Annexin VI Binds to a Synaptic Vesicle Protein, Synapsin I

Abstract: Annexin VI bound to >14 species of proteins in the whole homogenate of rat forebrain in a Ca²⁺/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M_r 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I. Annexin VI bound to the head domain of synapsin I. When the binding of annexin VI to synapsin I was characterized in the native state, the affinity of the binding for Ca²⁺ ( K _Ca) was 12.6 µ M , and the affinity for annexin VI ( K _D) was ∼270 n M . Phosphorylation of synapsin I by cyclic AMP-dependent protein kinase and by Ca²⁺/calmodulin-dependent protein kinase II inhibited the annexin VI binding. The mode of the inhibition was different between the two kinases. These results indicate that annexin VI may modulate the function of synapsin I in a Ca²⁺- and phospholipid-dependent manner.     

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II by Brain Gangliosides

Abstract: Purified rat brain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca²⁺/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca²⁺-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC₅₀=0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca²⁺/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.     

Occurrence of Two Types of Ca2+-Dependent Protein Kinases in the Cytosol Fraction of the Brain

Abstract— Two types of Ca²⁺-dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE-cellulose, Sephadex G-200, and calmodulin-affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a K _a value of 16.7 n m . The K _a values of peaks I and II for Ca²⁺ were 2.4 and 1.6 μ m , respectively.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Multiple specificities of brain Ca2+- and calmodulin-dependent protein kinase for substrate

The purified Ca2+- and calmodulin-dependent protein kinase from rat brain, which has a M.W. of 120,000 by gel filtration analysis, showed a broad substrate specificity. In addition to myosin light chain from chicken gizzard, the enzyme phosphorylated myelin basic protein, casein and two endogenous substrates in a Ca2+- and calmodulin-dependent manner. In contrast, chicken gizzard myosin light chain kinase exclusively phosphorylated myosin light chain.     

Sphingosine, W-7, and Trifluoperazine Inhibit the Elevation in Cytosolic Calcium Induced by High K+Depolarization in Synaptosomes

Abstract: A possible role for protein kinases in the regulation of free cytosolic Ca²⁺ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca²⁺ (monitored with the Ca²⁺-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K⁺. The ability of various drugs to inhibit the cytosolic Ca²⁺ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca²⁺ induced by high K⁺ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 m/W ), staurosporine (1μ M ), and K252_a(1μ M ), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca²⁺ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca²⁺ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca²⁺ increases after depolarization.     

Tyrosine Hydroxylase in "Leaky" Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP-Dependent Systems

Abstract: The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca²⁺, the other on cyclic AMP. The Ca²⁺-dependent system is half-maximally activated by 1-2 μ M Ca²⁺ and 0.5 m M ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 m M ) does not inhibit significantly Ca²⁺-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 μ M cyclic AMP and about 0.3 m M ATP. Ca²⁺-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10⁻⁸ M Ca²⁺ plus 2 × 10⁻⁵ M cyclic AMP or of 10⁻⁵ M Ca²⁺, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca²⁺-dependent system, whereas the other site(s) are phosphorylated by both the Ca²⁺- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.     

Calcium/Calmodulin-Dependent Kinase II Phosphorylates Drosophila Visual Arrestin

Abstract: Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca²⁺ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca²⁺/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca²⁺/calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca²⁺/calmodulin-dependent kinase II such as Ca²⁺ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca²⁺/calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca²⁺/calmodulin-dependent kinase II activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca²⁺/calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca²⁺/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

A calcium and calmodulin-dependent protein kinase present in differentiating Dictyostelium discoideum

Abstract A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca²⁺/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum . The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca²⁺/calmodulin-dependence upon standing at 4°C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [γ-³² P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca²⁺ and calmodulin but not Ca²⁺ alone. Ca²⁺/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

S-100 and Other Acidic Proteins Promote Ca2+-Independent Phosphorylation of Protamine Catalyzed by a New Protein Kinase from Brain

Abstract: A new protein kinase modulated by S-100 (tentatively referred to as protein kinase X) was partially purified from pig brain extracts. The activity of protein kinase X, which was independent of Ca²⁺, was demonstrated when protamine (free base), but not protamine sulfate and other proteins (including histone), was used as substrate. The enzyme activity, found to distribute in both soluble and particulate fractions and to occur at the highest level in brain compared with other tissues (heart, kidney, liver, skeletal muscle, spleen, and testis) of rats, was also modulated by other acidic proteins (calmodulin, troponin C, and stimulatory modulator) in a Ca²⁺-independent manner. S-100 and other acidic proteins appeared to function as "substrate modifiers" by interacting with protamine (a highly basic protein), but not with the enzyme, thus rendering protamine in the complex a superior phosphate acceptor. The two isoforms of S-100 (i.e., a and b) were equally effective. Although the enzyme was not inhibited by many agents (trifluoperazine, melittin, cytotoxin I, polymyxin B, and spermine) shown to inhibit markedly phospholipid/Ca²⁺- or calmodulin/Ca²⁺-stimulated protein kinase, gossypol was found to inhibit specifically protein kinase X. The present findings suggest that S-100, a major acidic protein specific to nervous system, may promote phosphorylation by protein kinase X of certain neural proteins resembling protamine or containing protamine-like domains, in addition to its presumed role of a low-affinity Ca²⁺-binding protein.     

Protein kinase activities in ripening mango (Mangifera indica) fruit tissue. II. Purification and characterization of a casein kinase I

A protein kinase (PK‐II), phosphorylating casein, was purified from ripening mango, Mangifera indica L., fruit tissue. The purification procedure consisted of ammonium sulphate fractionation and sequential anion exchange‐, dye‐ligand, and gel filtration chromatography. The enzyme was purified over 500‐fold to near homogeneity with a recovery of 4%. The purified enzyme had a specific activity of ca 1 µmol mg⁻¹ protein min⁻¹ with ATP as phosphoryl donor. SDS‐PAGE results indicated a monomeric enzyme with molecular mass of 35 kDa. The protein kinase phosphorylated the acidic substrates casein and phosvitin, but had a very low activity with histones and protamine sulphate. The optimum pH and temperature for catalysis were determined to be 9.6 and 35°C, respectively. Mn²⁺ could not substitute for the Mg²⁺ needed for activity and Ca²⁺ had a slight stimulatory effect. Phospholipids, cAMP, calmodulin and the calmodulin inhibitor, calmidazolium, did not have any significant effect on activity, but the enzyme was inhibited by heparin and the specific inhibitor, CKI‐7, ( N ‐[2‐aminoethyl]‐5‐chloroisoquinoline‐8‐sulphonamide). Autoradiographic studies revealed the ability of the protein kinase to autophosphorylate as well as the presence of endogenous protein substrates in the crude extract. Initial velocity studies with casein as substrate and product inhibition studies with ADP indicated a K_m (ATP) and K_m (casein) of 14 µ M and 0.18 mg ml⁻¹, respectively, with a K_i (ADP) of 3.2 µM. The enzyme can be classified as a casein kinase I type of protein kinase (EC 2.7.10).     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Glutamate-Induced Loss of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons

Abstract: The exposure of cultured rat hippocampal neurons to 500 µ M glutamate for 20 min induced a 55% decrease in the total Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca²⁺-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N -methyl- d -aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca²⁺ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.