶ by CaM kinase II is involved in the desensitization of the μ-opioid receptor.