Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p38 Mitogen-activated protein kinase alpha (p38 MAPKalpha) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPKalpha, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1-10) that at pH 7.5, 10 mm Mg2+ and 27 degrees C p38 MAPKalpha phosphorylates ATF2Delta115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (beta,gamma-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPKalpha with dissociation constants of KA = 360 microm, KI = 240 microm, and KI > 2000 microm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPKalpha-ATF2Delta115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd approximately 70 microm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2Delta115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a beta-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPKalpha inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations > 100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor.
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PMID:Kinetic mechanism for p38 MAP kinase alpha. A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate. 1615 85

N-methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine (PCP) can induce positive and negative symptoms of schizophrenia in humans and related effects in rodents. PCP treatment of developing rats induces apoptotic neurodegeneration and behavioral deficits later in life that mimic some symptoms of schizophrenia. The precise mechanism of PCP-induced neural degeneration is unknown. This study used selective antagonists, siRNA, and Western analysis to investigate the role of the Akt-glycogen synthase kinase-3beta (GSK-3beta) pathway in PCP-induced neuronal apoptosis in both neuronal culture and postnatal day 7 rats. PCP administration in vivo and in vitro reduced the phosphorylation of Akt Ser427 and GSK-3beta Ser9, decreasing Akt activity and increasing GSK-3beta activity. The alteration of Akt-GSK-3beta signaling parallels the temporal profile of caspase-3 activation by PCP. Reducing GSK-3beta activity by application of selective inhibitors or depletion of GSK-3beta by siRNA attenuates caspase-3 activity and blocks PCP-induced neurotoxicity. Moreover, increasing synaptic strength by either activation of L-type calcium channels with BAY K8644 or potentiation of synaptic NMDA receptors with either a low concentration of NMDA or bicuculline plus 4-aminopyridine completely blocks PCP-induced cell death by increasing Akt phosphorylation. These neuroprotective effects are associated with activation of phosphoinositide-3-kinase-Akt signaling, and to a lesser extent, the MAPK signaling pathway. Overall, these data suggest that PCP-induced hypofunction of synaptic NMDA receptors impairs the Akt-GSK-3beta cascade, which is necessary for neuronal survival during development, and that interference with this cascade by PCP or natural factors may contribute to neural pathologies, perhaps including schizophrenia.
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PMID:The role of Akt-GSK-3beta signaling and synaptic strength in phencyclidine-induced neurodegeneration. 1763 6

c-jun-N-Terminal kinase 3alpha1 (JNK3alpha1) is a mitogen-activated protein (MAP) kinase family member expressed primarily in the brain that phosphorylates protein transcription factors including c-jun and activating transcription factor 2 (ATF2) upon activation by a variety of stress-based stimuli. In this study, the kinetic mechanism for JNK3alpha1 was determined via initial velocity patterns in the presence and absence of both ATP and ATF2 competitive inhibitors. Peptide inhibitors from both ATF2 (peptide 1) and JNK-interacting protein 1 (JIP-1) (peptide 3), derived from the homologous delta-domain JNK docking sequence, inhibited JNK3alpha1 activity in a competitive fashion versus ATF2 while being pure noncompetitive toward ATP. In contrast, peptides derived from the phosphoacceptor activation domain on ATF2 (peptides 4 and 5) were recognized neither as substrates nor as inhibitors of JNK3alpha1. AMP-PCP and compound 6, a small molecule analinopyrimidine, exhibited pure noncompetitive inhibition versus ATF2 and competitive inhibition versus ATP. Peptide inhibitors based on the delta-domain sites of JIP ( 3) and ATF2 ( 1) were not recognized by p38, also of the MAPK family, which may give insight into finding more selective inhibitors for the JNK family of kinases. Collectively these data showed that JNK3alpha1 proceeded by a random sequential kinetic mechanism and that the ATP and ATF2 substrate sites were non-interacting. Moreover, these results established the 11-mer JIP peptide ( 3) as a potent ( K i = 25 +/- 6 nM) competitive inhibitor versus ATF2 in JNK3alpha1.
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PMID:Kinetic mechanism and inhibitor characterization for c-jun-N-terminal kinase 3alpha1. 1826 48

Repeated administration of phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor blocker, produces schizophrenia-like behaviors in humans and rodents. Although impairment of synaptic function has been implicated in the effect of PCP, the molecular mechanisms have not yet been elucidated. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity, we examined whether exposure to PCP leads to impaired BDNF function in cultured cortical neurons. We found that PCP caused a transient increase in the level of intracellular BDNF within 3 h. Despite the increased intracellular amount of BDNF, activation of Trk receptors and downstream signaling cascades, including MAPK/ERK1/2 and PI3K/Akt pathways, were decreased. The number of synaptic sites and expression of synaptic proteins were decreased 48 h after PCP application without any impact on cell viability. Both electrophysiological and biochemical analyses revealed that PCP diminished glutamatergic neurotransmission. Furthermore, we found that the secretion of BDNF from cortical neurons was suppressed by PCP. We also confirmed that PCP-caused downregulation of Trk signalings and synaptic proteins were restored by exogenous BDNF application. It is possible that impaired secretion of BDNF and subsequent decreases in Trk signaling are responsible for the loss of synaptic connections caused by PCP.
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PMID:Phencyclidine-induced decrease of synaptic connectivity via inhibition of BDNF secretion in cultured cortical neurons. 2246 67

Many acute treatments transiently reverse the deficit in novel object recognition (NOR) produced by subchronic treatment with the N-methyl-d-aspartate receptor non-competitive antagonist, phencyclidine (PCP), in rodents. Treatments which restore NOR for prolonged periods after subchronic PCP treatment may have greater relevance for treating the cognitive impairment in schizophrenia than those which restore NOR transiently. We examined the ability of post-PCP subchronic lurasidone, an atypical APD with potent serotonin (5-HT)1A partial agonism and subchronic tandospirone, a selective 5-HT1A partial agonist, to enable prolonged reversal of the subchronic PCP-induced NOR deficit. Rats treated with subchronic PCP (2mg/kg, twice daily for 7 days) or vehicle, followed by a 7day washout period were subsequently administered lurasidone or tandospirone twice daily for 7 days (day 15-21), and tested for NOR weekly for up to two additional weeks. Subchronic lurasidone (1, but not 0.1mg/kg) or tandospirone (5, but not 0.6mg/kg) significantly reversed the PCP-induced NOR deficit at 24h and 7days after the last injection, respectively. The effect of lurasidone persisted for one more week (day 36, 14 days after the last lurasidone dose), while tandospirone-treated rats were able to perform NOR at 7, but not 14, days after the last tandospirone dose. Co-administration of WAY100635 (0.6mg/kg), a 5-HT1A antagonist, with lurasidone, blocked the ability of lurasidone to restore NOR, suggesting that 5-HT1A receptor stimulation is necessary for lurasidone to reverse the effects of PCP. The role of dopamine, GABA and the MAPK/ERK signalling pathway in the persistent, but not indefinite, restoration of NOR is discussed.
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PMID:Prolonged reversal of the phencyclidine-induced impairment in novel object recognition by a serotonin (5-HT)1A-dependent mechanism. 2634 83