Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Microtubule-associated protein tau is a neuronal phosphoprotein that promotes microtubule assembly in vitro and has been shown to play a role in the development of axonal morphology. Tau can be phosphorylated in vitro by several kinases, some of which cause a change in the conformation and activities of tau. Here we report the consequences of converting two of the protein kinase A phosphorylation sites (positions 156 and 327), first to alanine to eliminate phosphorylation, and second to aspartate, to mimic phosphorylation. We show that a serine to aspartate mutation at position 327 results in a conformational change similar to that caused by phosphorylation of this residue. This mutation does not affect the activities of tau in microtubule assembly as compared with wild-type tau. However, an additional mutation at position 156 to aspartate drastically decreases the microtubule nucleation activity of tau but does not affect the activity of tau to promote microtubule growth. All constructs are similarly bound to microtubules and promote process formation when expressed in cytochalasin-treated PC12 cells. We conclude that serine to aspartate mutations provide a useful system for analyzing the effect of individual phosphorylation sites on the conformation and function of tau in vitro and in cells. The results provide evidence that microtubule growth and nucleation can be differentially affected by phosphorylation of individual residues in a region amino-terminally flanking the microtubule binding domain of tau.
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PMID:Conversion of serine to aspartate imitates phosphorylation-induced changes in the structure and function of microtubule-associated protein tau. 907 70

Axonal microtubules have two essential roles: providing the track for organelle transport and forming the cytoskeletal framework to maintain axonal morphology. Microtubule-associated proteins (MAPs) are essential for the formation of cytoskeletal architecture. However, they may have additional roles on the regulation of organelle transport by their interaction with motor proteins on the microtubules. We first examined the effects of axonal MAPs on the organelle movement along microtubules in a heterologous system using COS fibroblasts, which express no axonal MAPs, such as tau or MAP2C. Transfection of tau or MAP2C gene suppressed organelle movement almost completely in this cell type, hence interaction of axonal MAPs with microtubules interferes with organelle transports. It is known that the phosphorylation of MAPs reduces their interaction with microtubules. In this sense, phosphorylation of MAPs can be a good candidate for the molecular switch to regulate the organelle transport. As a second set of experiments, we investigated the effects of modulating cAMP dependent protein kinase pathway on organelle transports in primary sensory neurons, where high-molecular-weight tau protein is the major MAP. We found that the application of dibutyryl cAMP enhanced transports of large organelles in the axon. Furthermore, this drug treatment phosphorylated endogenous tau protein and thus reduced the affinity of tau to microtubules. These results indicate that axonal MAPs can work as a phosphorylation-dependent regulator of organelle transport. Local activation of protein kinase pathways in the axon might play an important role on the segregation of microtubules serving for either organelle transport or cytoskeletal architecture.
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PMID:Microtubule-associated proteins regulate microtubule function as the track for intracellular membrane organelle transports. 911 34

To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of L-[35S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.
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PMID:Increased solubility of high-molecular-mass neurofilament subunit by suppression of dephosphorylation: its relation to axonal transport. 916 53

Certain unc mutants in the nematode Caenorhabditis elegans, such as unc-14 and unc-51, show abnormal axonal elongation and axonal structures. We cloned the unc-51 gene previously and predicted that it encodes a novel serine/threonine protein kinase. In this study, we precisely localized the activity to rescue an unc-14 mutation. Also, we identified four cDNA clones encoded by the unc-14 rescuing region, in screens for proteins that bind to UNC-51 using a yeast two-hybrid system. A mutation site in the cDNA was identified for each of the six unc-14 mutants, establishing that the unc-14 gene was cloned. The unc-14 gene encodes a novel protein of 665 amino acids, and is coexpressed with the unc-51 gene in the cell bodies and axons of almost all neurons including DD/VD and hermaphrodite-specific neurons. Another clone recovered in the two-hybrid screen encodes a carboxy-terminal region of UNC-51. Analysis using the yeast two-hybrid system suggested that a central region of UNC-14 bound to a carboxy-terminal region of UNC-51, and that the UNC-51 carboxy-terminal region oligomerized. In in vitro binding studies using recombinant fusion proteins, UNC-14 interacted with UNC-51 directly. We propose that UNC-51 protein kinase acts as an oligomer, and that UNC-14 is a regulator of UNC-51, in axonal elongation and guidance.
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PMID:The UNC-14 protein required for axonal elongation and guidance in Caenorhabditis elegans interacts with the serine/threonine kinase UNC-51. 924 88

We carried out experiments to investigate the mechanisms of serotonin-induced axonal excitability changes using isolated dorsal columns from young (seven to 11-day-old) Long-Evan's hooded rats. Conducting action potentials were activated by submaximal (50%) and supramaximal constant current electrical stimuli and recorded with glass micropipette electrodes. In experiment 1, to study Ca(2+)-mediated mechanisms, we superfused the preparations with Ringer solutions containing varying Ca2+ concentrations. Following superfusion with Ca(2+)-free Ringer solution for 4 h, we tested initial responses to serotonin agonists. Studies then were repeated after preparations had been washed for 1 h with Ringer solution containing 1.5 mM Ca2+ and 1.5 mM Mg2+. After 4 h superfusion of Ca(2+)-free Ringer solution, quipazine (a serotonin2A agonist, 100 microM) did not induce significant axonal excitability changes (amplitude change of 1.4 +/- 1.3%, percentage of predrug control level, +/-S.D., n = 6). A 100 microM concentration of 8-hydroxy-dipropylaminotetralin (a serotonin1A agonist) reduced response amplitudes by 36.3 +/- 4.2% (+/-S.D., P < 0.0005, n = 7) and prolonged latencies by 22.3 +/- 4.3% (+/-S.D., P < 0.0005, n = 7). Application of serotonin (100 microM) decreased amplitudes by 6.6 +/- 5.0% (+/-S.D., P < 0.05, n = 6). Extracellular calcium concentration ([Ca2+]e) was measured at various depths in the dorsal column with ion-selective microelectrodes. Four hours' superfusion with Ca(2+)-free Ringer solution reduced [Ca2+]e to less than 0.1 mM in dorsal columns. In 1.5 mM Ca2+ Ringer solution, quipazine increased the amplitudes by 38.3 +/- 5.8% (P < 0.0005, n = 6). Likewise, serotonin increased the amplitudes by 13.8 +/- 4.9% (P < 0.005, n = 6). In contrast however, 8-hydroxy-dipropylaminotetralin still reduced amplitudes by 35.0 +/- 6.4% (P < 0.0005, n = 7) and prolonged latencies by 24.1 +/- 4.5% (P < 0.0005, n = 7). In experiment 2, we investigated calcium-dependent and cAMP-mediated protein kinase signalling pathways to evaluate their role as intracellular messengers for serotonin2A receptor activation. Two protein kinase inhibitors, 50 microM H7 (an inhibitor of protein kinase C and c-AMP dependent protein kinase) and 100 microM D-sphingosine (an inhibitor of protein kinase A and C) effectively eliminated the excitatory effects of the serotonin2A agonist. 100 microM cadmium (a Ca2+ channel blocker) also blocked the effects of quipazine. Neither these protein kinase inhibitors nor cadmium alone affected action potential amplitudes. These results suggest that replacing Ca2+ with Mg2+ blocks the excitatory effects of quipazine but does not prevent the inhibitory effects of 8-hydroxy-dipropylaminotetralin, and calcium-mediated protein kinase mechanisms modulate axonal excitability changes induced by serotonin and its agonist.
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PMID:Calcium-mediated intracellular messengers modulate the serotonergic effects on axonal excitability. 933 Mar 59

A role for protein phosphorylation in the process of neurite outgrowth has been inferred from many studies of the effects of protein kinase inhibitors and activators on cultured neurotumor cells and primary neuronal cells from developing brain or ganglia. Here we re-examine this issue, using a culture system derived from a fully differentiated neuronal system undergoing axonal regeneration--the explanted goldfish retina following optic nerve crush. Of the relatively non-selective protein kinase inhibitors employed, H7, staurosporine and K252a were found to block neurite outgrowth, whereas HA1004 had no effect, a result which appears to rule out a critical role for protein kinase A. The more selective protein kinase C inhibitors, sphingosine, calphostin C and Ro-31-8220 were all inhibitory, as was prolonged treatment with phorbol ester and the protein phosphatase inhibitor okadaic acid. These results are in support of a role for protein kinase C in axonal regrowth.
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PMID:Protein kinase inhibitors block neurite outgrowth from explants of goldfish retina. 934 20

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 IIalpha and IIbeta 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 IIbeta 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 IIalpha, 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 IIalpha immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIalpha 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 IIalpha 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 IIbeta down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIalpha appears to support axonal regrowth.
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PMID:Calcium/calmodulin-dependent protein kinase II expression in motor neurons: effect of axotomy. 936 52

The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. approximately 15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10-20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation.
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PMID:Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments. 938 59

Crushing nerves, which contain the axons of central sensory neurons, in Aplysia causes the neurons to become hyperexcitable and to sprout new processes. Previous experiments that examined the effects of axonal injury on Aplysia sensory neurons have been performed in the intact animal or in the semi-intact CNS of Aplysia. It therefore has been unclear to what extent the long-term neuronal consequences of injury are due to intrinsic or extrinsic cellular signals. To determine whether injury-induced changes in Aplysia sensory neurons are due to intrinsic or extrinsic signals, we have developed an in vitro model of axonal injury. Isolated central sensory neurons grown for 2 days in cell culture were axotomized. Approximately 24 h after axotomy, sensory neurons exhibited a greater excitability-reflected, in part, as a significant reduction in spike accommodation-and greater neuritic outgrowth than did control (unaxotomized) neurons. Rp diastereoisomer of the cyclic adenosine 3',5'-monophosphorothiate (Rp-cAMPS), an inhibitor of protein kinase A, blocked both the reduction in accommodation and increased neuritic outgrowth induced by axotomy. Rp-cAMPS also blocked similar, albeit smaller, alterations observed in control sensory neurons during the 24-h period of our experiments. These results indicate that axonal injury elevates cAMP levels within Aplysia sensory neurons, and that this elevation is directly responsible, in part, for the previously described long-term electrophysiological and morphological changes induced in Aplysia sensory neurons by nerve crush. In addition, the results indicate that control sensory neurons in culture are also undergoing injury-related electrophysiological and structural changes, probably due to cellular processes triggered when the neurons are axotomized during cell culturing. Finally, the results provide support for the idea that the cellular processes activated within Aplysia sensory neurons by injury, and those activated during long-term behavioral sensitization, overlap significantly.
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PMID:Long-term effects of axotomy on excitability and growth of isolated Aplysia sensory neurons in cell culture: potential role of cAMP. 949 18

Intracerebral administration of the excitotoxin ibotenate to newborn mice induces white matter lesions mimicking periventricular leukomalacia, the most frequent brain lesion occurring in premature human babies. In this model, coinjection of vasoactive intestinal peptide prevents white matter lesions. In the present study, coadministration of ibotenate, vasoactive intestinal peptide, and selective transduction inhibitors showed that protein kinase C and mitogen-associated protein kinase pathways were critical for neuroprotection. In vivo and in vitro immunocytochemistry revealed that vasoactive intestinal peptide activated protein kinase C in astrocytes and neurons, and mitogen-associated protein kinase in neurons. In vitro neuronal transduction activation was indirect and required medium conditioned by astrocytes in which protein kinase C had been activated by vasoactive intestinal peptide. Although vasoactive intestinal peptide did not prevent the initial in vivo appearance of white matter lesion, it promoted a secondary repair of this lesion with axonal regrowth. Through protein kinase C activation, vasoactive intestinal peptide also prevented ibotenate-induced white matter astrocyte death. These data support the following hypothetical model: Vasoactive intestinal peptide activates protein kinase C in astrocytes, which promotes astrocytic survival and release of soluble factors; these released factors activate neuronal mitogen-associated protein kinase and protein kinase C, which will permit axonal regrowth.
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PMID:Regulation of neuroprotective action of vasoactive intestinal peptide in the murine developing brain by protein kinase C and mitogen-activated protein kinase cascades: in vivo and in vitro studies. 960 24


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