Distribution of Calmodulin- and Cyclic AMP-Stimulated Protein Kinases in Synaptosomes

The subcellular location of calmodulin- and cyclic AMP stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [gamma-32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin- or cyclic AMP-stimulated protein phosphorylation. When assessed by calmodulin-stimulated autophosphorylation of the alpha subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin-stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP-stimulated phosphorylation of endogenous substrates, no cyclic AMP-stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP-stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin- and cyclic AMP-stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.     

Neurobiological consequences of acute footshock stress: effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla

Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40‐min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone, corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, ventral tegmental area and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31 phosphorylation than the mPFC, presumably because of their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma adrenocorticotropic hormone at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal‐regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min. TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, pre‐synaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the hypothalamic‐pituitary‐adrenal axis.

     

Autophosphorylation of neuronal calcium/calmodulin-stimulated protein kinase II

A unique feature of neuronal calcium/calmodulin-stimulated protein kinase II (CaM-PK II) is its autophosphorylation. A number of sites are involved and, depending on the in vitro conditions used, three serine and six threonine residues have been tentatively identified as autophosphorylation sites in the alpha subunit. These sites fall into three categories. Primary sites are phosphorylated in the presence of calcium and calmodulin, but under limiting conditions of temperature, ATP, Mg2+, or time. Secondary sites are phosphorylated in the presence of calcium and calmodulin under nonlimiting conditions. Autonomous sites are phosphorylated in the absence of calcium and calmodulin after initial phosphorylation of Thr-286. Mechanisms that lead to a decrease in CaM-PK II autophosphorylation include the thermolability of the enzyme and the activity of protein phosphatases. A range of in vitro inhibitors of CaM-PK II autophosphorylation have recently been identified. Autophosphorylation of CaM-PK II leads to a number of consequences in vitro, including generation of autonomous activity and subcellular redistribution, as well as alterations in conformation, activity, calmodulin binding, substrate specificity, and susceptibility to proteolysis. It is established that CaM-PK II is autophos-phorylated in neuronal cells under basal conditions. Depolarization and/or activation of receptors that lead to an increase in intracellular calcium induces a marked rise in the autophosphorylation of CaM-PK II in situ. The incorporation of phosphate is mainly found on Thr-286, but other sites are also phosphorylated at a slower rate. One consequence of the increase in CaM-PK II autophosphorylation in situ is an increase in the level of autonomous kinase activity. It is proposed that the formation of an autonomous enzyme is only one of the consequences of CaM-PK II autophosphorylation in situ and that some of the other consequences observed in vitro will also be seen. CaM-PK II is involved in the control of neuronal plasticity, including neurotransmitter release and long-term modulation of postreceptor events. In order to understand the function of CaM-PK II, it will be essential to ascertain more fully the mechanisms of its autophosphorylation in situ, including especially the sites involved, the consequences of this autophosphorylation for the kinase activity, and the relationships between the state of CaM-PK II autophosphorylation and the physiological events within neurons.     

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.     

Microhabitats of sharknose goby (Elacatinus evelynae) cleaning stations and their links with cleaning behaviour

Coral Reefs - Coral reefs are renowned for the complexity of their habitat structures and their resulting ability to host more species per unit area than any another marine ecosystem. Dedicated...     

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.     

Dominance and diversity in the primary human CD4 T cell response to replication-competent vaccinia virus

Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication-incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relationship between Ab and CD4 T cell specificity for complex pathogens.     

Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.