cDNA cloning and sequencing of Ca2+ calmodulin-dependent protein kinase IIα subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity, known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, se.) produces first mild ataxia followed by paralysis in 7-14 days in hens. DFP treatment also increases in vitro autophosphorylation of Ca²⁺ calmodulin-dependent protein kinase II (CaM kinase II) and the phosphorylation of several cytoslceletal proteins in the hen brain. To investigate whether increase in CaM kinase II activity is associated with increased expression of its mRNA, we cloned and sequenced CaM kinase II a subunit cDNA, and used it to study CaM kinase II expression in brain regions and spinal cord. Hen CaM kinase II subunit differs in 7 amino acids from that of rat CaM kinase II. Its mRNA occurs predominantly as a 6.7 kb message, which is very close to that of human CaM kinase II a subunit. Northern blot analysis showed a transient increase in CaM kinase II subunit mRNA in the cerebellum and spinal cord of DFP-treated chickens. The increase in CaM kinase II mRNA expression is consistent with the previously reported increase in its activity in brain and spinal cord, and its increased expression only in cerebellum and spinal cord, which are sensitive to the Wallerian-type degeneration characteristic of OPIDN, suggests the probable role of this enzyme in delayed neurotoxicity.     

C-fos mRNA Induction in the Central and Peripheral Nervous Systems of Diisopropyl Phosphorofluoridate (DFP)-Treated Hens

A single dose of diisopropyl phosphorofluoridate (DFP), an organophosphorus ester, produces delayed neurotoxicity (OPIDN) in hen. DFP produces mild ataxia in hens in 7–14 days, which develops into severe ataxia or paralysis as the disease progresses. Since, OPIDN is associated with alteration in the expression of several proteins (e.g., Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) -subunit, tau, tubulin, neurofilament (NF) protein, vimentin, GFAP) as well as their mRNAs (e.g., NF, CaM kinase II -subunit), we determined the effect of a single dose of DFP on the expression of one of the best known immediate-early gene (IEG), c-fos. C-fos expression was measured by Northern hybridization in cerebrum, cerebellum, brainstem, midbrain, spinal cord, and the sciatic nerves of hens at 0.5 hr, 1 hr, 2 hr, 1 day, 5 days, 10 days, and 20 days after a single 1.7 mg/kg, sc. injection of DFP. All the tissues (cerebrum, 52%; cerebellum, 55%; brainstem, 49%; midbrain, 23%; spinal cord, 80%; sciatic nerve, 157%;) showed significant increase in c-fos expression in 30 min and this elevated level persisted at least up to 2 hr. Expressions of -actin mRNA and 18S RNA were used as internal controls. The significant increase in c-fos expression in DFP-treated hens suggests that c-fos may be one of the IEGs involved in the development of OPIDN.Both of them equally contributed towards this work     

Neurofilament phosphorylation and [125I]Calmodulin binding by Ca2+/calmodulin-dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)-treated hen

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animal species, e.g., adult chicken. The chickens were sacrificed 18 days after a single dose of DFP (1.7 mg/kg, sc.), which produced severe ataxia or paralysis in 10–14 days. We studied Ca²⁺/calmodulin-dependent in vitro neurofilament phosphorylation by the brain subcellular fractions of control and DFP-treated hens. There was enhanced phosphorylation of all three NF subunits by the brain supernatant of treated hens. This was accompanied by enhanced autophosphorylation of both Ca²⁺/CaM-dependent protein kinase II (CaM-kinase II) subunits and increased calmodulin binding using either¹²⁵I-CaM or biotinylated calmodulin to only subunit without concomitant increase in the amount of this enzyme. This enhanced phosphorylation of neurofilament subunits was completely and partially inhibited by mastoparan and KN-62, respectively. There was no alteration in the distribution of CaM-kinase II activity in treated hens and the activity was not related to its concentration in different subcellular fractions. The difference in¹²⁵I-CaM binding to CaM-kinase II subunit in the brain supernatants of control and DFP-treated hens was not altered by its phosphorylation or dephosphorylation. The increased CaM-kinase II activity in the soluble fraction of DFP-treated hen brain may be involved in the aberrant phosphorylation of axonal neurofilaments, and thus play a role in OPIDN.Abbreviations CaM calmodulin - CaM-kinase II Ca²⁺/calmodulin-dependent protein kinase II - DFP diisopropyl phosphorofluoridate - ECL enhanced chemiluminescence - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid - MAP-2 microtubule-associated protein-2 - MBP myelin basic protein - OPIDN organophosphorus ester-induced delayed neurotoxicity - PIPES 1,4-piperazinediethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Comparison of Ca2+/calmodulin-dependent protein kinase II purified from control and diisopropyl phosphorofluoridate (DFP)-treated hens

Diisopropyl phosphorofluoridate (DFP) produces type I organophosphorus ester-induced delayed neurotoxicity in humans and sensitive animal species. This is accompanied by enhanced Ca²⁺/CaM-dependent protein kinase II (CaM-kinase II) activity, and [¹²⁵I]calmodulin binding to CaM-kinase II in DFP-treated hen brain supernatant without increase in the enzyme quantity. We have purified CaM-kinase II from control and DFP-treated hen whole brains and compared various physical and biochemical properties. The two enzymes exhibited similar properties in many respects. However, there was a decrease in calcium-independent protein kinase II activity after autophosphorylation, and an increase in K_0.5 for free calcium and calmodulin of enzyme purified from DFP-treated hen brains. This change in kinetic parameters may result in greater percentage of total CaM-kinase II present in unphosphorylated form, which is consistent with the increased autophosphorylation of CaM-kinase II and [¹²⁵I]calmodulin binding in the brain supernatant of DFP-treated hens.Abbreviations used CaM calmodulin - CaM-kinase II Ca²⁺/calmodulin-dependent protein kinase II - MAP-2 microtubule associated protein-2 - DFP diisopropylphosphorofluoridate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - NEPHGE nonequilibrium pH gradient electrophoresis - OPIDN organophosphorus ester-induced delayed neurotoxicity - PIPES 1,4-piperazinediethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dedecyl sulfate-polyacrylamide gel electrophoresis - St. aureus V₈ protease Staphylococus aureus V₈ protease - TOCP tri-O-cresyl phosphate - TPCK N-tosyl-I-phenylalanine chloromethyl ketone     

Effect of prevention and potentiation of diisopropyl phosphorofluoridate (DFP)-induced delayed neurotoxicity on the mRNA expression of neurofilament subunits in hen central nervous system.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces mild ataxia in 7-14 days and severe ataxia or paralysis in about 20 days (OPIDN) in hens. Previous studies in this laboratory have shown enhanced temporal expression of neurofilament (NF) subunit mRNAs in the spinal cord (SC) of DFP-treated hens. The main objective of this investigation was to study the effect of DFP administration on NF subunit mRNAs expression, when OPIDN is protected or potentiated by pre-treatment or post-treatment, respectively, with phenylmethylsulfonyl fluoride (PMSF). The hens were sacrificed 1, 5, 10, and 20 days after the last treatment. In contrast with enhanced mRNA expression of NF subunits reported in OPIDN, there was no alteration in the expression of NF subunits in the SC of PMSF-protected hens that did not develop OPIDN. PMSF post-treatment of DFP-treated hens, which enhanced delayed neurotoxicity produced by a low dose of DFP, exhibited decrease in the mRNA expression of NF subunits in SC at all time periods (1-20 days) of observation. The expression of NF subunits was also studied in the degeneration-resistant tissue cerebrum of treated hens. The results from protected hens suggested that temporal enhanced expression of NF subunit mRNAs in DFP-treated hens might be contributing to the development of OPIDN in hens. By contrast, PMSF post-treatment seemed to potentiate OPIDN by a mechanism different from that followed by DFP alone to produce OPIDN.     

Alteration in Cytoskeletal Protein Levels in Sciatic Nerve on Post-Treatment of Diisopropyl Phosphorofluoridate (DFP)-Treated Hen with Phenylmethylsulfonyl Fluoride

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, and a single dose (1.7 mg/kg, sc.) of this compound produces mild ataxia in hens in 7–14 days and a severe ataxia or paralysis (OPIDN) in three weeks. OPIDN is associated with axonal swelling and their degeneration. We have previously observed alteration in neurofilament (NF) protein levels in the spinal cord of DFP-treated hens. The main objective of this investigation was to study NF protein levels in the sciatic nerves (SN) of hens, in which OPIDN has been potentiated by phenylmethylsulfonyl fluoride (PMSF) post-treatment. PMSF is known to protect DFP-treated (1.7 mg/kg) hens from developing OPIDN if injected before, and potentiate OPIDN if injected after the administration of DFP (0.5 mg/kg). The potentiation of OPIDN was accompanied by earlier elevation of NF proteins in the SN particulate fraction. In contrast, SN supernatant fraction showed a transient fall in NF protein levels in potentiation OPIDN. Out of the two other cytoskeletal proteins (i.e., tubulin, tau) studied in this investigation, tubulin also showed earlier elevation in its level in the particulate fraction in potentiated OPIDN. The earlier elevation of NF protein levels in SN particulate fraction in potentiated OPIDN suggested the possible involvement of NFs in delayed neurotoxicity.     

Tau phosphorylation by diisopropyl phosphorofluoridate (DFP)-treated hen brain supernatant inhibits its binding with microtubules: role of Ca2+/Calmodulin-dependent protein kinase II in tau phosphorylation.

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hen, human, and other sensitive species. This is characterized by mild ataxia, which progresses to severe ataxia or paralysis in a few days. Ultrastructurally, OPIDN is associated with the degeneration of axons in central and peripheral nervous systems. Bacterially expressed longest human tau protein (htau40) phosphorylated by DFP-treated hen brain supernatant showed a decrease in microtubule binding in a shorter time than that phosphorylated by control hen brain supernatant. The decrease in htau40-microtubule binding observed on htau40 phosphorylation by the recombinant Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) alpha-subunit showed that CaM kinase II present in brain supernatant could participate in tau phosphorylation even in the absence of Ca2+/CaM and decrease tau-microtubule binding. In addition, use of htau40 mutants, htau40m1 (Ala416) and htau40m6 (Asp416), suggested that replacement of Ser416 by neutral or acidic amino acid produced some change in htau40 conformation that caused diminished binding with microtubules phosphorylated by brain supernatant in the presence of ethylene glycol bis(beta-aminoethyl ether) N, N'tetraacetic acid (EGTA). The change in conformation produced by Ser416 phosphorylation, however, was different from that produced by mutants since only nonmutated htau40 showed a significant decrease in binding with microtubules on phosphorylation by recombinant CaM kinase II in the presence of Ca2+/CaM compared to that obtained by phosphorylation in the presence of EGTA. This study showed that enhanced Ca2+/CaM-dependent protein kinase activity in DFP-treated hen brain supernatant may cause decreased tau-microtubule binding and destabilization of microtubules and may be involved in axonal degeneration in OPIDN.     

Differential alteration of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP).

A single dose (1.7 mg/kg, s.c.) of diisopropylphosphorofluoridate (DFP) causes organophosphorus ester-induced delayed neurotoxicity (OPIDN) in susceptible species. We studied the effects of DFP administration on the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic protein at different time points (1, 2, 5, 10 and 20 days) post-treatment. Total RNA was extracted from cerebrum, cerebellum, brainstem, midbrain, and spinal cord of the control and DFP-treated hens, and northern blots were prepared using standard protocols and hybridized with GAPDH, as well as beta-actin and 28S RNA cDNA (control) probes. There was a distinct spatial/temporal mRNA expression pattern for the different tissues studied. Non-susceptible tissue, cerebrum showed a dramatic increase in GAPDH mRNA at day 1, post-treatment and levels remained high at all time points, suggestive of protective mechanisms from the beginning. In contrast, highly susceptible tissues like brainstem, spinal cord and midbrain showed either no elevation or slight down-regulation at day 1, suggesting trauma and cell injury/cell death. Overall, there was moderate level of induction during the subsequent time points in these tissues, indicative of pathways of either recovery or degeneration. Cerebellum being the less susceptible tissue showed moderate increase initially, followed by higher induction, suggestive of rapid recovery. Our current data on GAPDH provides an important link in this complex network of molecular changes involving pathways identified by our group and others, such as nitric oxide (NO), CaM kinase-II (CaMK-II), protein kinase-A (PKA), c-fos, and phosphorylated-CREB (p-CREB) in DFP-induced OPIDN.     

In vivo and in vitro effects of diisopropyl phosphorofluoridate (DFP) on the rate of hen brain tubulin polymerization

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in sensitive species. We have investigated the in vivo and in vitro effects of DFP on hen brain tubulin polymerization. Hens were treated with a single dose of DFP (1.7 mg/kg, sc.), and were sacrificed after 18–21 days. Tubulin from DFP-treated hen brains showed small but significant decrease (14.42%) in the rate of polymerization and 11.05% decrease in rise in O.D. at 340 nm in 30 min. DFP in vivo treatment also resulted in decreased concentration of tau and an enhanced concentration of two peptides (45 kDa, 35 kDa) in the brain supernatant. These peptides seemed to be the degradation products of MAP-2. The decrease in the rate of brain tubulin polymerization in treated hens is consistent with neurochemical alterations and the focal degeneration and aggregation of these filamentous structures in OPIDN.Abbreviations DFP Diisopropyl phosphorofluoridate - DMSO dimethyl sulfoxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid - EDTA ethylenediaminetetraacetic acid - 2, 5-DH 2, 5-hexanedione - DMHD 3, 4-dimethyl-2, 5-hexanedione - OPIDN organophosphorus ester-induced delayed neurotoxicity - PMSF phenylmethylsulfonyl fluoride - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - TOCP tri-o-cresyl phosphate     

Altered Time Course of mRNA Expression of Alpha Tubulin in the Central Nervous System of Hens Treated with Diisopropyl Phosphorofluoridate (DFP)

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of single dose of DFP (1.7 mg/kg/s.c.) on the expression of alpha tubulin which is one of the major sub-unit of tubulin polymers that constitute an important constituent of cellular architecture. The hens were sacrificed at different time points i.e. 1, 2, 5, 10, and 20 days. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, and brainstem as well as spinal cord. Northern blots prepared using standard protocols were hybridized with alpha tubulin as well as with -actin and 28S RNA cDNA (controls) probes. The results indicate a differential /spatial /temporal regulation of alpha tubulin levels which may be the result of perturbed microtubule dynamics not only in the axons but also in perikarya of neurons in the CNS of DFP treated hens. In the highly susceptible tissues like brainstem and spinal cord the initial down-regulation of mRNA levels could be attributed to DFP induced stress response resulting in inhibited cell metabolism and or cell injury / cell death. Increase in levels of mRNA at 5 days and thereafter coincided with increased tubulin transport which may be due to increased phosphorylation of tubulins in both axons and perikarya and other intraaxonal changes resulting in impaired axonal transport. DFP induced decreased rate of tubulin polymerization resulting in increased levels of free tubulin monomers may be involved in the altered alpha tubulin mRNA expression at different time points by autoregulatory circuits. Cerebellum being the less susceptible tissue showed only a moderate decline at day 2, while the alpha tubulin remained at near control levels at day 1. Delayed down-regulation may be due to the co-ordinated up or down- regulation of different sub-types of alpha and beta tubulins as well as the differential response of specialised cell types in cerebellum. Continuous overexpression of alpha tubulin in cerebrum from the beginning may be its effective protective strategy to safeguard itself from neurotoxicity. Differential expression pattern observed could be due to the differential susceptibility and variability in the rate of axonal transport of different regions besides the tubulin heterogenity of CNS. Hence our results indicte differential expression of alpha tubulin is either one of the reasons for the development of OPIDN or the result of progressive changes taking place during OPIDN.     

Protein phosphatase 1 is involved in the dissociation of Ca2+/calmodulin-dependent protein kinase II from postsynaptic densities

Autophosphorylation-dependent translocation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) to postsynaptic densities (PSDs) from cytosol may be a physiologically important process during synaptic activation. We investigated a protein phosphatase responsible for dephosphorylation of the kinase. CaM kinase II was shown to be targeted to two sites using the gel overlay method in two-dimensional gel electrophoresis. Protein phosphatase 1 (PP1) was identified to dephosphorylate CaM kinase II from its complex with PSDs using phosphatase inhibitors and activators, and purified phosphatases. The kinase was released from PSDs after its dephosphorylation by PP1.     

Altered glycine transport by cerebral tissue and decreased Na+ and Ca++ pump activities during organophosphorus-ester–induced delayed neurotoxicity development period

Uptake of [U-¹⁴C] glycine during the organophosphorus-ester-induced delayed neurotoxicity (OPIDN) development period was studied. Diisopropyl fluorophosphate (DFP), a delayed neurotoxic organophosphorus ester was administered to adult rats and hens. Results showed a decreased accumulation of glycine in hen cerebral cortex slices during the delayed neurotoxicity development period. An altered sensitivity toward transport inhibitors 2,4-dinitrophenol and ouabain was observed in DFP-treated hens. An altered neuronal membrane function during the OPIDN development period is reported in the present work. Brain Na⁺, K⁺-ATPase and Ca⁺⁺-ATPase activities decreased during the neurotoxicity development period. The decrease in Ca⁺⁺-ATPase activity persisted in hens until the complete development of neurotoxic symptoms. Decreased Ca⁺⁺ pump activity is correlated with altered membrane function during OPIDN. © 1996 John Wiley & Sons, Inc.     

Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of Ca2+/calmodulin-dependent protein kinases in cultured rat hippocampal neurons

Calcium/calmodulin-dependent protein kinases (CaM kinases) are major multifunctional enzymes that play important roles in calcium-mediated signal transduction. To characterize their regulatory mechanisms in neurons, we compared glutamate-induced phosphorylation of CaM kinase IV and CaM kinase II in cultured rat hippocampal neurons. We observed that dephosphorylation of these kinases followed different time courses, suggesting different regulatory mechanisms for each kinase. Okadaic acid, an inhibitor of protein phosphatase (PP) 1 and PP2A, increased the phosphorylation of both kinases. In contrast, cyclosporin A, an inhibitor of calcineurin, showed different effects: the phosphorylation and activity of CaM kinase IV were significantly increased with this inhibitor, but those of CaM kinase II were not significantly increased. Cyclosporin A treatment of neurons increased phosphorylation of Thr196 of CaM kinase IV, the activated form with CaM kinase kinase, which was recognized with an anti-phospho-Thr196 antibody. Moreover, recombinant CaM kinase IV was dephosphorylated and inactivated with calcineurin as well as with PP1, PP2A, and PP2C in vitro. These results suggest that CaM kinase IV, but not CaM kinase II, is directly regulated with calcineurin.     

Biochemical changes in sciatic nerve of hens treated with tri-o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins.

Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1-2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri-o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [gamma-32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.     

Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle

The relation between CaM kinase II activity and high Ca²⁺-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 M) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 M); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 M) or ATP (100 M) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca²⁺-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca²⁺-overload induced cellular stress response in cells.     

Inactivation and Subcellular Redistribution of Ca2+/ Calmodulin-Dependent Protein Kinase II Following Spinal Cord Ischemia

Abstract: Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II α and β subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the a and β subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (≤25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.     

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.     

Rat Brain Protein Phosphatase 2A: An Enzyme that May Regulate Autophosphorylated Protein Kinases

Abstract: Protein phosphatase 2A (PP2A) isolated from whole rat brain homogenate supernatants has been compared with that extracted from rat synaptosomal membranes. Both purified enzymes are comprised of the three known PP2A polypeptide chains of 65 (A subunit), 55 (B/B' subunit), and 38 (C subunit) kDa and have okadaic acid inhibition curves ( K _i = 0.05 n M ) nearly identical to that reported for skeletal muscle PP2A. The isolated 38-kDa subunit of rat brain PP2A appears to contain phosphotyrosine based on cross-reactivity with a specific monoclonal antibody (PY-20). Amino acid compositions and sequences of peptides isolated from the 65- and 38-kDa species correspond to regions of the cDNA-deduced sequences of the regulatory and catalytic subunits of protein phosphatase 2A from several sources. Studies reported here also demonstrate that autophosphorylated protein kinases, particularly Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), are excellent substrates for brain PP2A. Furthermore, Ca²⁺-dependent K⁺-depolarization of hippocampal synaptosomes was accompanied by a sequential increase, then decrease, in CaM kinase II phosphorylation level over a 45-s time course. The decrease was blocked by 1 n M okadaic acid. These data demonstrate that the type 2A protein phosphatase is present at the synapses of CNS neurons where its localization could alter the functions of phosphoproteins involved in synaptic plasticity.     

Aberrant activation of CDK5 is involved in the pathogenesis of OPIDN

Exposure to triorthocresyl phosphate (TOCP) may result in a late neurological complication, i.e. organophosphate-induced delayed neuropathy (OPIDN). The aim of this study was to examine changes in levels of cyclin-dependent kinase 5 (CDK5) and of its activator, p35/p25, in the spinal cord of hens treated by TOCP. After exposure to a single dose of TOCP, groups of adult hens were examined in 3, 5, 7, 9, 14, and 18 days after exposure. CDK5, p35/p25 expression and distribution in the lumbar spinal cord were evaluated by immunohistochemistry and Western blotting. The hens showed signs of OPIDN around day 9 after exposure. The number of p (phosphorylated) -CDK5 and p35 positive cells increased significantly. Co-localization and mislocalization of p-CDK5 and p35/p25 was identified and became evident in neurons around the 9th day. Meanwhile, CDK5, p-CDK5, p35, p25 protein levels and p25/p35 ratio were increased, and peaked around the 9th day, then decreased. Some hens' unilateral common peroneal was treated by roscovitine 3 days after TOCP exposure. Axonal transport of these nerves was faster than of their opposite side and of those simply treated by TOCP. These findings indicate aberrant activation of CDK5 may be involved in the pathogenesis of OPIDN.