Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An afterdischarge in the bag cell neurons of Aplysia was previously shown to be associated with calcium entry into these cells and with changes in the phosphorylation state of at least two bag cell proteins (BC-I and BC-II). We have now investigated the role of calcium plus calmodulin (Ca/CaM) in the control of phosphorylation of Aplysia nervous system proteins, including those of the bag cell neurons. In cell-free preparations of Aplysia CNS, we demonstrated Ca/CaM-stimulated protein phosphorylation that could be inhibited by the calmodulin-blocking drugs R24571 , trifluoperazine, chlorpromazine, and W7 . A number of substrate proteins for Ca/CaM-dependent protein phosphorylation with Mr values from 17,000 to 310,000 were consistently observed in homogenates of the Aplysia CNS. In the bag cells, we found that a major substrate for Ca/CaM-dependent protein phosphorylation was the bag cell-specific, Mr = 21,000 protein (BC-II). BC-I (Mr = 33,000), on the other hand, appeared not to be a substrate for a Ca/CaM-dependent protein kinase. We found that there are a minimum of two Ca/CaM-dependent protein kinases in the Aplysia nervous system. These enzymes were distinguished on the basis of their subcellular distribution and their ability to phosphorylate distinct sites on synapsin I, an exogenous neuronal protein from vertebrates. Phosphorylation by one of these kinases (calmodulin kinase I) was on a site recovered in an Mr = 10,000 proteolytic fragment of synapsin I, and phosphorylation by the other (calmodulin kinase II) was on a site recovered in an Mr = 30,000 fragment. The predominant enzyme in the Aplysia CNS, as in the mammalian nervous system, was calmodulin kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calcium/calmodulin-dependent protein phosphorylation in the nervous system of Aplysia. 672 49

Previous work has shown that serotonin induces an increase in membrane K+ conductance in Aplysia neuron R15 and that this response is mediated by cAMP. The present study examines the role of protein phosphorylation in the response to serotonin. A specific inhibitor of cAMP-dependent protein kinase was injected intracellularly into neuron R15. The injection blocked the serotonin-induced increase in K+ conductance completely for at least 4 hours. The blockage was selective because the cell's response to dopamine was not inhibited. Furthermore, the blockage was specifically produced by protein kinase inhibitor because injection of other proteins (alpha-bungarotoxin and bovine serum albumin) did not affect the serotonin response. The serotonin response recovered fully 5-13 hours after the injection, presumably as a result of intracellular proteolysis of the protein kinase inhibitor. The results indicate that protein phosphorylation is a necessary step in the process that leads to activation of K+ channels by serotonin in neuron R15.
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PMID:Intracellular injection of protein kinase inhibitor blocks the serotonin-induced increase in K+ conductance in Aplysia neuron R15. 695 30

Increases in activity of both protein kinase A (PKA) and protein kinase C (PKC) contribute to short-term facilitation of Aplysia sensorimotor synapses evoked by serotonin (5-HT). We report here that increasing levels of cAMP in sensory neurons evokes increases in both synaptic efficacy and in the number of sensory neuron varicosities contacting the major axons of motor cell L7 at intermediate times (3 hr) that persist for 24 hr. Treatment with phorbol esters results in a large transient increase in synaptic efficacy that is accompanied by a large transient increase in the number of sensory neuron varicosities with the newest varicosities most susceptible to elimination. The reversal of the synaptic facilitation and the structural changes does not appear to be the result of long-term inhibitory actions of persistent PKC activation by phorbol esters, since changes in synaptic efficacy can be evoked by additional applications of either phorbol esters or 5-HT. The short-lived changes in structure evoked by phorbol esters occur in preexisting sensory neurites and not by new growth, since increases in PKC activity with phorbol esters lead to reductions in neurite extension and to retractions by sensory neuron growth cones. The action of phorbol esters on growth cone extension is reversible with washout. The results suggest that increases in PKA and PKC activities by 5-HT contribute to short (minutes) and intermediate (hours) forms of facilitation of sensorimotor synapses while increases in PKA activity also mediate long-term (days) maintenance of synaptic facilitation.
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PMID:Transient versus persistent functional and structural changes associated with facilitation of Aplysia sensorimotor synapses are second messenger dependent. 747 3

In response to the facilitating neurotransmitter serotonin (5-HT), the cAMP-dependent protein kinase (PKA) acquires a special mnemonic characteristic in Aplysia sensory neurons. PKA becomes persistently activated at basal cAMP concentrations owing to a decreased regulatory (R) to catalytic (C) subunit ratio. We previously implicated ubiquitin-mediated proteolysis in this selective loss of R. Here we show that ubiquitin (Ub), Ub-conjugates and proteasomes are present in cell bodies, axon, neuropil and nerve terminals of Aplysia neurons. Because R subunits are not decreased in muscle exposed to 5-HT, comparison of the two tissues provides a tractable approach to determine how the Ub pathway is regulated. We compared the structure of M1, the muscle-specific R isoform, to that of N4, a major neuronal R isoform, to rule out the possibility that the differences in their stability result from differences in structure. We present evidence that N4 and M1 are encoded by identical transcripts; they also behave similarly as protein substrates for the Ub pathway in extracts of the two tissues. Nervous tissue contains 20-times more free Ub, but we present evidence that the susceptibility of R subunits to degradation in neurons relative to muscle results from the greater capacity of neurons to degrade ubiquitinated proteins through the proteasome. Thus, factors that regulate the activity of proteasomes could underlie the enhanced degradation of R subunits in long-term sensitization.
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PMID:Persistent activation of cAMP-dependent protein kinase by regulated proteolysis suggests a neuron-specific function of the ubiquitin system in Aplysia. 747 10

The unusually large (approximately 600 to > 3000 kDa) myosin-associated proteins of the titin/twitchin superfamily are considered to be important cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60-240 modular fibronectin-type-III and immunoglobulin-C2 repeats in these proteins which further contain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitchin was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19-kDa myosin regulatory light chains. The twitchin kinase phosphorylated purified light chains on Thr15 in a region which shared a high degree of similarity with the phosphorylation site for vertebrate smooth muscle myosin light chain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the first potential substrate for any of the giant protein kinases and support a dual role of twitchin in molluscan muscle as a cytoskeletal protein as well as a myosin light chain kinase.
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PMID:Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase. 758 84

The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C-terminus which bears similarity to the substrate. A calmodulin-binding sequence has also been identified in the C-terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin-binding site in the titin kinase C-terminal sequence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C-terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interaction of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt alpha-helical conformations in solution. Helicity increases upon binding of calmodulin in a calcium-dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin-binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.
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PMID:A calmodulin-binding sequence in the C-terminus of human cardiac titin kinase. 760 48

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits were monitored by confocal fluorescence microscopy. Bath application of serotonin produced a much greater elevation of [cAMP] in the processes than in the central bodies of the neurons. The resulting gradients must drive a sizable centripetal flux of cAMP because direct microinjection of cAMP showed that it diffused readily. Perinuclear increases in [cAMP] slowly caused the translocation of the freed catalytic subunit into the nucleus to an extent proportional to the percentage of its dissociation from the regulatory subunit.
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PMID:Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. 768 36

We have examined the absorbance of a charge-transfer transition near 760 nm, known as band III, in several hemoproteins and heme complexes. The band III position correlates with the rate of carbon monoxide binding to the heme. A band III present at 760 nm indicates an unfavorable geometry of the heme for carbon monoxide binding; a red-shift of the band III to 765 nm indicates a less-constrained geometry of the heme as evidenced by higher carbon monoxide association rates. The band III position correlates well with the Raman frequency of the Fe-His(F8) bond as suggested previously for normal hemoglobin A [Sassaroli, M. & Rousseau, D. L. (1987) Biochemistry 26, 3092-3098]. Aplysia myoglobin and the chimeric heme protein kinase FixL from Bradyrhizobium japonicum, hemoproteins with an apolar residue in place of the highly conserved polar histidine E7, do not fit the relationship between the band III position and the rate of binding of carbon monoxide to the heme. With these few exceptions, the measurement of band III appears to be a practical means to probe the stretch frequency of the Fe-His(F8) bond.
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PMID:Correlation of carbon monoxide association rates and the position of absorption band III in hemoproteins. 773 61

Both for Aplysia and Drosophila a key role in the molecular mechanism of learning and memory processes has been assigned to the cAMP cascade. In any learning process a short-time stimulus has to be translated into long-lasting changes. The molecular correlate must be a cascade of biochemical reactions with different kinetics, functionally interlinked and operating in overlapping time ranges. Biochemical studies in Drosophila have led to the suggestion that one of these steps is a proteolytic modification of the regulatory subunits of protein kinase A. A quantitative analysis of the relaxation kinetics of a system of protein kinase A, phosphatases and a calcium-dependent protease can give an image of essential characteristics of learning behaviour in Drosophila.
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PMID:Posttranslational modification of protein kinase A. The link between short-term and long-term memory. 775 4

1. In the pleural mechanoafferent sensory neurons of Aplysia, serotonin (5-HT)-induced spike broadening consists of at least two components: a cAMP and protein kinase A (PKA)-dependent, rapidly developing component and a protein kinase C (PKC)-dependent, slowly developing component. Voltage-clamp experiments were conducted to identify currents that are modulated by PKC and thus may contribute to the slowly developing component of 5-HT-induced spike broadening. 2. We compared the effects of phorbol esters, activators of PKC, on membrane currents with those of 5-HT. Bath application of 5-HT had complex modulatory effects on currents elicited by voltage-clamp pulses to potentials > 0 mV. The kinetics of both activation and inactivation of the membrane currents were slowed by 5-HT. This led to a decrease in an outward current at the beginning of the voltage-clamp pulse and an increase at the end of the pulse. Previous work has shown that these effects represent, in part, the modulation of a large, voltage-dependent K+ current (IK,V) by 5-HT. 3. Active phorbol esters mimicked some of the actions of 5-HT on membrane currents in that they slowed activation and inactivation kinetics of current responses to voltage-clamp pulses more positive than 0 mV. This led to a decrease in an outward current at the beginning of the pulse and an increase at the end of the pulse. Because inactive phorbols did not mimic the actions of 5-HT, the effects of active phorbol esters appeared to be PKC specific. In addition, preexposure of the sensory neurons to active phorbol esters appeared to occlude the modulatory actions of 5-HT on IK,V. Thus it is likely that modulation of IK,V by 5-HT is mediated, at lease in part, by PKC. 4. To further characterize which currents were modulated by PKC, low concentrations of tetraethylammonium (TEA, 2 mM) were used to block Ca(2+)-activated K+ current (IK,Ca). Low TEA partially blocked the phorbol ester-induced increase of the outward current at the end of voltage-clamp pulses. These results agreed with previous reports that activation of PKC enhanced a fast component of IK,Ca in these sensory neurons. Such an enhancement would lead to an increase in outward current that should be blocked by low TEA. Low TEA, however, did not affect phorbol ester-induced decrease of the outward current at the beginning of pulse, where the predominant current is IK,V, which is less sensitive to TEA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activators of protein kinase C mimic serotonin-induced modulation of a voltage-dependent potassium current in pleural sensory neurons of Aplysia. 780 8


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