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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.
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PMID:Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C. 1677 99

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.
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PMID:Calcium-mediated transient phosphorylation of tau and amyloid precursor protein followed by intraneuronal amyloid-beta accumulation. 1708 46

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca(2+) signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca(2+) signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca(2+) regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.
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PMID:DARPP-32 is a robust integrator of dopamine and glutamate signals. 1719 17

The stimulated dephosphorylation of the dephosphin group of endocytic proteins by calcineurin and their subsequent rephosphorylation by cyclin-dependent kinase 5 (cdk5) is required for synaptic vesicle (SV) retrieval in central nerve terminals. However, the specific endocytic pathway(s) controlled by these enzymes is unknown. To address this issue, we combined functional and morphological assays of endocytosis in primary neuronal cultures with pharmacological and molecular ablation of calcineurin and cdk5 activity. During strong stimulation, inhibition of calcineurin or cdk5 blocked uptake of the activity-dependent membrane marker FM1-43, but not the more hydrophilic FM2-10. However, FM2-10 uptake-measured poststimulation was sensitive to cdk5 and calcineurin inhibition, indicating that a slow form of endocytosis persists after termination of stimulation. In parallel EM studies, inhibition of cdk5 during strong stimulation greatly reduced horseradish peroxidase labeling of plasma membrane-derived nerve terminal endosomes, but not SVs. Furthermore, during mild stimulation, FM1-43 uptake was unaffected by cdk5 inhibition and the SV membrane was exclusively retrieved via a single SV route, suggesting that recruitment of the endosomal route of membrane retrieval is activity dependent. Thus, we propose that the calcineurin/cdk5-dependent phosphorylation cycle of the dephosphins specifically controls a slow endocytic pathway that proceeds via endosomal intermediates and is activated by strong physiological stimulation in central nerve terminals.
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PMID:Activity-dependent control of slow synaptic vesicle endocytosis by cyclin-dependent kinase 5. 1721 1

Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.
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PMID:Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5. 1740 May 54

In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca(2+)-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca(2+)-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca(2+)-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B'' or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca(2+)-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca(2+) binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca(2+), both in vitro and in vivo. Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca(2+) can activate serine/threonine phosphatase activity.
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PMID:The B''/PR72 subunit mediates Ca2+-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A. 1753 22

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M(r) 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca(2+) channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M(r) 32 000 required protein phosphatase 1/2A activity and extracellular Ca(2+). In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na(+) or Ca(2+) sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.
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PMID:Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization. 1786 22

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.
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PMID:Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation. 1846 Apr 67

The function of the D(3) dopamine (DA) receptor remains ambiguous largely because of the lack of selective D(3) receptor ligands. To investigate the function and intracellular signaling of D(3) receptors, we established a PC-12/hD3 clone, which expresses the human D(3) DA receptor in a DA producing cell line. In this model, we find that the D(3) receptor functions as an autoreceptor controlling neurotransmitter secretion. Pre-treatment with 3,6a,11, 14-tetrahydro-9-methoxy-2 methyl-(12H)-isoquino[1,2-b] pyrrolo[3,2-f][1,3] benzoxanzine-1-carboxylic acid, a D(3) receptor preferring agonist, dose-dependently suppressed K+-evoked [3H]DA release in PC-12/hD3 cells but not in the control cell line. This effect was prevented by D(3) receptor preferring antagonists GR103691 and SB277011-A. Furthermore, activation of D(3) receptors significantly inhibits forskolin-induced cAMP accumulation and leads to transient increases in phosphorylation of cyclin-dependent kinase 5 (Cdk5), dopamine and cAMP-regulated phosphoprotein of M(r) 32 000 and Akt. Because we observed differences in Cdk5 phosphorylation as well as Akt phosphorylation after DA stimulation, we probed the ability of Cdk5 and phosphatidylinositol-3 kinase (PI3K) to influence DA release. Cdk5 inhibitors, roscovitine, or olomoucine, but not the PI3K inhibitor wortmannin, blocked the D(3) receptor inhibition of DA release. In a complimentary experiment, over-expression of Cdk5 potentiated D(3) receptor suppression of DA release. Pertussis toxin, 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one and cyclosporine A also attenuated D(3) receptor-mediated inhibition of DA release indicating that this phenomenon acts through Gi/oalpha and casein kinase 1, and phosphatase protein phosphatase 2B (calcineurin), respectively. In support of previous data that D(3) DA receptors reduce transmitter release from nerve terminals, the current results demonstrate that D(3) DA receptors function as autoreceptors to inhibit DA release and that a signaling pathway involving Cdk5 is essential to this regulation.
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PMID:The D(3) dopamine receptor inhibits dopamine release in PC-12/hD3 cells by autoreceptor signaling via PP-2B, CK1, and Cdk-5. 1952 35

Dopamine- and cAMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP-32) is a key integrative molecule in the dopaminergic and glutamatergic signaling pathways in the striatum. Electroconvulsive shock (ECS), which induces massive neuronal depolarization, can activate various signaling pathways. In this study we investigated whether ECS could affect the phosphorylation status of DARPP-32. Male Sprague-Dawley rats underwent ECS and were sacrificed by decapitation at 0, 2, 10, 60, and 180 min after treatment. The phosphorylations of Thr34 and Thr75 residues of DARPP-32 and Ser159 residue of cyclin-dependent kinase 5 (CDK5) were investigated in the striatum. The activity of protein phosphatase 1 (PP1) and the binding between DARPP-32 and PP1 were also analyzed. Thr34 phosphorylation of DARPP-32 increased immediately after ECS and this state was maintained for more than 60 min. The activity of PP1 decreased and the binding between PP1 and DARPP-32 increased in accordance with this phosphorylation pattern. However, the phosphorylation at Thr75 showed no significant change except for an initial transient decrease. The phosphorylation of CDK5, which is responsible for Thr75 phosphorylation of DARPP-32, did not exhibit significant fluctuations. Our findings indicate that ECS increases Thr34 phosphorylation of DARPP-32, and thus inhibits the activity of PP1.
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PMID:Effects of electroconvulsive shock on the phosphorylation of DARPP-32 in rat striatum. 1981 13


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