Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factor FA has further been identified as a cAMP and Ca(2+)-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factor FA could phosphorylate synapsin I with a low Km value of about 2 microM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factor FA specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factor FA could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factor FA as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.
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PMID:Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules. 133 16

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.
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PMID:The phosphorylation of kinesin regulates its binding to synaptic vesicles. 142 30

Synapsin I, a neuron-specific, synaptic vesicle-associated phosphoprotein, is thought to play an important role in synaptic vesicle function. Recent microinjection studies have shown that synapsin I inhibits neurotransmitter release at the squid giant synapse and that the inhibitory effect is abolished by phosphorylation of the synapsin I molecule (Llinas et al., 1985). We have considered the possibility that synapsin I might modulate release by regulating the ability of synaptic vesicles to move to, or fuse with, the plasma membrane. Since it is not yet possible to examine these mechanisms in the intact nerve terminal, we have used video-enhanced microscopy to study synaptic vesicle mobility in axoplasm extruded from the squid giant axon. We report here that the dephosphorylated form of synapsin I inhibits organelle movement along microtubules within the interior of extruded axoplasm and that phosphorylation of synapsin I on sites 2 and 3 by calcium/calmodulin-dependent protein kinase II removes this inhibitory effect. Phosphorylation of synapsin I on site 1 by the catalytic subunit of cAMP-dependent protein kinase only partially reduces the inhibitory effect. In contrast to the inhibition of movement along microtubules seen within the interior of the axoplasm, movement along isolated microtubules protruding from the edges of the axoplasm is unaffected by dephospho-synapsin I, despite the fact that the synapsin I concentration is higher there. Thus, synapsin I does not appear to inhibit the fast axonal transport mechanism itself. Rather, these results are consistent with the possibility that dephospho-synapsin I acts by a crosslinking mechanism involving some component(s) of the cytoskeleton, such as F-actin, to create a dense network that restricts organelle movement. The relevance of the present observations to regulation of neurotransmitter release is discussed.
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PMID:Phosphorylation-dependent inhibition by synapsin I of organelle movement in squid axoplasm. 251 74

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.
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PMID:Properties of several protein kinases that copurify with rat spinal cord neurofilaments. 275 40

Sensitization of the gill- and siphon-withdrawal reflex in Aplysia is thought to result from a set of molecular processes with different time courses: short-term sensitization is explained by cyclic AMP-dependent modulation of ion-channel function in sensory neurons lasting minutes; memory that endures for hours or longer, by the expression and distribution within the neurons of new gene products. Because gene induction and axonal transport are relatively slow, there may also be a need for a distinct form of intermediate memory to bridge the short- and long-term processes. We now report that a protocol producing long-term sensitization results in a decrease in the amount of regulatory subunits of the cAMP-dependent protein kinase in animals 24 h after training, with no effect on the catalytic subunit. The loss appears to be post-translational. Because a decrease in the ratio of regulatory to catalytic subunits would result in elevated kinase activity after cAMP has returned to its unstimulated concentration in sensory cells, it could be the molecular mechanism of intermediate memory.
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PMID:A molecular mechanism for long-term sensitization in Aplysia. 304 Dec 25

The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. To study the effect of phosphorylation and substrate modulation on tau's distinct activities to promote growth of existing microtubules and nucleation of new ones, we phosphorylated bacterially expressed human tau by cAMP-dependent protein kinase in the absence or presence of heparin, an acidic substrate modulator. We found that heparin increased phosphorylation of tau by a factor of more than 2 and produced tau bands with decreased electrophoretic mobility. We demonstrate that phosphorylation of tau in the absence or presence of heparin similarly reduced tau's activity to promote microtubule growth, whereas tau's activity to promote microtubules was suppressed much more after phosphorylation in the presence of heparin. Using recombinant tau fragments we showed that heparin-induced phosphorylation caused a specific shift in electrophoretic mobility indicative of a change in tau's conformation. By aminoterminal sequencing of a tau fragment starting at residue 154 we provide evidence that phosphorylation of serine 156 is responsible for this mobility shift and for the effect on tau's nucleation activity. We conclude that tau's activities to promote growth of existing microtubules and nucleation of new ones are differentially affected by the phosphorylation of specific tau residues. Regulation of the phosphorylation state by substrate modulation may play an important role in regulating tau's function.
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PMID:Differential effect of phosphorylation and substrate modulation on tau's ability to promote microtubule growth and nucleation. 816 74

In the present study the role of cAMP for axonal outgrowth and Schwann cell proliferation was studied using the cultured frog sciatic nerve. An intrinsic rise in nerve and ganglionic cAMP could be measured as a response to nerve injury, both in vitro and in vivo. Treatment with 0.1-1.0 microM forskolin, an activator of the cAMP-generating enzyme adenylyl cyclase, increased the cAMP content up to 13-fold, but was yet without effect on axonal outgrowth during an 8-day culturing period. HA-1004, an inhibitor of cAMP-dependent protein kinase, also lacked effect on the regeneration. In contrast, the proliferation of Schwann cells, measured as [3H]thymidine incorporation, was inhibited to about 70% of control by forskolin, whereas HA-1004 stimulated proliferation to approximately 130% of control. The results suggest that cAMP is involved in the injury-induced proliferation of Schwann cells of an adult peripheral nerve but that it lacks a central role in the regeneration of sensory axons of such nerves.
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PMID:Increased cyclic AMP in in vitro regenerating frog sciatic nerves inhibits Schwann cell proliferation but has no effect on axonal outgrowth. 853 Dec 26

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.
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PMID:Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons. 995 2

During the development of neuronal circuits, axonal growth cones can contact many inappropriate targets before they reach an appropriate postsynaptic partner. Although it is well known that the contact with synaptic partners upregulates the secretory machinery of the presynaptic neuron, little is known about the signaling mechanisms involved in preventing the formation of connections with inappropriate target cells. Here, we show that the contact with a nonphysiological postsynaptic target inhibits neurotransmitter release from axonal terminals of the Helix serotonergic neuron C1 by means of an active mechanism requiring ongoing protein synthesis and leading to the inhibition of cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (Erk) pathways. The reversal of the inhibitory effect of the nonphysiological target by blockade of protein synthesis was prevented by cAMP-PKA or MAPK-Erk inhibitors, whereas disinhibition of neurotransmitter release promoted by cAMP-PKA activation was not affected by MAPK-Erk inhibitors. The data indicate that the inhibitory effect of the nonphysiological target on neurotransmitter release is an active process that requires protein synthesis and involves the downregulation of the MAPK-Erk and cAMP-PKA pathways, the same protein kinases that are activated after contact with a physiological target neuron. These mechanisms could play a relevant role in the prevention of synapse formation between inappropriate partners by modulating the neurotransmitter release capability of growing nerve terminals according to the nature of the targets contacted during their development.
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PMID:Inhibition of neurotransmitter release by a nonphysiological target requires protein synthesis and involves cAMP-dependent and mitogen-activated protein kinases. 1516 98

Mammalian neuronal cells abundantly express a de-ubiquitinating isozyme, ubiquitin carboxy-terminal hydrolase L1 (UCH L1). Loss of UCH L1 function causes dying-back type of axonal degeneration. However, the function of UCH L1 in neuronal cells remains elusive. Here we show that overexpression of UCH L1 potentiated ATP-induced currents due to the activation of P2X receptors that are widely distributed in the brain and involved in various biological activities including neurosecretion. ATP-induced inward currents were measured in mock-, wild-type or mutant (C90S)-UCH L1-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in both wild-type and C90S UCH L1-transfected cells, suggesting that hydrolase activity was not involved but increased level of mono-ubiquitin might play an important role. The increased currents were dependent on cAMP-dependent protein kinase (PKA) and Ca2+ and calmodulin-dependent protein kinase (CaMKII) but not protein kinase C. In addition, ATP-induced currents were likely to be modified via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) that is regulated by PKA and phosphatases. Our finding shows the first evidence that there is a relationship between UCH L1 and neurotransmitter receptor, suggesting that UCH L1 may play an important role in synaptic activity.
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PMID:Potentiation of ATP-induced currents due to the activation of P2X receptors by ubiquitin carboxy-terminal hydrolase L1. 1571 57


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