Protein Kinase C and Its 80-Kilodalton Substrate Protein in Neuroblastoma Cell Neurite Outgrowth

A potential role of the protein kinase C (PKC) system in differentiation of human neuroblastoma cell line LA-N-5 was investigated. It was found that neurite outgrowth induced by 12-O-tetradecanoylphorbol 13-acetate (TPA, 81 nM) was associated with a down-regulation of PKC as determined independently by immunocytochemistry, immunoblot, and enzyme activity assay. Down-regulation of PKC in cells induced to differentiate by retinoic acid (1 microM) was less pronounced, whereas it was undetected in cells induced to differentiate by nerve growth factor (100 ng/ml). The in vitro phosphorylation of an 80-kilodalton protein present in control LA-N-5 cells or in cells treated with TPA, retinoic acid, or nerve growth factor for 1 day decreased to various extents at days 4 or 7 concomitant with neuritogenesis. Pretreatment of LA-N-5 cells with a high concentration (1 microM) of TPA to deplete cellular PKC rendered the cells unresponsive to the differentiating effect of the agents. It was observed that CHP-100 cells, another human neuroblastoma line shown to be resistant to differentiation induced by the agents, had a reduced PKC level and the amount of in vitro phosphorylation of the 80-kilodalton protein was greatly reduced in control cells and remained relatively unchanged when the cells were treated with the agents for up to 7 days. The present studies suggested that PKC and its 80-kilodalton substrate protein were likely involved in initiation and/or progression of LA-N-5 cell differentiation induced by TPA and that separate PKC-independent pathways might also be involved in the differentiating effect of retinoic acid or nerve growth factor.     

MCC and PSC, the Putative Protein Import Channels of Mitochondria

All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecytoplasm and imported into the organelle. Water-filled channels are integral to the process oftranslocating proteins since channels can provide an aqueous pathway through the hydrophobicenvironment of the membrane. The MCC (multiple conductance channel) and PSC(peptide-sensitive channel) are two high-conductance channels previously identified inelectrophysiological studies of mitochondrial membranes. MCC and PSC are the putative pores of the importcomplexes of the inner and outer membranes, respectively. The genetic, biochemical, andbiophysical evidence regarding these assignments are summarized herein. These findingssupport the identification of MCC and PSC as the protein import channels of mitochondria.     

Regulation of Sodium and Calcium Channels by Signaling Complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.     

Protein Kinase C Modulates Calcium Channels in Isolated Presynaptic Nerve Terminals of Rat Hippocampus

Abstract: Nerve terminals (“synaptosomes”) isolated from rat brain hippocampus were loaded with the fluorescent Ca²⁺ indicator fura-2 and were subjected to depolarization with an elevated K⁺ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca²⁺ channels in the presynaptic membrane. Three components of Ca²⁺ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca²⁺ channels with different inactivation kinetics (τ of ～60 ms and 1 s, respectively) and Na⁺/Ca²⁺ exchange working in the “reverse” mode. The activity of both classes of voltage-dependent Ca²⁺ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca²⁺ channel activity, but the effect of DHS could be showed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca²⁺ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.     

Conversion of a Chaperonin GroEL-independent Protein into an Obligate Substrate

Chaperones assist protein folding by preventing unproductive protein aggregation in the cell. In Escherichia coli, chaperonin GroEL/GroES (GroE) is the only indispensable chaperone and is absolutely required for the de novo folding of at least ∼60 proteins. We previously found that several orthologs of the obligate GroE substrates in Ureaplasma urealyticum, which lacks the groE gene in the genome, are E. coli GroE-independent folders, despite their significant sequence identities. Here, we investigated the key features that define the GroE dependence. Chimera or random mutagenesis analyses revealed that independent multiple point mutations, and even single mutations, were sufficient to confer GroE dependence on the Ureaplasma MetK. Strikingly, the GroE dependence was well correlated with the propensity to form protein aggregates during folding. The results reveal the delicate balance between GroE dependence and independence. The function of GroE to buffering the aggregation-prone mutations plays a role in maintaining higher genetic diversity of proteins.     

Calcium Influx Through AMPA Receptors and Through Calcium Channels Is Regulated by Protein Kinase C in Cultured Retina Amacrine-Like Cells

Abstract: The functional modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors by protein kinase C (PKC) was investigated in cultures enriched in retinal amacrine-like cells. The kainate-evoked [Ca²⁺]_i increase is due to Ca²⁺ entry through open AMPA receptor channels, because it was blocked by the active isomer of a 2,3-benzodiazepine (LY 303070), an AMPA receptor antagonist. The AMPA receptor response to kainate was potentiated by phorbol 12-myristate 13-acetate, which specifically stimulates PKC, and it was decreased by bisindolylmaleimide I, a selective inhibitor of PKC, as well as by PKC down-regulation. The results indicate not only that the AMPA receptor activation has a PKC requirement, but also that PKC amplifies maximal receptor activation by 100 µ M kainate. The effect of PKC activation or inhibition on voltage-gated Ca²⁺-channel activity was also investigated. Activation of PKC caused inhibition of Ca²⁺ channels, and the same effect was produced by inhibition of PKC, whereas the inactive analogue of the phorbol ester did not affect channel activity. Our results show an important role for PKC in regulating the function of both AMPA receptors and Ca²⁺ channels in cultured retina cells.     

Substrate Competitive GSK-3 Inhibitors strategy and Implications

Glycogen synthase kinase-3 (GSK-3) is a highly conserved protein serine/threonine kinase ubiquitously distributed in eukaryotes as a constitutively active enzyme. Abnormally high GSK-3 activity has been implicated in several pathological disorders, including diabetes and neuron degenerative and affective disorders. This led to the hypothesis that inhibition of GSK-3 may have therapeutic benefit. Most GSK-3 inhibitors developed so far compete with ATP and often show limited specificity. Our goal is to develop inhibitors that compete with GSK-3 substrates, as this type of inhibitor is more specific and may be useful for clinical applications. We have employed computational, biochemical, and molecular analyses to gain in-depth understanding of GSK-3's substrate recognition. Here we argue that GSK-3 is a promising drug discovery target and describe the strategy and practice for developing specific substrate-competitive inhibitors of GSK-3.     

Novel Type of Signaling Molecules: Protein Kinases Covalently Linked with Ion Channels

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyosteliummyosin heavy chain kinases. Here we cloned, sequenced, and analyzed the tissue distribution of five new putative mammalian -kinases: melanoma -kinase, kidney -kinase, heart -kinase, skeletal muscle -kinase, and lymphocyte -kinase. All five are large proteins of more than 1000 amino acids with an -kinase catalytic domain located in the carboxyterminal part. We expressed the catalytic domain of melanoma -kinase in Escherichia coli, and found that it autophosphorylates at threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that long aminoterminal portions of melanoma and kidney -kinases represent new members of the TRP ion channel family, which are thought to mediate the capacitative Ca²⁺entry in nonexcitable mammalian cells. This suggests that melanoma and kidney -kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca²⁺influx in mammalian cells.     

A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K(+) current (I (Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I (Kr) by cAMP.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary

Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 +/- 11 fmol/mg protein and a KD value of 1.4 +/- 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 microM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and NT lost its capacity to release PRL in the absence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Catecholamine Synthesis Inhibition by Neuropeptide Y: Role of Ca2+ Channels and Protein Kinases

Abstract: We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca²⁺ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca²⁺ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by ～90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca²⁺ channel blocker ω-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca²⁺/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.     

The C-terminal Tails of the Bacterial Chaperonin GroEL Stimulate Protein Folding by Directly Altering the Conformation of a Substrate Protein

Many essential cellular proteins fold only with the assistance of chaperonin machines like the GroEL-GroES system of Escherichia coli. However, the mechanistic details of assisted protein folding by GroEL-GroES remain the subject of ongoing debate. We previously demonstrated that GroEL-GroES enhances the productive folding of a kinetically trapped substrate protein through unfolding, where both binding energy and the energy of ATP hydrolysis are used to disrupt the inhibitory misfolded states. Here, we show that the intrinsically disordered yet highly conserved C-terminal sequence of the GroEL subunits directly contributes to substrate protein unfolding. Interactions between the C terminus and the non-native substrate protein alter the binding position of the substrate protein on the GroEL apical surface. The C-terminal tails also impact the conformational state of the substrate protein during capture and encapsulation on the GroEL ring. Importantly, removal of the C termini results in slower overall folding, reducing the fraction of the substrate protein that commits quickly to a productive folding pathway and slowing several kinetically distinct folding transitions that occur inside the GroEL-GroES cavity. The conserved C-terminal tails of GroEL are thus important for protein folding from the beginning to the end of the chaperonin reaction cycle.     

Calcium/Diacylglycerol-Dependent Protein Kinase and Its Major 87-Kilodalton Protein Substrate Are Differentially Distributed in Rat Basal Ganglia

When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol-dependent protein kinase (protein kinase C) and an 87-kilodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chemical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87-kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal ganglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87-kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87-kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Dengue Virus M Protein C-Terminal Peptide (DVM-C) Forms Ion Channels

A chemically synthesized peptide consisting of the C-terminus of the M protein of the Dengue virus type 1 strain Singapore S275/90 (DVM-C) produced ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to sodium and potassium ions than to chloride ions and more permeable to chloride ions than to calcium ions. Hexamethylene amiloride (100 μM) and amantadine (10 μM), blocked channels formed by DVM-C. Ion channels may play an important role in the life cycle of many viruses and drugs that block these channels may prove to be useful antiviral agents.     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.     

Phosphoglycerates and Protein Phosphorylation: Identification of a Protein Substrate as Glucose-1,6-Bisphosphate Synthetase

We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate [( 1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [gamma-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP72 preparation.